s41392-024-01888-z by Biogenomics

cellcommunication:newinsightsandclinical implications

JimengSu1,2,3,YingSong1,2,ZhipengZhu1,2,XinyueHuang4,JibiaoFan3,JieQiao5,6,7,8 ✉ andFengbiaoMao1,2 ✉

Multicellularorganismsarecomposedofdiversecelltypesthatmustcoordinatetheirbehaviorsthroughcommunication.Cell–cell communication(CCC)isessentialforgrowth,development,differentiation,tissueandorganformation,maintenance,and physiologicalregulation.Cellscommunicatethroughdirectcontactoratadistanceusingligand–receptorinteractions.Socellular communicationencompassestwoessentialprocesses:cellsignalconductionforgenerationandintercellulartransmissionof signals,andcellsignaltransductionforreceptionandprocessionofsignals.Decipheringintercellularcommunicationnetworksis criticalforunderstandingcelldifferentiation,development,andmetabolism.First,wecomprehensivelyreviewthehistorical milestonesinCCCstudies,followedbyadetaileddescriptionofthemechanismsofsignalmoleculetransmissionandthe importanceofthemainsignalingpathwaystheymediateinmaintainingbiologicalfunctions.Thenwesystematicallyintroducea seriesofhumandiseasescausedbyabnormalitiesincellcommunicationandtheirprogressinclinicalapplications.Finally,we summarizevariousmethodsformonitoringcellinteractions,includingcellimaging,proximity-basedchemicallabeling,mechanical forceanalysis,downstreamanalysisstrategies,andsingle-celltechnologies.Thesemethodsaimtoillustratehowbiological functionsdependontheseinteractionsandthecomplexityoftheirregulatorysignalingpathwaystoregulatecrucialphysiological processes,includingtissuehomeostasis,celldevelopment,andimmuneresponsesindiseases.Inaddition,thisreviewenhancesour understandingofthebiologicalprocessesthatoccuraftercell–cellbinding,highlightingitsapplicationindiscoveringnew therapeutictargetsandbiomarkersrelatedtoprecisionmedicine.Thiscollectiveunderstandingprovidesafoundationfor developingnewtargeteddrugsandpersonalizedtreatments.

SignalTransductionandTargetedTherapy (2024)9:196 ;https://doi.org/10.1038/s41392-024-01888-z

INTRODUCTION

Thecoordinationofcellularactivities,essentialformulticellular existence,iscontingentuponcell–cellinteractions(CCIs)amonga varietyofcelltypesandtissuesthroughoutanorganism.1–3 Cell–cellcommunication(CCC)isanessentialprocessthat profoundlyinﬂuencesanorganism’shomeostasis,development, anddiseaseprocesses.4 Typically,CCCinvolvesinteractionswith secretedligandsandplasmamembranereceptors,yetitalso includessecretases,extracellularmatrixproteins,transporters,and directcell-to-cellcontactmechanisms.5 Differentcellsemploy differentCCCstoensurebiologicaldevelopment,homeostasis, andtissuerepair.

Essentially,CCCisafundamentalcharacteristicofmulticellular organisms.6 Thedynamiccommunicationnetworkestablished betweencellsthroughcollaborationplaysapivotalroleinvarious biologicalprocesses.6–8 Thisinteractionisintegraltothe functioningoflivingorganisms,inﬂuencingcellularmetabolism, energytransformation,maintenanceofphysiologicalfunctions, regulationofgrowth,development,immuneresponses,single-cell functions,andothercriticallifeprocesses.9 Forexample,during immuneresponses,CCCsenableimmunecellstorecognizeand

combatpathogens.Ingrowthanddevelopment,CCCsregulate cellproliferationanddifferentiation,facilitatingthenormal developmentoforgansandtissues.Diseasestypicallymanifest whencellsfailtointeractcorrectlyormisinterpretmolecular information.5

CCCsreflectthefundamentallevelofphysiologicalcommunication,triggeringresponsestointernalorexternalenvironments essentialforsurvival.Whencellscommunicatewitheachother, extracellularsignalstypicallyinduceintracellularsignaltransductioncascades,leadingtocellularresponsessuchaschangesinthe cytoskeleton,metabolism,orgeneexpression.10 Theregulation andfeedbackmechanismsatvariouslevelsofthesetransduction cascadesmodulatethepathway’sactivityovertime.11 Signal pathwaysarethebasisofinternalcommunicationandresponseto theexternalenvironmentinorganisms.Theyareresponsiblefor convertingextracellularsignalsintointracellularresponses, therebyregulatingcellbehaviorandfunction.Thesepathways involveaseriesofprecisemolecularevents,includingthe receptionofsignals,amplification,distribution,andthetriggering ofspecificcellularresponses. 12,13 Criticalcellulardeterminations, suchascytoskeletalreorganization,cellcyclecheckpoints,and

1InstituteofMedicalInnovationandResearch,PekingUniversityThirdHospital,Beijing,China; 2CancerCenter,PekingUniversityThirdHospital,Beijing,China; 3CollegeofAnimal ScienceandTechnology,YangzhouUniversity,Yangzhou,Jiangsu,China; 4BiomedicalResearchInstitute,ShenzhenPekingUniversity-theHongKongUniversityofScienceand TechnologyMedicalCenter,Shenzhen,China; 5StateKeyLaboratoryofFemaleFertilityPromotion,DepartmentofObstetricsandGynecology,PekingUniversityThirdHospital, Beijing,China; 6NationalClinicalResearchCenterforObstetricsandGynecology(PekingUniversityThirdHospital),Beijing,China; 7KeyLaboratoryofAssistedReproduction (PekingUniversity),MinistryofEducation,Beijing,Chinaand 8BeijingKeyLaboratoryofReproductiveEndocrinologyandAssistedReproductiveTechnology,Beijing,China Correspondence:JieQiao(jie.qiao@263.net)orFengbiaoMao(fengbiaomao@bjmu.edu.cn) Theseauthorscontributedequally:JimengSu,YingSong.

Received:29December2023Revised:9May2024Accepted:2June2024

Fig.1 MilestoneeventsofCCCresearch.KeyeventsinthedevelopmentofCCCwereretrospectivelysummarizedfrom1951tothepresent day.Detailedinformationonmilestoneeventsarenarratedinthisreview

programmedcelldeath,arecontingentuponthestringent temporalregulationandthespeciﬁcspatialdistributionof activatedsignaltransducers.14 Understandinghowthesepathwaysaredisruptedindiseasesoffersthepossibilityfordeveloping newtherapeuticapproaches.15–17

ThecomplexityofCCChasbeenrecognizedaspartofthe molecularmechanismsofdevelopmentalbiology,carcinogenesis, andorgandysfunction.18 ExploringCCCdynamicchangesunder differentconditionsprovidesdeeperinsightsintotheunderlying mechanismsofdiversebiologicalprocessesandhelpselucidate themechanismsbehindtheonsetandprogressionofdiseases. Overthedecade,single-cellRNAsequencinghasgainedwidespreaduseacrossmultipleresearch ﬁeldstoinvestigatethecritical roleofligand–receptordynamicsinintercellularcommunication.19 Technologieslikesingle-cellRNAsequencing(scRNA-Seq) empowerresearcherstoexploretheintricatecommunication patternsbetweendifferentcelltypeswithinmulticellularorganisms,offeringfreshperspectivesoncellcommunicationmechanisms,cellfunctions,andtheorganizationofcellpopulations.The analysisofintercellularcommunicationassistsinunderstanding theinterplaybetweencells,dissectingcommunicationnetworks, uncoveringvariouscellinteractionsinthedevelopmentalprocess, exploringthetumorimmunemicroenvironment,andidentifying potentialtherapeutictargetsfordiseases.20 Therefore,identifying andquantifyingintercellularsignalingpathwayshavebecome standardpracticesacrossdiversedisciplines.

Activatingspeciﬁccellsignalingpathwaysthrough ligand–receptorinteractions(LRIs)constitutesafundamental modeofcellcommunicationandisintricatelylinkedtovarious degenerativeprocessesanddiseases.Differentcelltypesshare commonbiologicalelementsfacilitatingtheseinteractions, encompassingligands,surfacereceptors,adhesionproteins, intracellularadaptors,aswellasglycans,lipids,cytoskeletons, andscaffoldingproteins.21 Comprehendingtheorchestrationof biophysical,genetic,andbiochemicaleventsinCCCsbythese sharedcomponentsacrossvariouscelltypesiscrucialfor developingclinicaltherapiesbasedonproteinsandcellsthat eithermodulateorutilizeintercellularcommunication.22,23 The analysisofLRIsprovidesthefoundationforcomprehendingcell behaviorandresponsestoneighboringcells.24

Historically,CCCresearchhasprimarilybeenconﬁnedto experimentsconductedinvitroinvolvingoneortwotypesof cellsandalimitedsetofgenes.Withadvancementsinscienceand technology,dataacquisitionatthesingle-celllevelenablesthe detectionoflow-abundancegenesandprovidesarobust foundationforcellcommunicationstudy.Inrecentyears,multiple researcheffortshaveconcentratedonintercellularsignalingby employingeithertheco-expressionofallgenesorparticularcell markers,25,26 theresemblanceinexpressionpatterns.27 orthe characteristicsofregulatorynetworks.28 UnderstandingLRIsisan

effectiveapproachtounderstandcellularcommunicationatthe single-celllevel,andamultitudeofresearchendeavorsare dedicatedtoformulatingstrategiestoconstructcellularcommunicationnetworksbasedontheseinteractions.Harnessingthese technologies,manylaboratorieshavedevelopedvariousalgorithmsandsoftwaresforcellcommunicationresearch.

Thereviewcomprehensivelyoutlinestheexperimentaland computationalCCCmethodsrootedinchemistryandbiologyto decodethecomplexitiesofCCCs.Itextensivelyexamineshow biologicalfunctionsrelyonCCCstoregulatecrucialphysiological process,includingtissuehomeostasis,celldevelopmentaswellas immuneresponses.5,24 Furthermore,thisreviewshedslightonthe roleofCCCmechanismsinregulatingvariousdiseases,which havenotonlyexpandedourunderstandingofCCCbutalsopaved thewayforinnovativeclinicaltreatments.

RESEARCHHISTORYANDMILESTONEEVENTSOFCCC

Inmulticellularorganisms,cellshaveevolveddifferentintercellular communicationmodestodevelopandregulatetheircoordinated functions.29 Atthemacroscopiclevel,directphysicalcontacts betweenadjacentcellsleadtotheformationoftissuesandbarrier structures,whileatthemicroscopicscale,theydrivechangesin cellularsignalingpathwaysandactivationstates.24 Comprehendinghowbiologicalcomponentssynergizetoorchestratebiochemical,genetic,andbiophysicallymediatedcellinteractionevents amongdiversecelltypesconstitutestheessenceofenhancingour understandingofthebiologyunderlyingCCCs(Fig. 1).

Identifyingcellsurfacereceptorsandtheirligands,suchas growthfactors,iscrucialforunderstandinghowcellsperceiveand respondtoexternalsignals.Duringaresearchendeavorexploring thespecificgrowth-stimulatingimpactsofmousesarcomaonthe sensoryandsympatheticnervoussystemsofchickembryos,itwas unveiledthatmousesarcomahadtheabilitytogenerateaspecific factortospecificallypromotethegrowthanddevelopmentof nervecells.30 Later,thisfactoriswell-knownasnervegrowth factor(NGF)andstandsastheinauguralgrowthfactortobe discovered,unveilingthepivotalroleofextracellularfactorsin modulatingcellgrowthanddifferentiation.Thisrevelationhas exertedaprofoundimpactontheevolutionofneuroscienceand cellularbiology.Withadeepeningunderstandingofcellular signalingmolecules,researchershavebeguntoinvestigatehow signaltransductionpathwaysalterinhumandiseases.Disrupting and/oralteringthesecellinteractioneventscanleadtosevere downstreampathophysiologicaleffects.Researchonobese hyperglycemicmousemodelshasrevealedthatinsulinresistance isassociatedwithdysfunctionofinsulinreceptors.31 Insulinexerts itseffectsbybindingtoitsreceptorsonthecellsurface.Insulin resistancemaybecausedbyareductionofinsulinreceptorsor receptordysfunction,leadingtodecreasedefficiencyofinsulin

signaltransduction.Evenifinsulinsuccessfullybindstoits receptor,certaincomponentsofthesignalingpathwaymaybe impaired,affectingthebiologicaleffectsofinsulin.Thestudiesof themolecularmechanismsofinsulinresistanceenhanceour understandingofhowcellfunctionanddiseasestatescanbe inﬂuencedatthesingle-celllevelbyregulatingtheinteractions betweensignalingmoleculesandreceptors,offeringpotential newtargetsforthetreatmentofmetabolicdiseases.Communicationerrorscanleadtodiseasessuchascancermetastasis,motor neurondiseases,virus-hostinteractions,anddiabetes.Therefore, researchintoCCCscanenhanceunderstandingofdisease mechanismsandfacilitatethedevelopmentof pharmaceuticals.32–35

Opticalmicroscopyhasbeenwidelyusedasapowerfulmeans foroveracenturytovisualizethesitesofCCCsandunderstand thespatialororganizationalstructuresunderlyingtheseinteractions.Earlyexamplesofstudyingcellcontactinterfacesbasedon microscopyincludethedirectobservationofcelldissociationin spongesandtheaggregationofcellsintotissue-likestructuresin higheranimalsusingopticalmicroscopy.36,37 Withtheimprovementof fluorescentdyesandopticalsystems,scientistsbeganto utilize fluorescentmaterialstostudycellsandtissues.Anearly study firstreportedtheprimarystructureofthegreen fluorescent protein(GFP)fromthejellyfishAequoreavictoria.38 Itnotonly identifiedtheaminoacidsequenceofGFPbutalsolaidthe foundationforsubsequentresearchutilizingGFPasareporter genetovisualizeandtrackspecificproteins,organelles,and intercellularcommunicationwithincells.GFPnotonlyenablesthe directobservationofgeneexpressioninlivingcellsbutalsoallows forthetrackingofspecificprocesseswithincellsthroughGFPtaggedproteins.Withoutaffectingthegrowthanddevelopment ofthehostcell,the fluorescencecanbestablyinheritedby offspring,makingitanidealtoolfortrackingandstudyingthe dynamicsofgeneexpression.39

Super-resolutionmethodologieshavemadesignificantstrides, transcendingthislimitationthroughtechniquessuchasnearfield,40 stimulatedemissiondepletion,41 structuredillumination,42,43 andreversiblesaturableoptical fluorescencetransitions microscopy.44 Yet,theobjectiveremainstoachievea fluorescence technologycapableofattainingresolutionsclosertothe molecularscale.AtechniqueknownasGFPreconstitutionacross synapticpartners(GRASP)employsthefusionoftwononfluorescentsplitGFPfragmentsontointeractingpartnersonopposing cellstodetectCCCs.45 Uponclosecontactbetweencells,thesplit proteinsassociate,reconstitutingtheGFP.Thismethodhasbeen expandedtoincludeothersplit fluorescentproteinfragments,YFP (yellow)andCFP(cyan),forthesimultaneousimagingofmultiple synapticinteractionfactors.46 Anadvancedimagingtechnique knownasopticalsuper-resolutionmicroscopy,specificallyphotoactivatedlocalizationmicroscopy,enablestheobservationof fluorescentproteinswithincellsatnanometerresolution.47 Ontheotherhand,high-throughputtechnologiesarepowerful andeconomicaltoolsforultra-high-throughputtranscriptionand proteinanalysis,48–52 whichhavegreatlyacceleratedourunderstandingofthegeneexpression,regulationandnetwork complexityofmammaliancells.53–59 Forexample,thedevelopmentofDrop-Seq.60 andinDrop.61 hasenabledsimultaneous analysisofalargenumberofsinglecells,significantlyimproving sequencingefficiencyandenhancingourunderstandingof complextissuesandcellularbiology.Understandingthespatial organizationofcellswithintissuesandhowtheycommunicateis essentialfordecipheringtheprinciplesoftissuearchitectureand organfunction.62,63 Thus,spatialtranscriptometechnology, namelymultiplexederror-robust fluorescenceinsituhybridization (MERFISH),wasdevelopedtosimultaneouslymeasurethecopy numbersandspatialdistributionofhundredstothousandsofRNA speciesinindividualcellsbyusingsingle-moleculeFISH fluorescentprobesthatbindwithhighspecificitytothedesiredRNA

targets.64 Duetoitshighspeciﬁcity,sensitivity,andspatial resolution,MERFISHhasawiderangeofapplicationsinbasic biologyandmedical ﬁelds.65,66

Meanwhile,spatialproteomeapproachesareemerging research fieldsfocusingonunderstandingthequalitativeand quantitativeaspectsofproteincompositionwithinsinglecells. Immunohistochemistryandimmunofluorescencetechniques, combinedwithmicroscopicimagingtechnology,candisplaythe distributionanddensityofcellsintumortissuesamples,aswellas thephysiologicalandbiochemicalactivitiesinvolvedbydifferent cells.67,68 Forexample,CODEX(CO-DetectionbyindEXing)62 and CellDIVE69 arecutting-edgehigh-dimensionalimagingtechnologiesthathaverevolutionizedthestudyofcellcommunication andtissueanalysis.ThecoredesignprincipleofCODEXistolabel eachantibodywithaspecificoligonucleotide “barcode”,ofwhich thecomplementarysequenceisboundwiththe fluorescentdyes usedforsubsequentimaging.62,70 Incontrast,eachantibodyused inCellDIVEisdirectlylabeledwith fluorescentdyes,followedby multipleroundsofstaining,imagingand fluorescencequenching. Therefore,bothCODEXandCellDIVEofferhigh-dimensional imagingofdozensofproteinswithinindividualcells,enabling researcherstoanalyzethespatialorganizationofcells,their interactions,andsignalingstateswithintissues.Furthermore, novelsingle-cellspatialinsituimagingtechnologies,suchas GeoMxDSPspatialmulti-omicstechnology,breakthroughthe limitationsofthenumberofdetectedproteinsandenable spatiallyprofiling570+ proteintargetsandthewholetranscriptomeseparatelyorsimultaneously.63,71 Collectively,these advancedspatialsingle-cellimagingtechnologieswilldrive deeperinsightsforcelltyping,tissuephenotyping,cell–cell interactions,cellularprocesses,andbiomarkerdiscovery.

MOLECULARMECHANISMOFCCC

Cellsignaling,whichinvolvestransmittinginformationbetween cellsorsubcellularcomponents,isaninherentcharacteristicof livingorganisms.Indiversetissuesandorgans,cellsignaling facilitatescommunicationandhomeostasis,whicharevitalfor cellularinteractionswithintheirlocalenvironment.Signalscan propagatethroughvariousmechanisms,includingchemical alterations,mechanicalforces,ortheirsynergisticeffects.A multitudeofextracellularsignalsandcellularmembraneproteins triggerselectiveintracellularpathways,influencingcrucialcellular outcomessuchassurvival,apoptosis,growth,motility,differentiation,andspecificfunctionslikemuscularcontraction,synaptic activity,orthrombocyteactivation.72 Typicalexamplesarethe triggeringofthetransforminggrowthfactor-β (TGF-β)signaling pathwayinthecontextoftissue fibrosis.73 andtheexcessive activationoftheRassignalingpathwayinnumerouscancer types.74 Overthelastdecades,extensiveresearchintocell signalingpathwayshasculminatedinthecreationoftherapeutics rootedinbiologicalscience,andthecomplexityrevealedbydrugs currentlyinclinicalusecontinuestouncoverfurtherinsightsinto theextentofinteractionsbetweensignalingnetworks.75 Cellular communicationencompassestwoessentialprocesses:cellsignal conduction,focusingonthegenerationandintercellulartransmissionofsignals,andcellsignaltransduction,whichemphasizesthe receptionofsignalsandhowsignalsareconvertedandprocessed uponreceipt(Fig. 2).

MechanismsofCCCinphysiologicalhomeostasis

Cellularcommunicationinvolvescellsrecognizing,receiving,and respondingtoexternalsignalmolecules,whichcanbelight, electricity,orchemicalmolecules.Finally,theinteractionof receptorscanactivateaseriesofdownstreamphysiologicaland biochemicalprocesses,whichplayanimportantroleincoordinatingcellfunction.Cellularcommunicationinvolvesthetransmissionofsignalsfromsignalgenerationtosignaltransduction.Cell

Fig.2 RepresentativesignalpathwaysofCCC.Cellularcommunicationistheprocessofsignalconstructiontosignaltransduction.The interactionsofligandsandreceptorsusuallyaffectcellmetabolismandenergytransformationofdifferentcelltypestomaintainthenormal functionoforganisms.Ligandsareactivesubstancescapableofspecificallybindingtoreceptors.Receptorsspecificallyrecognizeandbindto signalingmolecules,convertingthemintointracellularsignalstoperformspecificphysiologicalfunctions.Oneofthemajorsignaling pathwayswithinthesignaltransductionpathwayareGPCRspathways,includingPKAandPKCsystems.Theothersareenzyme-coupled receptorpathways,includingPKGandMAPKsystems.ACadenylatecyclase,cAMPcyclicadenosinemonophosphate,cGMPcyclicguanosine monophosphate,COcarbonmonoxide,DAGdiacylglycerol,ERKextracellularregulatedproteinkinases,GCguanylatecyclase,GDPguanosine5’-diphosphate,GPCRG-protein-coupledreceptor,GTPguanosinetriphosphate,IP3inositoltrisphosphate,IP3Rinositoltrisphosphate receptor,MAPKmitogen-activatedproteinkinase,MEKmitogen-activatedextracellularsignal-regulatedkinase,NOnitricoxide,PiPIP2 phosphatidylinositol-4,5-bisphosphate,PKAproteinkinaseA,PKCproteinkinaseC,PKGproteinkinaseG,PLCphospholipaseC

signaltransductionistheprocessthroughwhichbiological information(excitationorinhibition)istransformedandtransmittedbetweencellsorwithincells,leadingtovariousbiological effects.Thistypicallyreferstotransmembranesignaltransduction, whereinbioactivesubstancesactivateorinhibitcellfunction throughreceptorsorionchannels.Generally,thechemical substancesinvolvedinintercellularsignalcommunicationor intracellularsignaltransductionareknownassignalmolecules, whilesmallmoleculesspecializedincarryingbiologicalinformationarecalledmessengermolecules.Thechainsofsignal moleculesthatcompletetheconversionandtransmissionof biologicalinformationwithinorbetweencellsarereferredtoas signaltransductionpathways.Therefore,theessenceofcellsignal transductionliesintheintracellulartransformationandtransmissionofbiologicalinformationthroughspeciﬁcsignaltransduction pathways,whichmayinvolveregulatingthegeneexpression processesofrelatedfunctionalproteins.

Thestepsofcellsignalconduction

Productionandreleaseofsignalingmolecules:Certaincells, suchasneuronsorendocrinecells,reacttoexternalstimulior internalneedsbycreatingandreleasingsignalingmoleculeslike neurotransmitters,hormones,orchemokines.Thesemolecules serveasmessengerstocommunicatemessagestoneighboring cells.

Neurons,astheprimarycellsthatparticipateininformation processing,comeinamyriadofdistinctcelltypesdifferentiated bymorphology,location,connectivity,andchemicalproperties.76 Thevariouschemicalsthattransmitinformationbetweenneurons areknownasneurotransmitters.Owingtothecentralroleof neurotransmittersincerebralfunction,neurotransmitterreceptors

alongwithotherproteinsengagedinthesynthesisand deactivationofneurotransmittersemergeascriticaltargetsin thedevelopmentofcurativemedicationsformentalandnerve disorders,acheandmanyotherconditions.77 Asgaseous neurotransmitters,suchasnitricoxide(NO)andcarbonmonoxide (CO),playaregulatoryroleinvasodilationandneuraltransmission.78 79 Thenervoussystemtypicallyenablesinformationtobe transmittedrapidlybetweendifferentregionsofthebody.

Incontrast,hormonalcommunicationispredicatedonthe synthesisanddisseminationofaplethoraofglandularhormones, coupledwiththeirtransportationviathebloodstream,makingit moresuitedtosituationsrequiringbroaderandmoresustained regulatoryactions.Thesetwosystemsofcommunicationare mutuallycomplementary,withneuralstimulicapableofaffecting thesecretionofcertainhormones,andconversely.80 Certain hormonesaretailoredtointeractexclusivelywithalimitedarray oftargetcells,whereasothersexertinﬂuenceacrossabroad spectrumofcelltypesthroughouttheorganism.Topreserve homeostasisandadapteffectivelytoenvironmentalalterations, thebiosynthesisandreleaseofhormonesaresubjecttorigorous regulation.Thisregulatorymechanismisachievedthrougha complexinterplayamongmultiplehormones,whichreciprocally regulateoneanother,ratherthanbeinggovernedbyasolitary hormone.Hormonesplayapivotalroleinorchestratinga multitudeofbodilyfunctions,encompassinggrowthanddevelopment,metabolicprocesses,electrolyteequilibrium,andreproductivefunctions.

Cytokinesareproducedbyspecificcells(suchasimmunecells, endothelialcells,etc.)inresponsetospecificstimuli,suchas infection,injury,inflammatoryresponses,ortheactionofother cytokines,andarereleasedintotheextracellularenvironment.

Cytokinesarecapableofactivatingavarietyofcelltypeswithina specifictissueorinitiatingdiversifiedsignalingpathwayswithina specificcelltype,exemplifiedbyinterleukinsandinterferons, regulatingvariousphysiologicalprocesses,encompassingimmunity,development,growth,andtissuerepair.81,82 Servingas pivotalmediatorsofintercellularcommunicationwithinthe immunesystem,dysregulationincytokineexpressionortheir intracellularsignalingpathwaysdisruptsimmunehomeostasis, precipitatingtheonsetofpathologiessuchaschronicinflammation,autoimmunesyndromes,andmalignanttumors.83

Theproductionofsignalingmoleculesinitiateswithgene expression.Specificstimuli,suchasextracellularsignalsor changesininternalstates,triggerthetranscriptionandtranslation ofspecificgenes,leadingtotheproductionofproteinsorsmall moleculesassignalingmolecules.Priortomaturationand activation,theseproteinsignalingmoleculestypicallygothrough asequenceofpost-transcriptionalmodifications(suchassplicing) andposttranslationalmodifications(suchasphosphorylationand glycosylation).Meanwhile,somesmall-moleculesignalingsubstancesaresynthesizedwithinthecellthroughspecificbiochemicalpathways.Beforetheirrelease,signalingmolecules accumulateandarestoredinspecificorganelleswithinthecell. Forinstance,neurotransmittersareusuallystoredinsynaptic vesiclesofpresynapticneurons.84

Whencellsreceivestimulitoreleasesignalingmolecules,such aselectrical,chemical,ormechanicalsignals,thevesiclesstoring thesesignalingmoleculesmovetothevicinityofthecell membraneandfusewithit.Throughexocytosis,thesignaling moleculesarereleasedoutsidethecell,aprocessthatis particularlyimportantforproteinsandcertainlargemolecular signalingmolecules.Ontheotherhand,somesmallmolecules andlipophilicsignalingmoleculesdirectlypassthroughthecell membranetoenterorexitthecellwithoutvesicle-mediated processes.BesidesNOandCO,hydrogensulﬁdeisanotherwellrecognizedgaseoussignalingmolecule.85 Thesegaseousmoleculesareuniqueintheirmodeofactionastheycanfreelydiffuse acrosscellmembranes,enablingrapidsignalingwithouttheneed forspeciﬁcreceptorsortransportmechanismsfortheirrelease andaction.

Transmissionofsignalingmolecules:Signalingmoleculescan reachtargetcellsviadiffusion,bloodcirculation,directcontact,or intercellularjunctions.Inthecontextofmulticellularorganisms, fourfundamentalformsofintercellularsignalingexist:autocrine signaling,paracrinesignaling,signalingthroughgapjunctions, andendocrinesignaling.Autocrinesignalingisprevalentintumor cells,whereincellssecreteligandstoinduceresponsesvia homologousreceptorsexpressedonthesamecell.Paracrine signalingaffectsnearbycellsbysecretinglocalchemical mediatorsintotheextracellular ﬂuid,whichactonadjacenttarget cellsthroughlocaldiffusion.Signaltransmissionacrossgap junctionsinvolvesmoleculespassingdirectlybetweencells. Endocrinesignalingoccurswhenendocrinecellssecretesubstancesintothebloodstream,distributingthemtovariousbody partsviacirculation.

Forlong-distancesignals,thebloodstreamistheprimarymode oftransmission.Endocrinecellsregulatetheproductionand releaseofhormonesbymonitoringtheconcentrationofspeciﬁc substancesintheblood,suchasglucose,electrolytes,andother hormones.Thesehormonesarethentransportedtovariousparts ofthebodythroughthebloodstream,exertingregulatoryeffects ondistanttargetcells,therebymaintainingphysiologicalequilibriumandadaptingtochangesintheexternalenvironment.86 For localsignals,transmissionthroughintercellularspacesordirect cellcontactismorecommon.Neurotransmittersprimarilytransmit informationthroughparacrinesignaling.Theyareproducedand releasedbyneuronsatthepresynapticend,andtransmitted

acrossthesynapticgaptotheadjacentpostsynapticneuronor effectorcell.Oncereleased,neurotransmittersdiffuseacross synapses.Inconjunctionwiththeprimarilychemicalsynapses, electricalsynapsesalsoexist,facilitatingtheintercellular flowof ionsviagapjunctions.Electricalsynapsesenablethetransmission ofsimpleelectricalsignalsamongneurons,whilechemical synapsesallowexcitation,inhibition,andcomplexityofbiochemicalinformationtobepassedamongcells.77 Cytokinestypicallyact onneighboringcellsthroughparacrinesignaling,especially duringimmuneresponsesandinflammation.However,insome cases,theycanalsobereleasedintothebloodstreamtoregulate systemicimmuneresponsesandinflammation.Certaincytokines thatexistinamembrane-boundformrequiredirectcontactwith receptorsonadjacentcellstotransmitsignals.87,88

Bindingofsignalingmoleculestoreceptors:Signalingmolecules bindspecificallytoreceptorsonthesurfaceorinsidethetarget cell,formingreceptor-ligandcomplexesthatactivatethereceptor. Receptorsareproteinsorglycoproteinsdesignedtorecognizeand bindspecificsignalingmolecules,convertingexternalsignalsinto internalones.Hormonestraveltotargetcellsviathebloodstream andbindtospecificreceptorslocatedonthecellsurfaceorwithin thecell.Thesereceptorshavebindingsitesthatarehighly complementarytothehormonemolecules,allowinghormonesto specificallyrecognizeandbindtotheirreceptors.Uponbindingto itsreceptor,thehormonetypicallyinducesaconformational changeinthereceptor,therebyactivatingit.Forcellsurface receptors,thisconformationalchangecandirectlytrigger intracellularsignalingpathways.Forintracellularreceptorssuch assteroidhormonereceptors,thehormone–receptorcomplex oftentranslocatestothenucleus,interactswithDNA,and influencestheexpressionofspecificgenes.

Neurotransmittermolecules,liberatedfrompresynapticvesicles, traversethesynapticcleftandbindtoproteinsonthe postsynapticneuron’ssurfacemembraneknownasneurotransmitterreceptors,alteringthefunctionofthepostsynapticneuron. Therearetwotypesofneurotransmitterreceptors:ligand-gated ionchannelreceptors(LGICs)andG-protein-coupledreceptors (GPCRs).LGICreceptorsareproteinsspecificallydesignedto rapidlytransducechemicalsignalsofneurotransmittersdirectly intoelectricalresponses.89,90 Apartoftheproteinisdedicatedto bindingwithneurotransmittermoleculesontheextracellularside oftheprotein.Theportionoftheproteinembeddedwithinthe cellmembraneactsasanionchannel,creatinga fluid-filled passageinthemembraneforthetransitofchargedions,butions areunabletopassacrosslipidorothersolidmembrane constituents.Thissynaptictransmissionmodalityallowsforthe direct flowofionsacrosstheoutercellmembranerapidly.Whena neurotransmitterbindstothereceptor,theexchangeofnucleotideguanosine-5’-diphosphate(GDP)andguanosinetriphosphate (GTP)ontheprotein’sintracellularsideisexpedited,culminating inthesplitoftheGproteininto α-and β/γ-subunits,bothtwo typesofsubunitscanactupon “effector” proteins,alteringcellular biochemistry,physiology,andgeneexpression,initiatingintracellularchemicalsignalingevents.84,89,90

Cytokinesareclassifiedassecretedgrowthfactorsthatinstigate signaltransductionwithintargetcellsthroughbindingwiththe extracellulardomainsofcellsurfacereceptors,forcingreceptor dimerization.91 Mostcytokinescontainbindingsitesforbothhighaffinityandlow-affinityreceptors.92,93 Typically,thehigh-affinity receptorsubunitactsasacytokine-specific,privatereceptorthat determinescellspecificityaswellasthecytokine’sdose sensitivity.94 Incontrast,thelow-affinityreceptorsubunitsare commontobesharedamongvariouscytokinesandmainlyhave animpactontheefficiencyofcomplexassembly,thereby affectingthemaximumstrengthanddurationofreceptor signaling.92,95

Thestepsofcellsignaltransduction

Signaltransductionandamplification:Theactivationofreceptorssetsoffacascadeofcellularresponsesinternally.Signalsare transducedandamplifiedwithinthecellthroughmechanismslike secondmessengers,switchproteins,enzymecascades,etc. Secondmessengersaresmallmolecularcompoundsthatcarry informationwithinthecell.Thesecondmessengersplayacrucial roleincellsignaling,capableoftransformingtheactivationofa cellsurfacereceptorintotheactivationofmultiplemolecules withinthecell,therebyamplifyingandconveyingsignals internally.Thisprocessactivatesorinhibitsspecifictargetproteins andenzymes,triggeringacascadeofdownstreamreactions. Throughthesereactions,secondmessengersareinstrumentalin regulatingamyriadofcellularfunctions.Thediscoveryofthis signaltransductionpathwayhaslaidthefoundationalframework forunderstandinghowcellscommunicateviachemicalsignals.

Anearlystudyelucidatedforthe ﬁrsttimetheroleofcyclic adenosinemonophosphate(cAMP)asasecondmessengerwithin thecell,demonstratingitscapacitytotranslatethecellsurface receptor’sresponsetohormonesandneurotransmittersinto intracellularbiochemicalactivities. 96 Whenexternalsignaling moleculessuchasadrenalinebindtoGPCRs,theassociatedG proteinsareactivated.The α subunitoftheGproteinactivates adenylatecyclase(AC),convertingATPintocAMP.cAMP,serving asasecondmessenger,activatesproteinkinaseA(PKA).Incardiac cells,theincreaseincAMPleadstotheactivationofPKA,which phosphorylatesL-typecalciumchannels,increasingtheinﬂuxof calciumions,therebyenhancingthecontractileforceofthe heart.97–99

Calciumions(Ca2+)areimportantsecondmessengersinvolved invarioussignalingpathways.Whenexternalsignalssuchas neurotransmittersorhormonesactoncells,theopeningof calciumchannelsorthereleaseofCa2+ fromtheendoplasmic reticulumcancauseanincreaseinintracellularCa2+ concentration.Subsequently,Ca2+ bindstocalmodulinandthenactivates downstreamenzymeslikeCa/calmodulin-dependentprotein kinaseII(CaMKII),affectingvariousprocesseswithinthecell. Duringneurotransmission,thereleaseofneurotransmitterscauses theopeningofcalciumchannelsonthepostsynapticmembrane, allowingCa2+ to ﬂowintothecell,activatingsignalingpathways relatedtolearningandmemory,suchastheactivationofCaMKII, whichpromotesthestrengtheningofsynapses.100–102

Switchproteins,likeGproteinsandRas,regulatetheopening andclosingofsignalingpathways.RasproteinisasmallGTPase involvedinregulatingsignalingpathwaysforcellproliferationand differentiation.Whengrowthfactorssuchasepidermalgrowth factor(EGF)bindtotheirreceptorepidermalgrowthfactor receptor(EGFR),EGFRactivatesRas,causingRastoswitchfroma GDP-boundstatetoaGTP-boundactivatedstate.103 104 The activationofRaspromotestheactivationofthemitogen-activated proteinkinase(MAPK)orextracellularsignal-regulatedkinase (ERK)pathway,affectingtheexpressionofcellcycleregulatory proteinssuchascyclinD,andfacilitatingthecelltoentera proliferativestate.

Enzymecascadesinvolveaseriesofenzymesthatactivateor inhibiteachother,amplifyingandregulatingsignals(e.g.,PKA, MAPK).IntheMAPKpathway,Raf(MAPKKK)activatesMEK (MAPKK),whichinturnactivatesERK(MAPK).Eachstepof activationcanleadtothephosphorylationofmultipledownstreamproteins.Inresponsetocellularstress,thep38MAPK pathwayisactivated,leadingtoanincreaseintheexpressionof inﬂammatoryresponseproteinssuchastumornecrosisfactor α (TNF-α),participatinginthecell’sresponsetostressand inﬂammation.105

Cellresponse:Signaltransductionultimatelyinﬂuencesthecell’s physiologicalfunctions,includingchangesinmetabolicactivity, regulationofgeneexpression,alterationsinshape,ormovement.

Differentcelltypesandstatesmayexhibitvaryingresponsesto thesamesignalingmolecule.Insulinactivatesthephosphoinositide3-kinase(PI3K)/Akt(alsoknownasProteinKinaseB,PKB) signalingpathwaythroughtheinsulinreceptor.Then,PI3K/Akt signalingpathwaycanpromotethesurfaceexpressionofthe glucosetransporterGLUT4andincreasecellularglucoseuptake, supportingenergyproduction,andmusclecontraction.106–108 Similarly,inlivercells,insulinalsoenablestheactivationofthe PI3K/Aktsignalingpathwayandthuspromotestheactivationof glycogensynthase,increasingglycogensynthesis,andlowering bloodglucoselevels.109 Incontrast,TNF-α canactivatethenuclear factorkappa-light-chain-enhancerofactivatedBcells(NF-κB) signalingpathwaythroughitsreceptorandthenpromotethe expressionofinﬂammatoryresponseproteins,participatingin immuneresponsesandcellrepairprocesses.110 However,insome cancercells,thesamesignalingpathwaymaypromotethe survivalandproliferationofcancercellsbecausethesecellsmay haveacquiredresistancetoapoptosissignalsinducedbyTNF-α 111

Terminationorreductionofsignalingresponse:Tomaintainthe cell’ssensitivityandadaptabilitytoexternalstimuli,responsesto signalingmoleculesmustbeterminatedorreduced.Thiscanbe achievedthroughreceptordesensitization,receptordownregulation,degradation,hydrolysisofsecondmessengersorswitch proteins,andnegativefeedbackregulation,whichinvolves downstreameffectormoleculesinhibitingupstreamsignaling molecules,formingaclosedloop.Prolongedexposuretohigh concentrationsofagonists(suchasadrenaline)leadstodesensitizationanddownregulationofthecorrespondingGPCRs.The desensitizationprocessofteninvolvesphosphorylationof thereceptor,whichattracts β-arrestinproteinstobindwiththe receptor,preventingfurtheractivationofGproteinswhile promotingreceptorinternalization.112,113 Theinternalizedreceptorsmaybetransportedtolysosomesfordegradation(downregulation),orbedephosphorylatedandrecycledbacktothecell surface.Thisprocessleadstothedecreaseofreceptorsonthecell surface,reducingthecell’ssensitivitytotheagonist. TheriseandfallofcAMPlevelsarecrucialforsignal transmission.ThedegradationofcAMPiscatalyzedbycAMP phosphodiesterase,whichconvertscAMPtoAMP,thereby terminatingthesignaltransmissionmediatedbycAMP.114,115 TheMAPK/ERKsignalingpathwayactsimportantlyincell proliferationanddifferentiation.Activationofthispathway promotesthephosphorylationofasequenceofdownstream effectormolecules,consistingoftranscriptionfactors,thus affectinggeneexpression.Meanwhile,theMAPKpathwayalso activatescertainphosphatases,suchasDUSP(dual-speciﬁcity phosphatases),whichcandephosphorylateandinhibitcomponentsoftheMAPKpathway,suchasERK,forminganegative feedbacklooptosetlimitsonsignalstrengthandduration, preventingoverreaction.116 Thesemechanismstogetherensurea dynamicbalanceofsignaltransduction,allowingcellstomake adaptiveadjustmentstocontinuousorexcessivestimuli,maintainingthenormalfunctioningofphysiologicalfunctions.

Majorsignalingpathway

Withinthesignaltransductionpathway,areceptorisaprotein withinacellresponsibleforreceivingandtransducinginformation.Receptorsinthecellmembranearetermedmembrane receptors,whilethoseinthecytoplasmandnucleusareknownas cytoplasmicandnuclearreceptors,respectively.Ligandsareactive substancescapableofspecificallybindingtoreceptors.Receptors specificallyrecognizeandbindtosignalingmolecules,converting themintointracellularsignalstoperformspecificphysiological functions.Cellsignalstypicallybeginwithprimarymessengerslike growthfactors,hormones,andionsinstigateasequenceofsignal transductionprocessesviamembrane-boundorintracellular receptors.Thismechanismincorporatesmultiplefeedback

systemsandmanyintracellularchemicalsclassiﬁedassecond messengers,consistingofcAMP,cyclicguanosinemonophosphate(cGMP),calciumions,etc.117

Theconjunctionofaligandwithitsspeciﬁcreceptortriggersa distinctcellularsignalingroute.118 Therearetwotypesofsignal transductionpathwaysbasedonthenatureoftheligandand receptor.Oneisthenuclearreceptor-mediatedsignaltransduction,whereinlipid-solubleligandsenterthecellthroughsimple diffusionanddirectlybindtocytoplasmicornuclearreceptors, typicallyinﬂuencinggeneexpression.Anothertypeinvolves water-solubleligandsorphysicalsignals,whichinitiallyacton themembranereceptorandproduceeffectsthroughtransmembraneandintracellularsignaltransductionmechanisms.These signaltransductionreceptorsincludevarioustypes,including GPCRs,enzyme-coupledreceptorsandionchannel-linkedreceptors.Itshouldbenotedthatmostmembranereceptor-mediated signaltransductionpathwayscanalsoaltertranscriptionfactor activityandaffectgeneexpression.

GPCRspathway.GPCRsconstitutethemostextensiveandvaried typeofmembranereceptorsineukaryoticsystem.Gproteins composedofthreesubunitsseparately:alpha(α),beta(β),and gamma(γ)areuniqueproteinsthatbindtonucleotideslikeGTP andGDP.The α and γ subunitsaretetheredtothecellmembrane vialipidlinks.UponligandsattachmenttoaGPCR,ligandschange theGPCRconformation,leadingtotheactivationoftheGprotein. TheactiveG-proteindisassociatesfromthereceptor,splittinginto α and β/γ subunits.Thesesubunitsthenstimulatespeciﬁc effectors,leadingtothegenerationofsecondmessengers,which aredetectedbyvariousproteinkinases,ultimatelytriggeringa cascadeofsignalingeventsthatdrivecellularresponses.

GPCRsplayacriticalroleincellulardetectionofexternalstimuli, includingodorants,tastecompounds,photons,metals,neurotransmitters,biogenicamines,fattyacids,aminoacids,peptides, proteins,steroids,andlipids.Thevastarrayofpotentialligands andreceptorslinksGPCRstonumerousphysiologicaland pathologicalstates,includingpain,asthma,cancer,cardiovascular, gastrointestinal,andneurologicaldisorders.Thissigniﬁcance rendersGPCRsasprimetargetsforpharmaceuticalintervention.119 Forinstance,theidentiﬁcationofspontaneousGPCR mutationsinindividualswithvariousendocrinedisordershighlightstheirimportanceinendocrinology.120

TheGPCRpathwayregulatesmultiplesignalingcascades, notablyinvolvingthePKAsystem,inositoltrisphosphate(IP3) pathway,andthecalcineurin-dependentproteinkinase(Ca/CaM) pathway.Thesethreepathwaystogetherconstitutethemain frameworkofGPCRssignaltransduction.Throughdifferentsecond messengersandeffectorproteins,GPCRsparticipateinadjusting numerousphysiologicalfunctionsofthecell.

ThePKAsystemoperateswithinthecyclicnucleotidesystem, whereextracellularsignalsbindtocorrespondingreceptors.This activatesasignalingpathwaythatelicitsaresponsebyregulating thelevelofcAMP.Typically,thesignalingmoleculesare hormones,andthecAMPlevelsareregulatedbyadenylate cyclase.Followingtheactionofthesignalingmoleculeonthe membranereceptor,theG-protein-couplingsystemisactivated. OncecAMPisgenerated,itwillactivatePKAtoamplifythesignal. ThisPKAsignaltransductionpathwayregulatesmembrane proteinactivity,geneexpression,andhormonesynthesisaswell ashormonesecretion.121

IP3pathwayinvolvesinositoltrisphosphateasasecond messengerinG-protein-coupledreceptor-mediatedsignaltransduction.IntheIP3pathway,extracellularsignalingmoleculesbind tocorrespondingGPCRs,activatingtheGqproteinonthe membrane.Subsequently,theGqproteinactivatesCβ whichis theonekindoftheisoformsofproteinkinaseC(PKC)andcan dissolvephosphatidylinositol-4,5-bisphosphate(PIP2)intotwo secondmessengers.ThesecondmessengerIP3bindstoits

receptor,promotingthereleaseofCa2+.Anothersecond messenger,diacylglycerol(DAG),synergisticallyactivatesPKC alongwithCa2+ andphosphoacylserine,culminatinginacascade reactionthatdrivescellularresponses.122 PKCenzymestakea signiﬁcantpartintheprocessofcellproliferation,differentiation, andapoptosis.Ca2+ playsanimportantroleinneuronalcells, mediatingessentialphysiologicalprocesses.123 Thus,thispathway contributestotheregulationofcellproliferation,metabolism,and growth,makingitapotentialtargetfortumorinhibitionand myocardialprotection.

Ca/CaMpathwayisactivatedthroughaseriesofinteractions involvingcalciumions,calmodulin,andthephosphataseactivity ofcalcineurinitself.Thispathwayinvolvesthereleaseofcalcium ionsfromtheendoplasmicreticulumortheopeningofcalcium ionchannelsonthecellmembrane,resultinginanincreaseinthe concentrationofcalciumionsinthecytoplasm.Thecalciumions thenbindtocalmodulin,therebyactivatingthephosphorylation activityofproteinkinases.Thisactivationcanaffectthe physiologicalfunctionsofthecellbyregulatingthephosphorylationstateofsubstrates.124,125

Enzyme-coupledreceptorpathway.Enzyme-linkedreceptors, commonlysingle-passtransmembraneproteins,haveenzymatic activitywithintheirintracellularregionsordirectlyinteractwith proteinsthathaveenzymaticactivity.Theactivityofenzymelinkedreceptorsisstrictlyregulated,includingligand-bindinginducedreceptordimerization,phosphorylation,andnegative feedbackmechanisms.Ligands,suchasgrowthfactors,coupling withtheextracellulardomainofthereceptor,inducingreceptor dimerizationoroligomerization,therebyactivatingitsintracellular enzymaticactivity.Followingreceptordimerization,theintracellularenzymaticdomainsphosphorylateeachother,activatingthe receptor’senzymaticactivity.126–128 Theactivatedreceptortransmitssignalsbyphosphorylatingdownstreamproteins(suchas proteinkinasesandtranscriptionfactors),triggeringaseriesof signalcascadereactionsthatultimatelyleadtochangesinthe expressionofspeciﬁcgenesandregulationofcellbehavior.The terminationofsignaltransductionisusuallyachievedthrough mechanismssuchasreceptordephosphorylation,endocytosis, anddegradation,ensuringtheprecisionandtimelinessofsignal transmission.Byactivatingavarietyofdownstreamsignaling moleculesandpathways,enzyme-linkedreceptorsparticipatein thewidespreadregulationofphysiologicalfunctions,including cellproliferation,differentiation,migration,metabolism,and survival.

Enzyme-coupledreceptorspossessdistinctmolecularstructures andpropertiescomparedtoGPCRs.Forinstance,tyrosinekinase receptorspossessproteintyrosinekinase(PTK)activity.Whena hormonebindstothereceptor,thePTKsegmentinthe membraneisactivated.Thisactivationinitiatesaseriesof intracellularinformationtransmissioncascadereactionswith phosphorylationofpeptidechainsandtyrosineresiduesinthe membraneproteinsubstrate.129 Ultimately,genetranscription processeschangewithinthenucleusandresultincorresponding biologicaleffectswithinthecell.Mostgrowthfactors,insulin,and somepeptidehormonesaretransmittedthroughthisreceptor type.IthasbeenreportedthatPTKispivotalininitiating multicellularresponsesrelatedtoDNAsynthesisandcell proliferation.Theproteinsencodedbymanyretrovirusoncogenes andtheintracellularregionsofseveralgrowthfactortransmembranereceptorsexhibitPTKactivity.ThereceptorPTKnotonly playsaroleintransmittingextracellularinformation,suchas hormonesandgrowthfactors,butisalsoinvolvedinthe malignanttransformationandproliferationofcells.PTKhasbeen identiﬁedandcharacterizedasaselective,potent,andwelltoleratedkinaseinhibitorsuitableforcancertherapy.130 AsPTK takesacriticalpartinthedevelopmentandprogressionof tumors,itservesasapromisingtherapeutictargetincancercells.

Gene-targetingmedicationsavailablecommerciallycaneffectively reachtherapeuticobjectivesbysuppressingitsfunction.

Therearemanyothertypesofenzyme-linkedreceptors,among whichthemoreimportantonesincludereceptortyrosinekinases (RTKs)andguanylatecyclase(GC)receptors.RTKsreferto receptorswhoseintracellularpartofthemembraneitself possessestyrosinekinaseactivity.Theextracellularsignaling moleculesthatcanbindtothesereceptorsandcompletesignal transductionaremainlyvariousgrowthfactors,suchasepidermal growthfactor,platelet-derivedgrowthfactor, ﬁbroblastgrowth factor,andinsulin.Whentheextracellularpartofthereceptor bindstoaligand,theactivationofthetyrosinekinaseinthe cytoplasmicpartofthereceptormoleculeoccurs,thereby triggeringvarioussignalingproteinstotransmitsignalsalong differentpathwaysreviewedbelow.

TheRas-MAPKisanimportantsignalingpathwaymediatedby RTKs,mainlycomposedofthreekeykinases:Ras,Raf,andMAPK. WhencellsarestimulatedbygrowthfactorssuchasEGF,PDGF, thegrowthfactorreceptors(e.g.,EGFR,PDGFR)areactivated, leadingtotheaccumulationofGTP-boundRas(Ras-GTP).

DownstreamRafkinaseisthenactivatedbyRas-GTPonthe cellmembrane,phosphorylatingandactivatingMAPKK(MEK), whichinturnphosphorylatesandactivatesMAPK(ERK). 131 MAPKisabletophosphorylatedownstreamsubstrates,which areoftentranscriptionfactors(suchasElk1,Fos,Jun)orother proteinsthatchangecellbehavior.Theaberrantactivationof theRas-MAPKsignalingpathwayhasabearingonmany diseases,includingcancerandneurodegenerativediseases. 132 , 133 Moreover,thispathwayalsointeractswithother signalingpathwayssuchasPI3K – Akt,Januskinase(JAK)-signal transducersandactivatorsoftranscription(STAT),etc.,jointly regulatingprocessessuchascel lgrowth,proliferation,and differentiation.

AnotherpathwayactivatedbyRTKsisthePI3K–Aktsignaling pathway.WhenligandssuchasgrowthfactorsbindtoRTKs,the RTKsundergoautophosphorylationandactivation,whichprompts therecruitmentandactivationofPI3Knearthereceptor.The activatedPI3KconvertsthemembranelipidPIP2intophosphatidylinositol-3,4,5-trisphosphate(PIP3).PIP3,actingasasecond messenger,activatesAkt,therebyinitiatingaseriesofdownstreamreactionsthataffectcellsurvival,proliferation,growth,and metabolism.134,135

GCreceptorshaveasingletransmembrane α-helix,withthe N-terminalligand-bindingsitelocatedontheextracellularside andtheC-terminalGCdomainlocatedontheintracellularside. Oncethereceptorbindswithaligand,GCactivityisactivated. UnliketheactivationofAC,thisprocessdoesnotrequirethe involvementofGproteins.Onceactivated,GCcatalyzesthe conversionofGTPtocGMPwithinthecytoplasm,whichthenbind andactivatecGMP-dependentproteinkinaseG(PKG).Similarto PKAandPKC,PKGisaserine/threonineproteinkinasethat mediatessignaltransductionthroughthephosphorylationof substrateproteins.UponactivationbyNOandCO,GCaugments theproductionofcGMP.ThecGMPbindsandactivatesPKG, whichphosphorylatessubstrateproteins,activatingdownstream signalingpathwaystoregulatecellgrowthandrenewal.136 The PKGsignaltransductionsystemregulatessmoothmusclerelaxation,nervoussystemfunction,andphysiologicalprocessessuchas intestinalsecretion,reninrelease,bonegrowth,andvisualsignal transduction.137,138 Besides,therearesignaltransductionpathwaysdirectlyinitiatedorpropagatedbyintracellularfunctional compartments.Forinstance,thereceptorforNOisatypeofGC locatedwithinthecytoplasm,knownassolubleGC.WhenNOacts onsolubleGC,itincreasestheconcentrationofcGMPandthe activityofPKGwithinthecytoplasm,leadingtoresponsessuchas therelaxationofvascularsmoothmuscle.139,140 Thesepathways areessentialforcoordinatingtheactivitiesofeachorganellewith othercellularcomponents.

Inadditiontothesignalingpathwaysmentionedabove, enzyme-linkedreceptorsalsomediatetheJAK/STATpathway andtheTGF-β-Smadpathway.TheJAK-STATsignalingpathwayis typicallyactivatedbycytokinereceptors,whichlacksintrinsic tyrosinekinaseactivity,butinteractswithmembersofthe intracellulartyrosinekinasefamilyJAKs.Whencytokines(suchas interferonsandinterleukins)bindtotheirspeciﬁcreceptors,they promotetheactivationofJAKs.TheactivatedJAKsphosphorylate thereceptors,providingdockingsitesforSTATstobindand becomeactivated.TheactivatedSTATsdimerizeandthen translocatetothenucleus,wheretheydirectlyregulatethe expressionoftargetgenes.141 Besides,theTGF-β signaling pathwayisprimarilymediatedbyaclassofreceptorsknownas Serine/ThreonineKinaseReceptors,whichpossessserine/threoninekinaseactivityintheirintracellularregion.Whenligandsof theTGF-β family,suchasTGF-β,bonemorphogeneticproteins (BMPs),activins,etc.,bindtothesereceptors,theypromptthe receptorkinasestophosphorylateSmadproteins.ThephosphorylatedSmadproteinsfurtherinteractwithotherSmadproteinsor DNA-bindingproteins,transmittingthesignalfromthecell membranetothenucleus,therebyregulatingtheexpressionof speciﬁcgenes.142,143

Otherpathways.Externalsignalingmoleculestriggertheproteolyticcleavageofapotentialgeneregulatoryprotein.Controlled proteolysismodulatestheexpressionoftargetgenes.Signal transductionpathwaysrelyingonregulatedproteolysisencompassNotchpathway,Hedgehog(Hh)pathway,Wntpathwayand NF-κBpathway.

TheNotchsignalingpathwayisahighlyconservedintercellular communicationmechanismthatisextensivelyinvolvedinvarious biologicalprocesses,includingcellfatedetermination,embryonic development,andtissueregeneration.144–146 Adistinguishingtrait ofthissignalingpathwayisitsrelianceondirectcell–cellcontact, obviatingtherequirementforsignalmoleculestotravellong distancesbetweencells.TheNotchsignalingpathwayisinitiated bythedirectbindingoftheNotchreceptorlocatedonthesurface ofthesignal-receivingcelltoitsligandsituatedonthesurfaceof neighboringcells.Notchreceptorsandligandsarebothtransmembraneproteins,withtypicalligandsbelongingtotheJagged andDeltafamilies.Afterligand–receptorbinding,theNotch receptorundergoesaseriesofcleavageprocesses.First,theADAM (adisintegrinandmetalloprotease)familymembermetalloproteasescleavetheNotchreceptorintheextracellularregion, followedbycleavagebythe γ-secretasecomplexinthe transmembraneregion,leadingtothereleaseoftheNotch intracellulardomain(NICD).147 ThereleasedNICDthenentersthe nucleus,whereitbindstotheCSL(CBF1/Su(H)/Lag-1)familyof DNA-bindingproteinsandotherco-activatorstoformatranscriptionalactivationcomplex,directlyregulatingtheexpressionof downstreamgenes.148,149

TheHhsignalingpathwayisakeyintercellularsignal transductionmechanism,extensivelyinvolvedinthedevelopment ofanimalembryosandthemaintenanceofadulttissues.This pathwayplaysacrucialroleinthegrowthandproliferationofcells duringembryonicdevelopmentandafterembryoformation. AberrantactivationoftheHhsignalingpathwayisassociatedwith variouscancersanddevelopmentalabnormalities.150 Beforebeing secretedoutsidethecell,Hhproteinsundergoaseriesof posttranslationalmodiﬁcations,includingautocleavageandcovalentattachmenttocholesterol.151 IntheabsenceofHhligands, thePatched(Ptch)receptorinhibitstheactivityofSmoothened (Smo).WhenaHhligandbindstoPtch,thisinhibitionislifted, allowingSmotobeactivated.TheactivationofSmotriggersa seriesofintracellularsignalingevents,ultimatelyaffectingthe activityofglioma-associatedoncogenehomolog(Gli)transcription factors.Intheabsenceofsignaling,Glifactorsarepartially degradedintoarepressorform.WhentheHhsignalisactivated,

theinhibitionofGliisremoved,allowingitsfull-lengthformto enterthenucleus.TheactivatedGlitranscriptionfactorentersthe nucleusandpromotestheexpressionofdownstreamtargetgenes whichtakepartinprocessessuchascellproliferation,differentiation,andsurvival.

TheWntsignalingpathwayisacomplexcellsignalingsystem, extensivelyinvolvedintheembryonicdevelopment,cellproliferation,migration,differentiation,andmaintenanceofadult tissuehomeostasisinanimals.Thenameofthispathway originatesfromagenediscoveredinfruit ﬂiescalled “wingless” anditsmousehomolog “Int-1”,collectivelyknownasWnt.152 The Wntsignalingpathwayisprimarilydividedintotwopathways:the β-catenin-dependentcanonicalpathwayandthe β-cateninindependentnoncanonicalpathways.IntheabsenceofWnt ligands, β-cateniniscapturedinthecytoplasmbyacomplex (includingproteinssuchasAxinandGSK-3β)andphosphorylated byGSK-3β,leadingtoitsubiquitinationanddegradation.When Wntsignalsarepresent,WntligandsbindtotheFrizzledreceptor andLRP5/6co-receptor,inhibitingthe β-catenindegradation complex,preventingthephosphorylationandsubsequentdegradationof β-catenin.Thestabilized β-cateninaccumulatesand translocatestothenucleus,whereitbindstotranscriptionfactors oftheTCF/LEFfamily,activatingtheexpressionofdownstream targetgenes.153 Thenoncanonicalpathwaysdonotrelyon β-cateninbutaremediatedbyothersignalingmoleculessuchas Ca²+,JNK,RhoGTPase,etc.,inducingvariouscellularresponses, includingcellpolarity,movement,andtissuemorphogenesis.153

TheNF-κBsignalingpathwayisakeycellularsignaltransductionmechanism,extensivelyinvolvedinregulatingimmune responses,inflammatoryreactions,cellsurvival,proliferation,and differentiationamongvariousbiologicalprocesses.154–156 Serving asanimmediateresponsemechanism,itcanrapidlyrespondtoa widerangeofexternalstimuli,suchascytokines,pathogens,free radicals,andotherstresssignals.TheNF-κBpathwaycanbe activatedbymultiplesignals,includingTNF-α,interleukin1(IL-1), lipopolysaccharides(LPS),viralinfections,andotherstress conditions.Wheninactive,NF-κBisboundtoitsinhibitoryprotein IκBinthecytoplasm.Uponactivationbytheaforementioned signals,theIκBkinase(IKK)complexisactivated,leadingtothe phosphorylationofIκBanditssubsequentdegradationviathe ubiquitin-proteasomepathway.ThedegradationofIκBreleases NF-κB,allowingittotranslocatetothenucleus,bindto κBsiteson DNA,andactivatethetranscriptionofspecificgenes.157 The terminationoftheNF-κBsignalinvolvesnewlysynthesizedIκBα, whichcanenterthenucleus,bindtoNF-κB,andexportitbackto thecytoplasm,therebyreturningNF-κBtoaninactivestate. AccuratecontroloftheNF-κBsignalingpathwayiscrucialfor maintainingnormalcellularfunctionsandpreventingthedevelopmentofdisorders,includingcancer,autoimmunediseases,and chronicinflammatorydiseases.158,159 Thisunderscoresitssignificanceasaprimetargetforpharmaceuticalinterventions.

Multi-levelregulationofCCCanditsimplications

Upregulationanddownregulation.TheactivityofCCCsignal transductioncanbemodulatedthroughupregulation(enhancing signaltransmission)anddownregulation(weakeningsignal transmission).Thisregulationcanbeachievedbyalteringthe expressionlevelofreceptors,modulatingreceptoractivity,or changingtheavailabilityofsignalingmolecules.Incertain inﬂammatoryresponses,cytokinessuchasTNF-α inducethe upregulationofadhesionmolecules,suchasintercellularadhesion molecule-1(ICAM-1)andvascularcelladhesionmolecule-1 (VCAM-1),whicharelocatedonthesurfaceofendothelial cells.160–162 Thisupregulationenhancestheinteractionbetween whitebloodcellsandendothelialcells,promotingthemigration ofwhitebloodcellsandinﬂammatoryresponses.163–165 The prolongedorexcessiveuseof β-adrenergicreceptor(β-AR) agonistsforasthmatreatmentleadstoareductioninthequantity

of β-ARsoncardiacandsmoothmusclecells(SMCs),achieved throughmechanismsofreceptorinternalizationanddegradation.166–168 GPCRsafterbeingactivatedoveralongperiodcanbe internalizedthrougha β-arrestin-mediatedpathway.Thisprocess givesrisetoadecreaseinthenumberofreceptorsonthecell surface,leadingtoadampenedreceptoractivityandultimately impairingsignaltransmission.113,169–171 Thisphenomenondiminishingthecellresponsivenesstotheagonistcommonlytermed receptordownregulation.172

Desensitization.Long-termorexcessivesignalstimulationcauses cellstobecomedesensitizedtocertainsignals.Desensitizationisa protectivemechanismtopreventoverreaction,achievedby reducingthesurfaceexpressionofreceptorsorinhibitingthe activityofsignaltransductioncomponents.Inpatientswithtype2 diabetes,prolongedhighlevelsofbloodglucoseandinsulincan leadtodesensitizationofinsulinreceptors,reducingtheir sensitivitytoinsulinandfurtherexacerbatinginsulinresistance.173 Inaddition,long-termalcoholconsumptioncanincreasethe nervoussystem’stolerancetoalcohol,achievedbyregulatingthe expressionandsensitivityofneurotransmitterreceptorssuchas GABAreceptorsandglutamatereceptors.174–177

Upstreamregulatorsanddownstreameffectors.Theregulationof signalingpathwaysinvolvesmultipleupstreamregulatorsand downstreameffectors.Upstreamregulatorsareresponsiblefor receivingandintegratingexternalsignals,whiledownstream effectorsexecutethebiologicaleffectsofthesesignals,suchas alteringgeneexpressionandregulatingcellbehavior.These signalingpathwaysarekeymechanismsforcommunication betweencells,regulatingcellbehaviorandcellfatethroughthe receptionandtransmissionofexternalsignals.Theyplayrolesina varietyofbiologicalprocesses,includingcellproliferation, differentiation,migration,celldeath,andthemaintenanceof tissueandorganhomeostasis.Theaberrantactivationor inhibitionofthesepathwaysiscloselyrelatedtothedevelopment ofvariousdiseases,especiallycancer,inﬂammatorydiseases, neurodegenerativediseases,andcongenitaldevelopmental abnormalities.Theydemonstratethediversityandcomplexityof signaltransduction,includingLRIs,subsequentactivationof signalingmolecules,intracellularsignaltransmission,andthe ultimateactivationofeffectors.Thereisalsocrosstalkand interactionamongthesesignalingpathways,allowingthemto inﬂuenceandregulateeachother,formingacomplexnetworkto adapttodifferentphysiologicalandpathologicalconditions.

Spatialdistribution.ThespatialdistributionofCCCcomponentsis crucialfortheefficiencyandspecificityofsignaltransmission.Cells achieveprecisesignallocalizationandtransmissionbyrestricting thedistributionofreceptors,enzymes,andothersignaling moleculestospecificregionswithinthecell.Inmanytypesof cells,specificreceptorsarelocalizedtoparticularmicrodomainsof thecellmembrane,suchaslipidrafts.Lipidraftsarecell membraneregionsrichincholesterolandsphingolipids,capable ofaggregatingspecificsignalingproteins,includingGPCRsand RTKs.178,179 Thislocalizationenhancestheinteractionbetween signalingmolecules,improvingtheefficiencyandspecificityof signaltransmission.Directedtransportallowscellstoregulatethe activityofsignalingmoleculeswithinspecifictemporalandspatial ranges.Certainsignalingproteinsaretransportedtothecellpoles duringspecificphasesofcelldivisionorconcentratedinthe leadingedgeduringcellmigration,ensuringthecorrectexecution ofcellfunctions.Thetransmissionofsignalswithinneurons dependsontheprecisereleaseandreceptionofneurotransmitters,whichoccurinhighlyspecificspatiallocations.Neurotransmittersarestoredinsynapticvesiclesattheaxonterminals,and uponsignalarrival,thesevesiclesfusewiththepresynaptic membrane,releasingneurotransmittersintothesynapticcleft.

Receptorstypicallylocatedonthepostsynapticmembraneensure rapidandaccuratesignaltransmission.

Thespatialdistributionofsignalingmoleculesalsoinvolvesthe localizationandtransferofnuclearreceptors.Steroidhormone receptors,suchasestrogenandandrogenreceptors,areusually locatedinthecytoplasmintheirinactivestate.Uponhormone binding,thereceptor–hormonecomplexmovesintothenucleus, directlyregulatingthetranscriptionoftargetgenes.Thistransfer fromthecytoplasmtothenucleusisakeystepinthesignal transmissionprocess,affectingchangesingeneexpression. Furthermore,thespatialdistributionofCCCcomponentsaffects theassemblyofsignalingcomplexes.IntheWntsignaling pathway,thestabilizationandnucleartransferof β-catenin dependontheinteractionofmultiplesignalingmoleculesin speciﬁccellularregions.IntheabsenceofWntsignals, β-cateninis capturedanddegradedinthecytoplasm.WhentheWntsignaling pathwayisactivated,proteinssuchasAxinarerecruitedtothecell membrane,wheretheyimpedethedegradationof β-catenin.This preservationenables β-catenintoamassandtranslocatetothe nucleus,inﬂuencinggeneexpression.

Otherregulatorymechanisms.Inaddition,CCCsignaltransductionisregulatedbyposttranslationalmodiﬁcations,synthesisand degradationofsignalingmolecules,etc.Phosphorylationisa commonposttranslationalmodiﬁcationthatiscrucialforthe regulationofsignalingpathways.Forexample,intheEGF signalingpathway,thebindingofEGFtoitsreceptorEGFR triggersautophosphorylationofthereceptor.Thisprocessboosts thereceptor’styrosinekinaseactivity,leadingtotheactivationof downstreamsignalingpathwayssuchasRas/MAPK,whichinturn stimulatescellproliferationanddifferentiation.Thedynamic balancebetweenphosphorylationanddephosphorylationregulatesthestrengthanddurationofthesignal,affectingthe determinationofcellfate.

Moreover,ubiquitinationisanotherkeyposttranslational modiﬁcationthatregulatessignaltransductionbytagging proteinsfordegradation,therebymodulatingsignaling.Inthe NF-κBsignalingpathway,theubiquitinationandsubsequent proteasome-dependentdegradationofIκBα arecriticalstepsfor activatingNF-κB.180

Furthermore,thesynthesisofsignalingmoleculessuchas neurotransmittersisessentialforthetransmissionofneuralsignals. Forinstance,serotoninissynthesizedfromtryptophancatalyzedby tryptophanhydroxylase.Theamountofserotoninsynthesized directlyaffectsthestrengthofneuralsignaltransmissionand psychologicalstates,suchasmoodandsleep.181 Thetimely degradationofsignalingmoleculesisalsocrucialtoensurethe temporarinessofthesignalandtherestorationoftherestingstate. Forexample,acetylcholineisrapidlydegradedbyacetylcholinesterase,endingitssignaltransmissionattheneuromuscularjunction.182 Thisprocessisvitalfortheproperrelaxationofmusclesandthe preventionofcontinuouscontraction(spasm).Thesemechanisms worktogethertoensurethedynamicregulationofsignal transmissionandthecell’sadaptabilitytoenvironmentalchanges.

THECLINICALAPPLICATIONANDRESEARCHPROGRESSOF CCC

Asthebodyadaptstointernalandexternalenvironmental changes,varioussystemsandorgansofthebodyneedto coordinatetocompletetheadaptiveresponse,includingnervous, humoral,andself-regulatorysystems.Atthemicro-level,these threeregulatorymechanismsrelyonthecoordinatedactivitiesof variousfunctionalcellsinthebody,necessitatingacomplexsignal communicationprocessbetweendifferentcells,namelycellsignal transduction.CCCissocrucialinthedevelopmentoftissues, organs,andimmuneresponsesthatdiseasescanemergewhen cellsfailtointeractcorrectlyormisinterpretmolecularinformation

(Fig. 3).Therefore,studyingthemechanismsandregulationofcell communicationholdsgreatscientiﬁcsigniﬁcanceforunderstandingthenatureofbiologyanddisease,andhaspractical applicationvalueforclinicaltrials.

Somepathwaysmaybeabnormallyactivatedorinhibitedin diseasestates,anddrugscanactbytargetingspecificcell signalingpathways.183,184 However,theuseofdrugsneedstobe strictlycontrolledtoenhancetheefficacyofexistingtreatments andreducesideeffects.TheUnitedStatesFoodandDrug Administration(FDA)conductsrigorousreviewsofmedications, anddrugsapprovedbytheFDAhavepassedaseriesofclinical trialsprovingtheirefficacyandsafetyintreatingspecificdiseases orconditions.185–188 Table 1 showssomeFDA-approveddrugs takingeffectsthroughtherapeuticCCCtargets.Inthefollowing sectionsofthischapter,wewillintroducetheroleofCCCin differenthumandiseases,includingdiseasediagnosis,prevention, treatment,andprediction.

Cancers

Canceriswidelyrecognizedasaclusterofdisordersmarkedby uncontrolledproliferationanddisseminationofaberrantcells.189 Cancerremainsamajorglobalchallengeeventhoughsignificant effortshavebeenmadetodevelopnewcancertreatments.Asa result,thediscoveryofnoveltherapeuticsspecificallytargeting diversecancerformsisimperative.190 Sincevarioussignal transductionpathwaysregulatecellgrowth,abnormalactivation orsuppressionofthesepathwaysdrivestumorigenesis.121 Oneof theprincipalpathwaysisthePI3K–AKT–mTORsignalingthat linkedtodrugresistanceandthemalignanttumorprocessinsolid cancerpatients.191 Itisvitaltousecellsignalingmoleculesto recognizecancercellstoinhibittheexpansionandproliferationof cancercells.Posttranslationalproteinmodificationplaysavital roleinthecontrolofcellularsignaling.Diverseproteinkinasesand phosphatasesregulatethephosphorylationanddephosphorylationofproteins.Tumorsfrequentlyexhibitirregularoruncontrolledactivationofsuchkinasesandphosphatases,makingthem asessentialtargetsfortargetedcancertherapies.Tocitean instance,ImatinibisaBCR–ABLfusiontyrosinekinaseinhibitor andrepresentsthe firstkinaseinhibitorsuccessfullyappliedin treatingchronicmyeloidleukemia(CML).192 Subsequently,inhibitorstargetingproteinkinasessuchasmTOR,VEGFR,MAPK,EGFR, CDK12,andERBB2havebeenemployedintreatingvarious commonmalignanttumors.193–197 Employinggene-editingtechnologieslikeCRISPR/Cas9tointerveneatthegeneticlevelin cancercells,whetherbyknockingoutormodifyingspecificgenes, holdsthepromiseofhinderingcancercellproliferationand metastasis.198,199

Whilegeneticorepigeneticalterationsareoftencitedasthe rootcauseofcancer,theprogressionofcancerisintricatelylinked withcrosstalkamongtumorcells,surroundingstromalcells,and theextracellularmatrix(ECM).200 Tumorcellspromotetheirown growthandproliferationbycommunicatingwithsurrounding normalcells,immunecells,andothercelltypeswithinthetumor microenvironment(TME),suchas ﬁbroblastsandendothelialcells. Cancercellsdonotmanifestthediseaseinisolationbutrather conscriptandcorruptresidentandrecruitednormalcelltypes.201 Cancercellscanselectnoncancerouscellstoengageinextensive chemicalandphysicalinteractions,withmanytypesofcellsbeing recruitedintosolidtumorsandparticipatingincomplexinteractionsthatenablecancercellstoinvade.202 Tumorinvasionisnota simpleautonomousprocessofcancercellsbutreliesona complexnetworkofparacrineinteractions.203 Moreover,this networkcanchangeascancercellsdisseminate.Asthe constituentcellsofbloodvesselsandlymphaticvessels, endothelialcellsnotonlysupplytumorwithnutritionandoxygen butalsoactasan “escaperoute” forcancercells,enablingthemto metastasizetodistantsites.204 Theintercellularcommunication betweentheseTMEcomponentsandcellsisadriverofcancer

progressionandsigniﬁcantlyimpactstheefﬁcacyoftherapeutic interventions.

ThestudyoftheTMEinvolvescellcommunicationanalysisto selectthemostinteractingcellsubsetsandfurtherinvestigate theirmechanisms.Forexample,byusingreceptor-ligandanalysis

SignalTransductionandTargetedTherapy(2024)9:196

ofdifferentsubpopulationsinbladdercancersamples,onestudy showedthatinﬂammatorycancer-associated ﬁbroblasts(iCAFs) speciallyinteractwithendothelialcellstopromoteangiogenesis andtumorproliferation,revealingtheroleofiCAFsintheimmune microenvironmentofbladdercancer.205 Inaddition,researchers

Fig.3 ExamplesofsomediseasescausedbyrepresentativeabnormalCCC.CCCisanessentialprocessthatprofoundlyinfluencesan organism’shomeostasis,development,anddiseaseprocesses.Whencellsfailtointeractcorrectlyormisinterpretmolecularinformation, diseasestypicallymanifest. a Tumorcellsinvadesurroundingtissuesandbloodvesselwalls,infiltrateintobloodvesselsandspreadtoother partsofthebodyalongthecirculatorysystem,theninteractwithoriginaltissuenichecellsandmigratetodistanttissuestocolonizeand grow. b Antineutrophilcytoplasmicantibody(ANCA)-associatedvasculitis:Apro-inflammatoryenvironmentinitiatestheproductionofANCA byplasmacellsaswellastheprimingofneutrophilsthroughcytokines. c Rolesofastrocytesandmicrogliainneurodegeneration:Danger signalsorinvadingpathogensactivatemicrogliatoreleasepro-inflammatorycytokines,whichactonastrocytes,whichinturnareactivatedto releasepro-inflammatorycytokines. d PathogenesisofrheumatoidarthritisII:Themajorcelltypesandcytokinepathwaysinvolvedinjoint destruction. e TheroleofILC2sinasthmapathogenesis:Allergens,virusesorhelminthsprovokethereleaseofalarminsfromthedamaged epitheliumandstimulatetheGATA3+/RORa+ ILC2stoexpresstype2cytokines.Interleukins-4,-5,and-13causeIgEincreasefromplasmacells, eosinophilexpansion,andairwayhyper-responsiveness,respectively. f MechanismofSARS-CoV-2viralentry:SARS-CoV-2usesitsspike(S) proteintoadsorbandpenetratecells.S1bindstothereceptorangiotensin-convertingenzymeII(ACE2)onthecellmembranethroughits receptorbindingdomain(RBD),andS2mediatesthefusionoftheviralenvelopewiththehost,allowingtheviralnucleocapsidtoenterthe cytoplasm. g TypeIvs.typeIIdiabetes:Thedestructionoftheisletcellspreventsthemfromproducinginsulin,preventingglucosefrom enteringthecellsandleadingtotype1diabetes.Thereducedresponsivenessofthebody ’scellstoinsulinleadstoinsulinresistance,andthe inabilitytoproperlyuseinsulintometabolizeglucoseresultsintype2diabetes. h Differentialrolesofmicrogliainthedevelopingbrain: Duringhealthybraindevelopment,microgliainitshomeostaticstatemediatesthematurationofoligodendrocyteprecursorcells(OPCs)into myelinatingmatureoligodendrocytes

foundthespeciﬁcexpressionofACKR1intumorendothelialcells isassociatedwithunfavorableprognosticoutcomesinagastric cancercohort,providinganewtargetfortreatinggastric cancer.206 Variouscharacteristicsoftumorsareprimarilyregulated bytheTME,includingdysregulatedECM,sustainedactivationof proliferativesignals,inhibitionofsuppressorsandapoptosis, activatedinvasionandmetastasis,metabolicdysregulation,and evasionofimmunedestruction.Furthermore,factorssecretedby theprimarytumorcanmodifythemicroenvironmentofdistant organs,renderingthemconducivetosubsequentlycolonizedby metastaticcancercells.207 Thegrowthandprogressionoftumors dependonangiogenesis,withCAFsbeingaprimarysourceofproangiogenicfactorssuchasVEGForPDGF.208,209 Tumorcells dischargepro-angiogenicfactorsintotheirsurroundingenvironment,contributingtothesecretionofPDGFbyendothelialcells, whichattractssupportingcellstosolidifythenascentblood vessels.210 ThePDGFreleasedbytumorcellsdirectlybindsto receptorsonrecruitedbonemarrowprogenitorcellsandinduces differentiationintoendothelialcellsorSMCsthroughsignal activation,promotingtheirgrowthandmigration.211,212

Mutationsingenesandtheirrespectivesignalingpathwaysare theprimaryconsequencesleadingtocellapoptosis,proliferation, cellsurvival,anddifferentiation.213,214 Asignificantnumberof genesfrequentlymutatedincancerareresponsibleforencoding componentsortargetsofthePI3K–AktandRas-ERKpathways. Typically,thesepathwaysaretransientlyactivatedinresponseto signalsfromgrowthfactorsorcytokinesandtheoccupancyof ligandsforintegrinadhesionreceptors.Subsequently,mutations inthetumorsuppressorgenesTSC1andTSC2leadtothe overactivationofmTORC1signaling,animportanttargetof PI3K–Aktsignaling.215 ThetranscriptionfactorMycisasignificant downstreamtargetoftheRas-ERKsignalaswellasnumerous otherpathways,anditisoftenamplifiedoroverexpressedin cancer.Furthermore,thetumormicrobiomemayalsoemergeasa criticalfactorinshapingthelocalimmuneresponseintheTME.216 Theycanenhanceanti-tumorimmunitythroughmechanisms suchasstimulatorofinterferongenessignalingactivation,Tand naturalkiller(NK)cellactivation,tertiarylymphoidstructure production,andpresentationoftumormicrobiome-derived antigens.Inaddition,theycanreduceanti-tumorimmune responsesandpromotecancerprogressionbyincreasingreactive oxygenspecies(ROS)levels,fosteringananti-inflammatorymilieu, deactivatingTcells,andinducingimmunosuppression. 217,218 Immunecheckpointblocking(ICB)isarevolutionarycancer treatmentthatblockstheinteractionofinhibitorymolecules expressedonmalignantcellswithTcells,rejuvenatingTcellsin theearlystagesofdysfunction.ThemaintypesofICBtherapy includePD-1/PD-L1inhibitorsandCTLA-4inhibitors.PD-L1(the

ligandofPD-1)ismainlyexpressedontumorcellsandtumorinﬁltratingimmunecells,anditfunctionstoinhibitT-cellactivity bybindingtoPD-1.219,220 TheactionofPD-1/PD-L1inhibitorsisto blockthebindingbetweenPD-1andPD-L1,restoringtheimmune cells’ abilitytorecognizeandkilltumorcells.221 CTLA-4inhibitors workbyblockingthebindingofCTLA-4totheB7molecules(B7-1 andB7-2)onthesurfaceofantigen-presentingcells,thereby relievingtheinhibitorystateofTcells.Inaddition,CTLA-4 inhibitorscanalsoreducethenumberofregulatoryTcells(Tregs) thathighlyexpressCTLA-4inthetumormicroenvironmentby blockingCTLA-4,therebyrelievingtheimmunosuppressiveeffects ofTregcellsandpromotingtheactivationandproliferationof effectorTcells.222–225 Thesetwotypesofinhibitorstargetdifferent immunecheckpointproteins,buttheirmechanismsofactionare similar,bothworkingbyblockingimmunecheckpointproteinsto activatetheimmunesystem.RecentstudieshaveidentiﬁedIGSF8 asaninnateimmunecheckpointandtumorimmunotherapeutic target.226 ThenewlydevelopedIGSF8.06antibodycanblockthe inhibitoryeffectof IGSF8 expressedontumorsonNKcellfunction, thusstimulatingNKcellstokillmalignantcellswithantigen presentationdefectsandstresssignals.226

Stemcells,avitalcomponentofcelltherapy,playacrucialrole inrestoringorgansandtissues,holdingimmensepromisefor variousapplications.Itshouldbenotedthatstemcellsderived fromdifferentsourcesexhibitvaryingcapabilitiesintermsof proliferation,migration,anddifferentiation.Thesedifferences inﬂuencetheirsuitabilityfordeploymentinanti-tumortherapy. Indetail,cancerstemcells(CSCs)representasmallfractionof cancerouscellscharacterizedbytheircapacityformultifaceted differentiation,highself-renewal,andtumorigenicity.227 TheCSC theorypostulatestheexistenceofaminoryetcrucialcadreofselfperpetuatingcancercellscriticalintumormetastasis,recurrence, andresistancetotreatment.227 Nevertheless,theprecisionand biologicalroleofCSCsarestillambiguous,promptingsome researcherstoexercisecautionandregardthetheoryas contentious.228,229 Despitetheongoingdebate,researchonCSCs continuestoevolveanduncovernewinsights.230 CSCswere originallyextractedfromcasesofacutemyeloidleukemia,231,232 possiblyemergingfromregulartissue-speciﬁcstemcellsor differentiatedcellsattheonsetofthetumor,triggeringsurvival pathwaysandperpetualproliferation.233 Mechanisticstudies suggestdysfunctioninsomedevelopmentalandhomeostasis signalingpathwayscouldfacilitateuncontrolledself-renewaland differentiationessentialforCSCfunctionality.234 Suchmolecular signalingpathways,includingNotch,17 Hedgehog,235 Wnt/ β-catenin,236 PI3K/PTEN,237 JAK/STAT,238 andNF-κB,239 areknown toregulatenormalstemcellproliferation.Furtherchangesin thesesignalingpathwayswillleadtotheformationofCSCsand

MoleculartargetIndication

FDA-approveddrugsonvariousdiseasescausedbyabnormalCCC

Table1.

FDA-approveddrugsonvariousdiseasescausedbyabnormalCCC

GenericnameofdrugActiveingredientsInitial approvaldate

Cancers

TALZENNATalazoparib03/07/2024PARPMetastaticbreastcancer

TRUQAPCapivasertib11/16/2023AKTBreastcancer

AUGTYRORepotrectinib11/15/2023ROS1,TRKA,TRKB,andTRKCROS1-positivenon-smallcelllungcancer

FRUZAQLAFruquintinib11/08/2023VEGFR-1,-2,-3Refractorymetastaticcolorectalcancer

ZEJULANiraparibTosylate04/26/2023PARP-1,-2Epithelialovarian,fallopiantube,orprimaryperitonealcancer

Metastaticbreastcancer

ORSERDUElacestrant01/27/2023Er α

KRAZATIAdagrasib12/12/2022KRASG12CKRASG12C-mutatedlocallyadvancedormetastaticnon-small celllungcancer(NSCLC)

Folatereceptoralpha(FR α )positive,platinum-resistant epithelialovarian,fallopiantube,orprimaryperitonealcancer

ELAHEREMirvetuximabsoravtansine-gynx11/14/2022FR α

IMJUDOTremelimumab10/21/2022CTLA-4Metastaticnon-smallcelllungcancer(NSCLC)withno sensitizingepidermalgrowthfactorreceptor(EGFR)mutation oranaplasticlymphomakinase(ALK)genomictumor aberrations

03/23/2022PSMAProstate-speci ﬁ cmembraneantigen(PSMA)-positive metastaticcastration-resistantprostatecancer(mCRPC)

PLUVICTOLutetiumlu177vipivotide tetraxetan

OPDUALAGNivolumab;Relatlimab-rmbw03/18/2022PD-1;LAG-3Unresectableormetastaticmelanoma RYBREVANTAmivantamab-vmjw5/12/2021EGFRandMETEGFRexon20insertion-mutatednon-smallcelllungcancer indications

CYTALUXPafolacianine11/29/2021FROvariancancer;Knownorsuspectedcancerinthelung TIVDAKTisotumabvedotin-tftv09/20/2021TFRecurrentormetastaticcervicalcancer

EXKIVITYMobocertinib09/15/2021EGFRexon20insertionmutationsLocallyadvancedormetastaticnon-smallcelllungcancer (NSCLC)withepidermalgrowthfactorreceptor(EGFR)exon 20insertionmutations

VonHippel-Lindau(VHL)dosease;Pancreaticneuroendocrine tumors(pNET)

JEMPERLIDostarlimab-gxly08/17/2021PD-1Endometrialcancer;solidtumors WELIREGBelzutifan08/13/2021HIF-2 α

LUMAKRASSotorasib05/28/2021KRASKRASG12C-mutatedlocallyadvancedormetastaticnon-small celllungcancer(NSCLC)

TEPMETKOTepotinib02/03/2021METMetastaticnon-smallcelllungcancer

ORGOVYXRelugolix12/18/2020PituitaryGnRHreceptorAdvancedprostatecancer RIABNIRituximab-arrx12/17/2020CD20Non-Hodgkin ’ sLymphoma(NHL)

MARGENZAMargetuximab-cmkb12/16/2020HER2MetastaticHER2-positivebreastcancer

MetastaticRETfusion-positivethyroidcancer;Metastatic rearrangedduringtransfection(RET)fusion-positivenon-small celllungcancer

GAVRETOPralsetinib09/04/2020 RET

ZEPZELCALurbinectedin06/15/2020GuanineresiduesMetastaticsmallcelllungcancer(SCLC) RETEVMOSelpercatinib05/08/2020RETMetastaticnon-smallcelllungcancer(NSCLC);Metastatic medullarythyroidcancer(MTC);Metastaticthyroidcancer; Metastaticsolidtumorsfusion

Table1. continued

MoleculartargetIndication

FDA-approveddrugsonvariousdiseasescausedbyabnormalCCC

GenericnameofdrugActiveingredientsInitial approvaldate

TABRECTACapmatinib05/06/2020METMetastaticnon-smallcelllungcancer(NSCLC)

TRODELVYSacituzumabgovitecan-hziy04/22/2020Trop-2Metastaticbreastcancer;Metastaticurothelialcancer

TUKYSATucatinib04/17/2020HER2Metastaticbreastcancer;Unresectableormetastaticcolorectal cancer

ENHERTUFam-trastuzumabderuxtecan-nxki12/20/2019HER2HER2-positivemetastaticbreastcancer;HER2-lowmetastatic breastcancer;UnresectableormetastaticHER2-mutantnon- smallcelllungcancer

PADCEVEnfortumabvedotin-ejfv12/18/2019Nectin-4Metastaticurothelialcancer(mUC)

NUBEQADarolutamide07/30/2019ARNon-metastaticcastration-resistantprostatecancer(nmCRPC); Metastatichormone-sensitiveprostatecancer(mHSPC)

RUXIENCERituximab-pvvr07/23/2019CD20Non-Hodgkin ’ slymphoma(NHL)

TRUXIMARituximab-abbs11/28/2018CD20Non-Hodgkin ’ slymphoma(NHL)

Metastaticnon-smallcelllungcancer(NSCLC)

LORBRENALorlatinib11/02/2018ALKandROS1aswellasTYK1,FER,FPS,TRKA, TRKB,TRKC,FAK,FAK2,andACK

LIBTAYOCemiplimab-rwlc09/28/2018PD-1Cutaneoussquamouscellcarcinoma;Basalcellcarcinoma; Non-smallcelllungcancer

BRAFV600EorV600Kmutation-positiveunresectableor metastaticmelanoma;BRAFV600Emutation-positive metastaticvolorectalcancer(CRC);BRAFV600Emutation- positivemetastaticnon-smallcelllungcancer(NSCLC)

BRAFTOVIEncorafenib06/27/2018BRAFV600E,aswellaswild-typeBRAFandCRAF ;JNK1,JNK2,JNK3,LIMK1,LIMK2,MEK4,and STK36

VERZENIOAbemaciclib02/26/2018CDK4andCDK6Metastaticbreastcancer

LYNPARZAOlaparib08/17/2017PARPOvariancancer;Breastcancer;Pancreaticcancer;Prostate cancer

06/22/2017CD20Follicularlymphoma(FL);DiffuselargeB-Celllymphoma (DLBCL)

RITUXANHYCELARituximab;HYALURONIDASE (HUMANRECOMBINANT)

Anaplasticlymphomakinase(ALK)-positivemetastaticnon- smallcelllungcancer(NSCLC)

ALUNBRIGBrigatinib04/28/2017ALK,ROS1,insulin-likegrowthfactor-1receptor (IGF-1R),andFLT-3aswellasEGFRdeletionand pointmutations

RUBRACARucaparib12/19/2016PARPBRCA-mutatedrecurrentovariancancer;BRCA-mutated metastaticcastration-resistantprostatecancer

TECENTRIQAtezolizumab10/18/2016PD-L1Metastaticnon-smallcelllungcancer;Locallyadvancedor metastaticurothelialcarcinoma

TAGRISSOOsimertinib11/13/2015EGFREGFRmutation-positivenon-smallcelllungcancer(NSCLC); EGFRmutation-positivemetastaticNSCLC

Cardiovasculardiseases

TRYVIOAprocitentan03/19/2024ET-1Hypertension

INPEFASotagli ﬂ ozin05/26/2023SGLT2andSGLT1Cardiovasculardeath,hospitalizationforheartfailure,and urgentheartfailure

TAVNEOSAvacopan10/07/2021C5aRSevereactiveantineutrophilcytoplasmicautoantibody (ANCA)-associatedvasculitis(granulomatosiswithpolyangiitis [GPA]andmicroscopicpolyangiitis[MPA])

VERQUVOVericiguat01/19/2021sGCCardiovasculardeathandheartfailure(HF)hospitalization

Table1. continued FDA-approveddrugsonvariousdiseasescausedbyabnormalCCC

MoleculartargetIndication

GenericnameofdrugActiveingredientsInitial approvaldate

NEXLETOLBempedoicacid02/21/2020ACLPrimaryhyperlipidemiainadultswithheterozygousfamilial hypercholesterolemia(HeFH)oratheroscleroticcardiovascular disease

VYNDAQELTafamidismeglumine05/03/2019TTRCardiomyopathyofwild-typeorhereditarytransthyretin- mediatedamyloidosis(ATTR-CM)

GIAPREZAAngiotensinII12/21/2017G-protein-coupledangiotensinIIreceptortype1Increasebloodpressure

BEVYXXABetrixaban06/23/2017FxaThromboemboliccomplications

ENTRESTOSacubitril/valsartan07/07/2015AT1Cardiovasculardeathandhospitalizationforheartfailure KENGREALCangrelor06/22/2015P2Y12Periproceduralmyocardialinfarction(MI),repeatcoronary revascularization,andstentthrombosis(ST)

CORLANORIvabradinehydrochloride04/15/2015HCN)Hospitalizationforworseningheartfailure

SAVAYSAEdoxaban01/08/2015FXaStrokeandsystemicembolism(SE)

Centralnervoussystemdiseases

Eplontersen12/21/2023TTRmRNAThepolyneuropathyofhereditarytransthyretin-mediated amyloidosis

WAINUA (AUTOINJECTOR)

ZAVZPRETZavegepant03/09/2023CGRPMigrainewithorwithoutaura

SKYCLARYSomaveloxolone02/28/2023Nrf2Friedreich ’ sataxia

LEQEMBILecanemab-irmb01/06/2023AmyloidbetaplaquesAlzheimer ’ sdisease

AMVUTTRAVutrisiran06/13/2022TTRmRNAThepolyneuropathyofhereditarytransthyretin-mediated amyloidosis

QULIPTAAtogepant09/28/2021CGRPMigraine

ADUHELMAducanumab-avwa06/07/2021AmyloidbetaAlzheimer ’ sdisease

DANYELZANaxitamab-gqgk11/25/2020GD2Relapsedorrefractoryhigh-riskneuroblastomaintheboneor bonemarrow

DETECTNETCopperCu64dotatateinjection09/03/2020SSTR2Positronemissiontomography(PET)forlocalizationof somatostatinreceptorpositiveneuroendocrinetumors(NETs)

ENSPRYNGSatralizumab-mwge08/14/2020IL-6Neuromyelitisopticaspectrumdisorder(NMOSD)

EVRYSDIRisdiplam08/07/2020SMN2Spinalmuscularatrophy(SMA)

UPLIZNAInebilizumab-cdon06/11/2020CD19Neuromyelitisopticaspectrumdisorder(NMOSD)

TAUVIDFlortaucipirF1805/28/2020AggregatedtauproteinAlzheimer ’ sdisease(AD)

ONGENTYSOpicapone04/24/2020COMTParkinson ’ sdisease(PD)

KOSELUGOSelumetinib04/10/2020MEK1/2Neuro ﬁ bromatosistype1(NF1)

NURTECODTRimegepant02/27/2020Calcitoningene-relatedpeptidereceptorMigraine VYEPTIEptinezumab-jjmr02/21/2020CGRPMigraine

UBRELVYUbrogepant12/23/2019Calcitoningene-relatedpeptidereceptorMigraine

REYVOWLasmiditan10/11/20195-HT1FMigraine NOURIANZIstradefylline08/27/2019A2AParkinson ’ sdisease(PD)

TEGSEDIInotersen10/05/2018TTRmRNAPolyneuropathyofhereditarytransthyretin-mediated amyloidosis

AJOVYFremanezumab-vfrm09/14/2018CGRPMigraine

Table1. continued

MoleculartargetIndication

FDA-approveddrugsonvariousdiseasescausedbyabnormalCCC

GenericnameofdrugActiveingredientsInitial approvaldate

AIMOVIGErenumab-aooe05/17/2018CGRPMigraine

BRINEURACerliponasealfa04/27/2017M6P/IGF2Infantileneuronalceroidlipofuscinosistype2(CLN2)

INGREZZAValbenazine04/11/2017VMAT2Huntington ’ sdisease;Tardivedyskinesia

AUSTEDODeutetrabenazine04/03/2017VMAT2Huntington ’ sdisease;Tardivedyskinesia

XADAGOSa ﬁ namide03/21/2017MAO-BParkinson ’ sdisease(PD)

SPINRAZANusinersen12/23/2016SMNSpinalmuscularatrophy(SMA)

NUPLAZIDPimavanserin04/29/2016serotonin5-HT2AParkinson ’ sdisease

BRIVIACTBrivaracetam02/18/2016SV2APartial-onsetseizures

UNITUXINDinutuximab03/10/2015GD2High-riskneuroblastoma Autoimmunediseases

BIMZELXBimekizumab10/17/2023IL-17A,IL-17F,andinterleukin17-AFcytokinesModerate-to-severeplaquepsoriasis

BRIUMVIUblituximab-xiiy12/28/2022CD20Relapsingformsofmultiplesclerosis(MS)

SOTYKTUDeucravacitinib09/09/2022TYK2Moderate-to-severeplaquepsoriasis

SPEVIGOSpesolimab-sbzo09/01/2022IL-36Generalizedpustularpsoriasis(GPP) VTAMATapinarof05/23/2022AhRPlaquepsoriasis

SAPHNELOAnifrolumab-fnia07/30/2021IFNModerate-to-severesystemiclupuserythematosus(SLE)

PONVORYPonesimod03/18/2021S1PMultiplesclerosis(MS)

ZEPOSIAOzanimod03/25/2020S1PMultiplesclerosis(MS);Ulcerativecolitis(UC)

RINVOQUpadacitinib08/16/2019JAKRheumatoidarthritis;Psoriaticarthritis;Atopicdermatitis

MAYZENTSiponimod03/26/2019S1PMultiplesclerosis(MS)

CABLIVICaplacizumab-yhdp02/06/2019A1-domainofvWFAcquiredthromboticthrombocytopenicpurpura(aTTP)

OLUMIANTBaricitinib05/31/2018JAKModeratelytoseverelyactiverheumatoidarthritis

TREMFYAGuselkumab07/13/2017IL-23Moderate-to-severeplaquepsoriasis

KEVZARASarilumab05/22/2017IL-6Rheumatoidarthritis

OCREVUSOcrelizumab03/28/2017CD20Multiplesclerosis(MS)

ZINBRYTADaclizumab05/27/2016IL-2Multiplesclerosis(MS) TALTZIxekizumab03/22/2016IL-17AModerate-to-severeplaquepsoriasis;Psoriaticarthritis; Ankylosingspondylitis;Activenon-radiographicaxial spondyloarthritis

COSENTYXSecukinumab01/21/2015IL-17AModeratetosevereplaquepsoriasis

Respiratorydiseases

BEYFORTUSNirsevimab-alip07/17/2023RSVRSVlowerrespiratorytractdisease

Nirmatrelvir,ritonavir05/25/2023SARS-CoV-2;CYP3ACOVID-19

PAXLOVID (COPACKAGED)

TEZSPIRETezepelumab-ekko12/17/2021TSLPSevereasthma

VEKLURYRemdesivir10/22/2020SARS-CoV-2COVID-19

Community-acquiredbacterialpneumonia(CABP)

XENLETALefamulin08/19/2019TheA-andP-sitesofthepeptidyltransferase

center(PTC)indomainVofthe23srRNAofthe 50Ssubunit

Table1. continued FDA-approveddrugsonvariousdiseasescausedbyabnormalCCC

MoleculartargetIndication

GenericnameofdrugActiveingredientsInitial approvaldate

PRETOMANIDPretomanid08/14/2019MycolicacidPulmonarytuberculosis(TB)

Travelers ’ diarrhea

AEMCOLORifamycin11/9/2018ThebetasubunitofthebacterialDNA- dependentRNApolymerase

Severeasthma

FASENRABenralizumab11/14/2017IL-5R α

CINQAIRReslizumab03/23/2016IL-5Severeasthma

NUCALAMepolizumab11/04/2015IL-5Severeasthma;Chronicrhinosinusitis

Infectiousdiseases

Nirmatrelvir,ritonavir05/25/2023SARS-CoV-2;CYP3ACOVID-19

PAXLOVID (COPACKAGED)

SUNLENCALenacapavirsodium12/22/2022p24HIV-1

LIVTENCITYMaribavir11/23/2021CMVPost-transplantCMVinfection/disease

01/21/2021Integraseactivesite;HIV-1reversetranscriptase (RT)

CABENUVAKITCabotegravirandrilpivirine (copackaged)

Zaireebolavirus

EBANGAAnsuvimab-zykl12/21/2020GlycancapandinnerchaliceoftheEBOVGP1 subunit

COVID-19

VEKLURYRemdesivir10/22/2020SARS-CoV-2RNA-dependentRNApolymerase (RdRp)

Zaireebolavirus

10/14/2020 Zaireebolavirus glycoprotein(GP)

INMAZEBAtoltivimab,maftivimab,and odesivimab-ebgn

RUKOBIAFostemsavir07/02/2020Gp120subunitwithintheHIV-1envelope glycoproteingp160

PIFELTRODoravirine08/30/2018HIV-1reversetranscriptase(RT)

TPOXXTecovirimat07/13/2018OrthopoxvirusVP37proteinHumansmallpoxdisease

07/18/2017HCVNS5BRNA-dependentRNApolymeraseHCV

TROGARZOIbalizumab-uiyk03/06/2018Domain2ofCD4HIV-1 MAVYRETGlecaprevirandpibrentasvir08/03/2017HCVNS3/4AproteaseHCV VOSEVISofosbuvir,velpatasvirand voxilaprevir

ZEPATIERElbasvirandgrazoprevi01/28/2016HCVNS5A;HCVNS3/4AproteaseHCV DAKLINZADaclatasvir07/24/2015NS5AHCV Metabolicdiseases

RIVFLOZANedosiran09/29/2023GalNAcaminosugarresiduesPrimaryhyperoxaluriatype1(PH1)

POMBILITICipaglucosidasealfa-atga09/28/2023M6PLate-onsetPompedisease

BRENZAVVYBexagli ﬂ ozin01/20/2023SGLT2Type2diabetes

TZIELDTeplizumab-mzwv11/17/2022CD3Type1diabetes(T1D)

MOUNJAROTirzepatide05/13/2022GIPandGLP-1Type2diabetes

LEQVIOInclisiran12/22/2021GalNAcPrimaryhyperlipidemia

NEXVIAZYMEAvalglucosidasealfa-ngpt08/06/2021M6PLate-onsetpompedisease

KERENDIAFinerenone07/09/2021MRType2diabetes(T2D)

ZEGALOGUEDasiglucagon03/22/2021HepaticglucagonreceptorsSeverehypoglycemia

HIV-1

Table1. continued

MoleculartargetIndication

FDA-approveddrugsonvariousdiseasescausedbyabnormalCCC

GenericnameofdrugActiveingredientsInitial approvaldate

OXLUMOLumasiran11/23/2020Hydroxyacidoxidase1( HAO1 )Primaryhyperoxaluriatype1(PH1)

LOKELMASodiumzirconiumcyclosilicate05/18/2018PotassiumHyperkalemia

STEGLATROErtugli ﬂ ozin12/19/2017SGLT2Type2diabetes

OZEMPICSemaglutide12/05/2017GLP-1Type2diabetes

MEPSEVIIVestronidasealfa-vjbk11/15/2017LysosomesMucopolysaccharidosisVII

ADLYXINLixisenatide07/27/2016GLP-1Type2diabetes

ZURAMPICLesinurad12/22/2015URAT1;OAT4Hyperuricemia

VELTASSAPatiromerfororalsuspension10/21/2015PotassiumHyperkalemia

TRESIBAInsulindegludecinjection09/25/2015CirculatingalbuminDiabetesmellitus

Developmentaldisorders

DUVYZATGivinostat03/21/2024HistonedeacetylaseDuchennemusculardystrophy(DMD) AGAMREEVamorolone10/26/2023GlucocorticoidreceptorDuchennemusculardystrophy(DMD)

NGENLASomatrogon-ghla06/27/2023GHGrowthfailureduetoinadequatesecretionofendogenous growthhormone

VOXZOGOVosoritide11/19/2021NPR-BAchondroplasiawithopenepiphyses

SKYTROFALonapegsomatropin-tcgd08/25/2021GHGrowthfailureduetoinadequatesecretionofendogenous growthhormone(GH)

AMONDYS45Casimersen02/25/2021Exon45ofdystrophinpre-mRNADuchennemusculardystrophy(DMD)

SOGROYASomapacitan-beco08/28/2020DimericGHreceptorGrowthfailureduetoinadequatesecretionofendogenous growthhormone(GH)

VILTEPSOViltolarsen08/12/2020Exon53ofdystrophinpre-mRNADuchennemusculardystrophy(DMD)

VYONDYS53Golodirsen12/12/2019Exon53ofdystrophinpre-mRNADuchennemusculardystrophy(DMD)

MACRILENMacimorelinacetate12/20/2017GrowthhormonesecretagoguereceptorsAdultgrowthhormonede ﬁ ciency

EMFLAZADe ﬂ azacort02/09/2017GlucocorticoidreceptorDuchennemusculardystrophy(DMD)

EXONDYS51Eteplirsen09/19/2016Exon51ofdystrophinpre-mRNADuchennemusculardystrophy(DMD)

TheFDA-approveddrugssummarizedfrom2015tothepresentday.Thesedrugsaremainlyusedtotreatcancers,cardiovasculardiseases,centralnervo ussystemdiseases,respiratorydiseases,infectious diseases,metabolicdiseases,anddevelopmentaldisorders

subsequentcancercells.Giventhat,biomarkersofCSCare instrumentalindiagnosingcancer,guidingtargetedtreatments, andforecastingdiseaseprogressionsincegrowingevidences indicateCSCsmayplaypivotalrolesincriticaldiseasestagesfrom cancerinitiationtometastaticspread.240

Cardiovasculardiseases

Vitalbodyfunctionssuchasheartbeatandbloodpressure maintenanceareunderthecontroloftheautonomicnervous system.Thecardiovascularsystemrespondtosympathetic stimulationofhormonessecretedfromnerveterminalsby adrenergicreceptors(ARs),whicharethedominantGPCRsin theheart.Invascularsmoothmuscle,catecholaminestimulation causesvasoconstrictionthrough α-ARsandcausesvasodilation through β2-ARs.Intheheart,catecholaminestimulationcauses increasedheartrateandmyocardialcontractilitythrough β-AR. Thesignalingpathwaymostextensivelyresearchedincardiac myocytesisactivatedinresponseto β-adrenergic stimulation.241,242

Thecontractionoftheheartisinitiatedbyanelevationin cytosolicCa2+ concentrationwithincardiacmyocytesfollowing theirelectricalactivation.Thisprocessisregulatedbyamultitude ofsignalingpathways,whichinvolvecascadesofsignaling moleculesculminatinginposttranslationalmodiﬁcation(PTM, e.g.,phosphorylation)oftargetproteins.243 Forexample,CaMKIIis apivotalregulatorofexcitation-contractioncouplingandCa2+ cycling,inchargeofnumerousessentialcardiacfunctions. Accordingtoreports,theexpressionlevelandactivityofthe maincardiacsubtypeCaMKIIδ areunregulatedinhumanheart failure.244,245 ChronicoveractivationofCaMKIIcanleadtoseveral otherpathologicalsymptoms,includingcardiachypertrophy,246 diastolicandsystolicdysfunction,247,248 arrhythmia,249,250,and ischemia/reperfusioninjury.251,252 DifferentPTMsofCaMKIIlead heartdiseasethroughdifferentpathologicalmechanisms.In details,oxidizedCaMKIIcontributestoapoptosispost-myocardial infarctionandatrial ﬁbrillation,246 whileO-GlcNAcylationcontributestohyperglycemia-inducedarrhythmia.253–256 Whereas, nitrosylationofCaMKIIconfersasex-dependentprotectiveeffect againstharmfromischemia/reperfusioninfemales.257

Cardiovasculardiseaseencompassesarangeofconditions impactingtheheartorcirculatorysystem,includingheartfailure, coronaryarterydisease,stroke,highbloodpressure,and atherosclerosis.Atherosclerosisisachronicinflammatorydisease characterizedbytheformationoflipid-richplaquesonthewallsof bloodvessels,whichcanleadtomyocardialinfarction,stroke, unstableangina,andsuddencardiacdeath.258–260 Atherosclerosis isnotconsideredsimplyasalipidstoragedisorderanymore,as researchhasreportedtheinvolvementofinflammatorymechanismsintheprogressionofthedisease,suchastheaccumulationof leukocytesatsiteoflesion.261,262 Leukocyteswithintheplaque producegrowthfactors,inducingSMCproliferationinadvanced lesions.263 The flowofatherosclerosistriggersNF-κBactivationin endothelialcells,leadingtotheproductionofinflammatory cytokines,therebyestablishinganenvironmentconduciveto atherosclerosis.264 Atypicalatheroscleroticplaquecontainsalipid core,withapoptoticmacrophagesconstitutinganecroticcore.265 Macrophageactivationtriggersthereleaseofvariouscytokines, transformationintofoamcells,andsubsequentnecrosis.266

Inaddition,coronaryheartdisease(CHD)accountsfor42.1%of allcardiovasculardiseasedeaths.High-densitylipoprotein-associatedcholesterol(HDL-C)islinkedtolowerriskandenhanced outcomesinCHDpatientsviaCCC.Cholesterolistransportedfrom peripheraltissuecellssuchasmacrophagesorvascularSMCsto theliverthroughHDL-Cforrecoveryorexcretionthroughbileor feces.267–269 ApoA-1isthemajorHDLstructuralprotein,whichhas beenrecognizedasananti-atheroscleroticmarkerforacquiring cholesterolandphospholipidsefﬂuxedbyhepatocytesand enterocytes.270 Thedepositionofcholesterolinarteriescan

initiatetheatheroscleroticprocess,givingrisetotheinﬁltration ofmultifariouscelltypesincludingmacrophages, ﬁbroblasts,and SMCs,allofwhichplayaroleinplaqueformation.267

Studieshavedocumentedthepresenceofextracellularvesicles (EVs)withindevelopingplaquesandintimallesionsofadvanced plaques,indicatingtheirroleatboththeinceptionandculminationofplaqueformationinhumans.271–273 EVsoriginatingfrom foamcellshavebeenidentiﬁedascatalystsforSMCmigrationand activationoftheERKpathway,therebyexacerbatinglesion progression.274 Researchhasshownthatfollowingexposureto anatherogenictriggerlikeoxidizedlow-densitylipoprotein, macrophageEVsareenrichedwithnumerousmiRNAs,including miR-146a,miR-128,andmiR-185.275 Furthermore,miR-146ahas beenimplicatedinacceleratingatherosclerosisprogression throughthepromotionofmacrophagemigrationtowardsthe vascularwall.275 Intercellularcommunicationbetweenendothelial cellsandSMCsiscrucialformaintainingvascularhomeostasis.The transferofmiR-155mediatedbyEVsfromSMCstoendothelial cells,drivenbyKLF5,leadstothedisruptionoftightjunctionsand endothelialbarrierintegrity,promotingatherosclerosis.276 The transferofmiR-143andmiR-145inendothelialcellEVsinducedby KLF2blocksthetransdifferentiationofSMCs,therebymediatinga protectiveeffectagainstatherosclerosisthroughendothelialcellSMCcommunication. 277

Intheheart,increasingevidencesuggeststhepresenceofCCC amongcardiomyocytesandnon-myocytecellssuchas ﬁbroblasts andmacrophages.Besides,cardiomyocytesandvascularendothelialcellssharenumeroussystemsofCCC,includingdirect communicationandparacrinesignalssuchaspansexins,hemichannels,andpurinergicsignals.Itisreasonabletobelievethat theycanregulateeachother’sbehaviorthroughCCC.For example,vasculardysfunctionisrelatedtoarrhythmia.278–280 WithourincreasingunderstandingofCCC,newopportunitieswill emergetopromotethetreatmentofvariouscardiovascular diseases.

Centralnervoussystemdiseases

Almostone-sixthofthepeopleintheworldsufferfromcentral nervoussystem(CNS)diseases,rangingfrommildnerveinjuryto comaandevenbraindeath.281 Themaincelltypeinneuraltissue isneurons.Theprimaryfunctionofneuronsistobecapableof communicatingwitheachotherandwithothercelltypes.The axonsofneuronsreleasecontentstosynapticintervalsthrough exocytosis,transmittingthesechemicalneurotransmittersto receptorsonanotherpostsynapticcell.282

ExosomesareactiveparticipantsinCCC,beingreleasedbya varietyofcelltypeswithinthebody,includingneurons.Presentin diversebody fluidslikeblood,cerebrospinal fluid,alveolarlavage fluid,ascites,andamniotic fluid,exosomesinfluenceothercells, triggeringarangeofphysiologicalorpathologicalresponses.283 For example,exosomessecretedbyoligodendrogliomacellscan induceneuronaldeath.Inconditionssuchasamyotrophiclateral sclerosis(ALS),frontotemporaldementia(FTD),FTD-ALS,tauprotein disease,Parkinson’sdisease(PD),andAlzheimer’sdisease(AD), exosomesmigratethroughthebloodandcerebrospinal fluid, carryingmisfoldedproteinsorpro-inflammatorymolecules.284–286 Exosomesreleasedbyneuronscanbeinternalizedbyother neurons,indicatinganovelavenueforinterneuronalcommunication.287 NeuronsintheCNSsecreteexosomestoorchestrate intricatecommunicationwithastrocytesandmicroglia,facilitating extensivecrosstalkthatgovernsneuronalregenerationand synapticfunctionthroughoutbothdevelopmentalstagesandadult life.288,289 Intheirroleofregulatingmicroenvironment,astrocytes andoligodendrocytesproduceEVstoenhanceintercellular communicationandtheactivityoftargetcells.290–292 Therelease ofexosomesisspeculatedasacriticalprocessinneurogenesis, essentialforproteinclearance,andistriggeredbythefusionoflate endosomesandlysosomesduringaxonalelongation.293–295

IntheCNS,exosomespotentiallyplayadualrole:theyarevital componentsessentialfortheCNSdevelopmentandprotection undernormalconditions;whiletheirparticipationinthe pathogenesismightworsentheconditionsofcertainneurodegenerationandneuroinflammation.296–298 Forinstance,elevated levelsofmicroglialexosomeshavebeenobservedinAlzheimer’s diseasepatients,andexosomesfromoligodendrocyteshavebeen implicatedininducingneuronaldeath.299 Theextensiveinteractionsbetweenglial-derivedexosomesandneuronsalsosuggest thatthesevesiclesareinstrumentalinboththeformationand sustenanceofneuralcircuits,evidencedbytheirpromotionof neuriteoutgrowthinhippocampalneuronsandenhancementof corticalneuronviability.292 Moreover,exosomesoriginatingfrom microgliaareknowntoregulatetheactivityofneuronby enhancingmyelinmetabolism.290 Theabilityofexosomesto accessthebloodstreamandcerebrospinal fluidrendersthese vesiclespotentialmeansforremotecommunicationandtransportation,facilitatingthedeliveryofbioactivemoleculesto specifictargets.300 Asexosomesarecapableoftraversingthe blood–brainbarrier(BBB)andpreservingthecharacteristicsof theiroriginatingcells,circulatingexosomescanofferinsightsinto theconditionoftheoriginatingtissue.301,302 Thisfeaturepresents apreciseandminimallyinvasiveapproach(viaperipheralblood sampling)fortheearlydiagnosisofneurologicaldisorders.303–305 . Inthischapter,wefocusontherelationshipbetweenCNSand exosomes,andreviewhowexosomesaffectCCCtoleadtoCNS diseases.Consequently,variousstrategiesareoutlinedfor diagnosingandtreatingCNSconditionsbyleveragingexosomes intherealmofCCC.

Alpha-synuclein(α-syn)playsacentralroleinthepathogenesis ofPD,anditselevatedlevelsareadequatetocausePD.306–309 Exosomesinthebloodcarry α-synandintensifiestheaccumulationandaggregationof α-synthroughvariousmechanismssuch asmiRNAs,consequentlytriggeringinflammation,inhibiting autophagy,andcontributingtothepathogenesisofParkinson’s disease.306,308,310–317 Exosomesderivedfromglialcellstransport αsynandinflammatoryfactorsfromglialcellstoneurons, exacerbatingtheprogressionofPD.318 Contentsofexosomes derivedfromvariousbodily fluidscanserveasbiomarkersfor diagnosingPD.Forexample,exosomesintheplasmaofPD patientswerefoundanelevationexpressionof α-synandtau proteins.Thepresenceof α-synincludesadditionalcharacteristics suchas β-sheet-richstructuresanda fibrillaryappearance, indicatingthepathologicaltransformationofthisprotein.308 319 320 AsforthemiRNA,theexpressionofmiR-128,miR-505,andmiR19bisdownregulated,whiletheexpressionofmiR-331-5p,miR24,andmiR-195isincreasedinthepatient’sbloodexosomes.321–323 Elevatedphosphorylationlevelsofleucine-rich repeatkinase2,aswellasincreasedlevelsofsynaptosomeassociatedprotein23andcalbindinproteinslinkedtoPD-related damage,wereidentifiedinexosomesisolatedfromtheurineof individualswithPD.324,325 Inaddition,leucine-richrepeatkinase2 and α-synasabiomarkerhasenteredtheclinicalstage.326 ADisthemostcommonformofdementia,ofwhichthe numberwillreach130millionby2050.327 DuetotheaccumulationofexosomesproteinsinamyloidplaquesinthebrainofAD patients,exosomesarereceivingincreasingattention.Exosomes derivedfromdifferentcelltypesplaydifferentfunctionsinAD. HighconcentrationsofmicroglialexosomeswerefoundinAD patients.299 Exosomesderivedfromneuronalcellscontain precursorsofamyloidproteinandenzymesusedforprecursor maturation.Plasmaexosomesaccumulateinamyloidplaquesand participateinplaqueformation.328 ExosomesderivedfromM1 microgliastimulateactivationofrestingmicrogliaandenhance pro-angiogenicresponsesviaIrf1/miR-155-5p/Socs1axisinthe retina.329 ExosomesspringfromM2microgliamitigateneuronal damageandmitochondrialdysfunctioninADthroughthePINK1/ Parkinpathway.330 MicroRNAsthataffecttheoccurrenceand

developmentofADarepresentinexosomesderivedfromboth peripheralbloodandcerebrospinal fluidinpatientswithAD.331 Forexample,exosomesaffecttheprogressionofADbyblocking thetranscriptionofamyloidprecursorprotein(APP)throughmiR185-5porexosomecontentmiR-193b.332,333 Incontrast,exosomes comefromhumancerebralspinal fluidorN2acellsenhancethe synapticplasticitydestructionactivityofsynthesisandADbrainderivedamyloid-β (Aβ)invivo.334 Inaddition,exosomesfrom astrocyteswithaccumulatedcholesterolsignificantlycontributeto thetransportofAPP/Aβ peptidesandtheinfluenceofneuronal viabilityintheaffectedADbrainregions.335

Theexcessivephosphorylationoftauproteinisalsoa characteristicofAD.Exosomessecretedbymicrogliaareinvolved inthetransportoftauprotein.Inhibitingthesynthesisorsecretion ofexosomespreventstheaggregationoftauproteininthe brain.336–338 Inhibitionofexosomebiosynthesisbyblockingthe activityofakeyenzymeregulatingceramidebiosynthesis,neutral sphingomyelinase2,reducedtheproliferationofAβ plaqueand tauinADmousemodel.339 Someenzymescarriedinexosomes, suchasneprilysinandinsulin-degradingenzymes,reduce intracellularandextracellularAβ levels.340 Insummary,exosomes participateinthepathogenesisofADbytransmittingdifferent substancesorinformation.Therefore,exosomesserveastransmissionfactorsanddiagnosticbiomarkersforAD.341,342

IntheexplorationofADdiagnosis,somescientistsbelievethat exosomesinthebloodofADpatientsarefewerandsmaller.343 whiletherearealsoreportsthatexosomesinADpatientsare bigger.344 Brain-derivedexosomesinADpatientsexhibited signiﬁcantalterationsinglycerophospholipidandsphingolipid levels,especiallyanelevatedlevelofplasmalogenglycerophosphoethanolamineandareductioninpolyunsaturatedfattyacylcontaininglipids.345 Morethan20exosomalmiRNAsinAD patientswerefoundtobesigniﬁcantlydifferentfromthosein controlgroupbyusingnext-generationsequencing(NGS).346–348 Thecontentsoftheseexosomesmayhavehighpotentialvaluein thediagnosisofAD.

Inrecentyears,therolesofmastandmicrogliainthenervous systemhavealsobeendiscovered.Afterrespondingtoenvironmentalsignals,mastcellssecretedifferentneurotransmittersor neurotrophicfactors.Thisparacrinesecretionleadstoacute activationand/orlong-lastingchangesinexcitabilityandphenotype,whichisassociatedwithneuroinflammation.349 Besides, exosomesderivedfromglialcellsbindtotoll-likereceptor2and thetoll-likereceptor4ofneurons,leadingtoneuroinflammation andevenneuronalapoptosis.Exosomesderivedfromglialcells transportabnormallyexpressedmiRNAs,triggeringandspreading neuroinflammation.311,315 Understandingneuroinflammationalso requiresrecognizingthatthenon-neuronalcell–cellinteractions betweenglialcells,mastcells,andtheglialcellsthemselvesare integralcomponentsoftheinflammatoryprocess.Inthiscontext, mastcellsplayacrucialroleinorchestratingtheinflammatory process,fromitsinitiationtoprolongedneuroinflammation.350

BBBcomposedofendothelialcellsconnectedbytightjunctions andadherentprocessesprotectspotentialintrudersunder physiologicalconditions.InthetreatmentofCNSdiseases,BBB isanobstaclefordrugdelivery.351–353 Inthepastfewdecades, researchershavecontinuouslyexploredmethodsfordelivering drugstothebrainthroughBBB.Exosomeshaveloadingand deliveryfunctions,andtheirlipidbilayercanfusewithmembranelikestructuresinthebodysuchastheBBB.Beingabletopass throughBBBandcarrygoods,exosomeshavebecomeastar substancefortreatingCNSdiseases.The ﬁrsttreatmentapproach involvesutilizingexosomesreleasedbyspeciallytreatedcells, suchasexosomesobtainedfromcellstreatedwithcurcumin. Theseexosomeshaveshownpromiseinpreventingneuronal deathbothinvitroandinvivo,alleviatingAlzheimer’sdisease symptomsbyinhibitingtauproteinphosphorylationthroughthe activationoftheAKT/GSK-3β pathway.354 Thesecondtreatment

avenueinvolvesinvestigatingtherapiesusingexosomessourced fromstemcells,suchasexosomesreleasedbyhumanumbilical cord-derivedmesenchymalstemcells(MSCs)andadipose-derived mesenchymalstemcells.Theseexosomesarebeingexploredfor theirpotentialtoaddressconditionslikeneuroinﬂammation, Alzheimer’sdisease,braininjury,andneurodegenerativedisorders byreducingAβ aggregation.355–357 Exosomesderivedfromstem cellssourcedfromthedentalpulpofshedhumandeciduous teethexhibitedaneuroprotectiveimpactondopaminergic neurons.Throughintranasaldelivery,theseexosomesdemonstratedanimprovementinmotorfunctionandareductionin dopaminergicneuronlossinParkinson’sdisease.326 358 Thethird approachinvolvesutilizingexosomesasvehiclestotransport varioustherapeuticagents,includingsiRNAandothermedicinal RNAs,peptides,dopamine,syntheticdrugs,bioactivecompounds fromplants,enzymes,proteins,andantisenseoligonucleotides designedtotargetthehuman α-synucleinsequencewithsuitable modiﬁcations.Thisstrategyaimstotreatcentralnervoussystem diseasesbyleveragingexosomesascarriersfordeliveringthese therapeuticpayloads.359–367

Autoimmunediseases

Autoimmunediseasesresultfromanaberrantimmuneresponse againstthebody’sowncellsandtissues,impactingconditionslike inflammatoryboweldiseaseandrheumatoidarthritis.Assoluble messengers,cytokinesfacilitatecommunicationamongimmune cells,playingakeyroleinregulatingthebody’sresponseto pathogens.368 Althoughthesedrugsoftenresultinadverse reactions,currenttreatmentsforautoimmunediseasestypically involvedrugswithanti-inflammatoryandimmunosuppressive properties.Thedevelopmentofdrugstargetingcytokinesor receptors,commonlyknownas “biologics”,representsasignificant advanceintreatingautoimmuneandinflammatorydiseases. Nevertheless,whilebiologicshavebeentherapeuticallysuccessful, theymaynotcompletelyeliminaterheumaticpathologyinall patients.Moreover,theefficacyofnumeroussuchagents diminishesgraduallyowingtotheirimmunogenicity.369 By modulatingcell-to-cellsignaling,itisfeasibletosuppressthe hyperactivationoftheimmunesystemanddiminishinflammatory reactionsinautoimmunediseases.Forexample,introducing moleculeslike “pseudochain” proteinsonthesurfaceofTcells candisruptsignalsbetweenTcellsandothercells,offeringan effectiveapproachtotreatingautoimmunediseases.Many cytokineshijackJAKandSTATsforintracellularsignalingin autoimmuneandinflammatorydiseases.370 Geneticmutationsin JAKandSTATgenesarelinkedtoarangeofimmunedeficiency syndromesandareconnectedtothedevelopmentofautoimmunediseases.Giventheirrolesindownstreamsignalingof cytokinereceptorsandgrowthfactors,JAK/STATpathwaysare consideredaspromisingtherapeutictargetsforbothcancerand autoimmuneconditions.371 Theefficacyofsmall-moleculeJAK inhibitorsintreatingrheumatologicconditionsillustratesthe potentialoftargetingintracellularsignalingpathwaysforautoimmunediseasetherapy.372

NKcellsarelymphocytesoftheinnateimmunesystemthat makearapidrespondtodiverseinsultsthroughcytokine secretionandcytolyticactivity.373–376 Theynotonlybringcellmediatedcytotoxicitytobearontumorcellsorinfectedcellsbut alsoregulatetheeffectofotherimmunecellsviathesecretionof cytokinesandchemokines,therebyplayingaregulatoryroleinthe immuneresponse.377–379.However,hyperactivationormalfunctionofNKcellsmightbeimplicatedintheetiologyofspecific diseases.380,381 Inviral-inducedmodelsofautoimmunediabetes, NKcellsarelikelytocontributepathogenicallyinthelaterphases ofautoimmunity.382 Thepresenceofalargenumberofcirculating NKcellsmayalsobeanonspecificbutsignificantcharacteristicof apredispositiontomiscarriage.380 ItisclearthatNKcellspossess dualroles,bothprotectiveandpathogenic,acrossvariousdisease

models,andoccasionallyevenwithinthesamedisease.The cytokineenvironmentandotherstimuliactinguponvariouscell surfacereceptorsintargetorgans,suchasKIR,maytriggerNKcell reactionsdistinctlyandinﬂuencetheircontributiontovarious autoimmunedisorders.383

EVscandirectlyinteractwithimmunecells,activatingor regulatingtheirfunctionsbycarryingimmune-activatingmolecules(IL-12/15/18).384 SomeEVscancarryimmune-suppressive molecules(TGF-β,PD-L1,etc.),385 exertinginhibitoryeffectson immunecells,helpingtumorcellsevadeimmunesystem surveillance.Bydeliveringspecificsignalingmolecules,EVscan promotethegenerationandfunctionofregulatoryTcells(Tregs), therebyplayingaroleinmaintainingimmunetoleranceand preventingautoimmuneresponses.EVscansecretesoluble mediators,bindtoreceptors,andactivateintracellularsignaling pathways.386 whiletheycanalsoactthroughdirectmembrane contact.Ultimately,thisinteractionmayleadtotheactivationof membranereceptorsonthetargetcells,therebyactivating differentsignaltransductionpathways.387,388 EVsexpressboth self-antigensandpeptide-MHCcomplexes.Therefore,EVsmay representasourceofself-antigensandcouldpotentiallyactivate autoreactiveTcellsinthecontextofMHC.389 Exosomesarea subtypeofEVsthatareknowntoplayasignificantrolein intercellularcommunicationandantigenpresentation.Exosomes secretedbyantigen-presentingcells(APCs)theoreticallypossess thecomponentsnecessaryforantigenpresentationandthe activationofautoreactiveTlymphocytes.390 Exosomesmayact indirectlythroughinteractionswithAPCs,391–396 especiallyforthe initialactivationofTcells.396,397 EVscanbindtoAPCsthrough adhesionmoleculesexposedontheirsurfacealongwithselfantigen/MHCcomplexes,allowingtheT-cellreceptortoengage withtheAPC.393 Theco-stimulatorymoleculesexpressedbyAPCs providethenecessarysecondsignalfortheactivationofT lymphocytes.Thisexplainstheirinvolvementinthepathophysiologyofautoimmunediseasesthroughparticipationinphenomena suchasinflammationandthrombusformation,vasculardysfunction,andthemaintenanceofautoimmuneresponses.398

Respiratorydiseases

Evidencessuggestthatmultiplecellpopulationsinthelungswork togethertoregulatetheresponseoflunginflammationtodirect andindirectstimuli.399 Afterinfectionortrauma,residentcells suchasalveolarmacrophagesandalveolarepithelialcellssecrete inflammatorycytokinesintothealveoli,whichinducesalarge numberofinflammatorycellstomigratetothealveolarspace. Fromthesemigratinginflammatorycells,inflammatorymediators arereleasedandfurtherleadtotissuedamageandthe developmentofacutelunginjuryandacuterespiratorydistress syndrome.400 Thecoordinatedparticipationofneutrophilsand macrophagesinantimicrobialimmunityservesasbothinducers andeffectorsofadaptiveimmunityagainstextracellularand intracellularmicrobialpathogens.Neutrophilsandmacrophages playcrucialrolesintheinnateimmuneresponsebyphagocytosingpathogensandactivatingadaptiveimmuneresponses throughantigenpresentationandcytokinesecretion.CCC betweenlungepithelialcellsandalveolarmacrophagesplaysan essentialroleinlunginflammationandinjury.401–404 Polymorphonuclearneutrophil(PMN)accumulationandrapidinfiltrationin interstitialandalveolarspacesofthelungsisahallmarkoflung inflammation.405 InteractionsofPMNwithlungvascularendothelialcellscontributetotheactivationofspecificendothelialcells responsesinvolvedininnateimmunity.406–408

Asthmaisthemostcommonchronicrespiratorydisorder.CCC ishighlyinvolvedinthepathogenesisofasthmainwhich epithelial-derivedcytokinesdrivedendriticcellactivationand phenotypicchangesintheairways.Theseactivateddendriticcells thenmigratetosecondarylymphoidtissues,wheretheypresent allergenstonaïveTcells,triggeringandperpetuatingtheallergic

immuneresponseinasthma.409–411 Theaboveprocessisatypical inflammatoryresponseinasthma-type2inflammation.Duringthis process,interleukin(IL)-5targetingeosinophilsandIL-4targeting lymphocytesarereleased.412,413 Inasthma,mastcellsand macrophagesproducehistamine,serotonin,andvariousinflammatorysubstances.Thesemediatorspromotesmoothmuscle contraction,increasemucusproduction,andenhancevascular permeability,ultimatelyleadingtoedemaandcontributingtothe characteristicsymptomsofasthma.414 Exosomessecretedfrom variouscells,includingrespiratoryepithelialcells,lymphocytes, mastcells,eosinophils,respiratorysyncytialvirus-infectedcells, andlungepithelialcells,havethepotentialtocontributetoor exacerbateasthma.Theseexosomescancarrybioactivemolecules andsignalingfactorsthatmayinfluenceimmuneresponses, inflammation,andairwayremodelinginasthma pathogenesis.415–426

Infectiousdiseases

Humanimmunodeﬁciencyvirustype1(HIV-1)causesachronic infectionleadingtoAIDSviainfectingCD4receptor-expressing (CD4+)immunecells.TheHIV-1envelopeglycoproteinmediates twokindsofinfection.Oneiscalledcell-freeinfectionthat infectedhostcellsreleasevirionstoinfectnon-adjacentuninfectedtargetcells.Theotheroneiscalledcell–cellinfectionthat infectedhostcellstransmitHIV-1toadjacentuninfectedtarget cellsviadirectcell–cellconnectionscalledvirological synapses.427–433 InadditiontoCD4+ immunecells,myeloidcells suchasmacrophages,dendriticcells,andosteoclastsare increasinglyrecognizedasimportanttargetcellsforHIV-1.These myeloidcellscanplayrolesinvariousstagesofthedisease, includingsexualtransmissionandearlyvirusdisseminationin bothlymphoidandnon-lymphoidtissues.Theycanserveas reservoirsforpersistentviralinfection,contributingtothe establishmentofapersistentvirallibrarywithinthehost.Atleast invitro,thesemyeloidcellsarerarelyinfectedbycell-free infection.Onthecontrary,virustransmissionthroughcell–cell infectionmaybethemainmodeofvirusreproductioninvivo throughtheformationoftunnelingnanotubes,homotypicor heterotypiccell–cellfusionandphagocytosis.434–438

ThechronicinfectionofhepatitisBvirus(HBV)impacts approximately257millionindividualsglobally,leadingtosevere liverdiseases,includingcirrhosisandlivercancer.Theinteraction betweenhealthyandHBV-infectedcells,alongwithothercellular playerssuchasinnateandadaptiveimmunecells,ismediated throughdirectcontactsandtheexchangeofdiversefactors.This intercellularcommunicationcanbefacilitatedthroughvarious mechanisms,suchasthereleaseofmetabolites,virions,protein complexes,andexosomes.Theseelementsplaycrucialrolesin modulatingtheimmuneresponse,viralreplication,andtheoverall pathogenesisofHBVinfection.ChangesassociatedwithHBV infectionaltertheCCCbetweenhepatocytesandadaptive immunecells,signiﬁcantlyinﬂuencingthedisease’sprogression.439–442 Understandingthesecommunicationpathwaysis criticalforunravelingthecomplexinterplaybetweenthevirus andthehostimmunesystem,whichcanultimatelyinformthe developmentofeffectivetherapeuticinterventionsagainstHBV infection.

Thecoronavirusdisease2019(COVID-19)spreadworldwideina shortperiod,resultinginnumerouscasesandassociateddeaths.A previousstudyrevealedthatmonocytesinsevereCOVID-19cases havethecapacitytoengagewithCD8+ Tcells,BcellsandCD4+ Tcells,chemokinereceptorswerealsoenrichedinmonocytes fromseverepatients.443 Chemokinesinteractwithchemokine receptorstoexerttheirbiologicaleffects,suggestingthatthese cytokinesortheirreceptorscouldbepotentialtreatmentsfor severeCOVID-19patientsandmaybecometherapeutictargetsfor COVID-19patients.Syncytiaarelargemulti-nucleatedcells producedbythefusionoftwoormorecells.Syncytial

pneumocyteshavebeenobservedinpatientswhohave succumbedtotheCOVID-19.ThespikeproteinofSARS-CoV-2 interactswiththeACE2receptorandisprimedbytheserine proteaseTMPRSS2onneighboringcells,resultinginsyncytia formation.444 SyncytiatransmitvirusesthroughCCCtoimmune cellsandprotectingthevirusfromneutralizingantibodies, therebypromotinginfection.Rapidsyncytialcollapsetrigger inﬂammatoryimmuneresponseswhichinturnleadstoviral pathogenicity.445–457 CCCamongmyeloid,epithelialandTcells candrivetissuedamage.458 HeterogeneousCCCpatternsexist amongmoderateandseverepatientsacrossepithelialand immunecellsinlungtissues.459 460 Exosomesparticipateinviral pathogenesisandspreadingbyCCCandinducecellulardamage andmultipleorgandysfunctionsuchasinﬂammation,complementpathway,immunemodulation,andcoagulationinCOVID-19. Moreover,theexosome-basedvaccinethatcontainsmRNA encodingtheproteinsofimmunogenicCOVID-19hasbeen developed.Furthermore,mesenchymalstemcell-derivedexosomesandconvalescentplasma-derivedexosomesarenew promisingtherapeuticstrategiesinseverelyaffectedCOVID-19 patients.461–467

Metabolicdiseases

Metabolicdysfunctionencompassesaspectrumofdiseaserisk factors,encompassinghyperglycemia,dyslipidemia,hypertension, obesity,andinsulinresistance.Thepathogenesisofmetabolic dysfunctioniscomplex,involvingadiverserangeofcelltypes, tissues,organs,inﬂammatorysignalingpathways,andhumoral factors.468 EVscancarrysubstancesoftheirparentcells(RNA, DNA,andlipids)andmayprovidethevalueofdiagnosisand prognosisinmetabolicdysfunction.469–479 Theycanmediatelocal communicationbetweenhomologouscellswithintissues(suchas endothelialcells,hepatocytes,immunecells,orpancreaticcells) andtraverseorgansystemsbyenteringtheperipheralbloodstream.480,481 Therefore,EVsholdpromiseasbiomarkersfor predictinganddiagnosingmetabolicdiseases.475,477,482,483 Speciﬁcally,platelet-derivedEVs,markedbythepresenceofCD41, CD42b,andphosphatidylserine,havebeenimplicatedinvarious physiologicalandpathologicalprocesses,includingexercise,484 485 acuteinjury,486 anddiabetes.487

Thehighlycomplexpathogenesisoftype1diabetes(T1D)is drivenbyseveralimmunecellswitheffectiveresponseand regulatorycharacteristics,ultimatelyleadingtothedestructionof insulin-producing β cells.Theinteractionsbetweenimmunecell groupsandpancreaticisletsaremultifaceted.Inbothhumansand mice,mutationsintheFOXP3gene,akeyregulatorofTregulatory (Treg)celldevelopment,maintenance,andfunctionality,can precipitatesevereautoimmunity,includingT1D.488,489 Anexpanding corpusofresearchindicatesthatdisruptionsinTreginduction, stability,andfunctionalityarecentraltotheonsetofislet autoimmunityandtheclinicalprogressionofT1D.490–492 Specifically, inthecontextofisletautoimmunitypathogenesis,thepopulationof insulin-specificTregsismarkedlydiminished,andboththeinduction andstabilityofTregsarecompromisedinhumansandmice.493 The pivotalfunctionofTregsinthwartingT1Dalignswiththeobserved rapiddiseaseadvancementinNODmicedevoidofTregs,494 andit hasbeenfoundthatTregdeficiencyleadstoheightenedT-celland NKcellinfiltrationinthepancreas.495 StudieshaveshownthatTregs fromT1DpatientsexhibitdysfunctionalIL-2receptorsignaling pathways.496 AdministeringalowdoseofIL-2for fiveconsecutive daysincreasedthenumberofpancreaticTregsinNODmiceatthe prediabetesstageby1.5times.Thisinterventionpreventthe progressionofT1Din60%ofthetreatedanimalsandrestoredblood glucoselevelstonormalcy.497 Consequently,thetargetedenhancementofTregsthroughlow-doseIL-2administrationemergesasa viabletherapeuticapproach.

RecentstudieshaveinvestigatedtheroleofEVsinregulating systemicmetabolism,revealingthatEVsoriginatingfrom

adipocytesserveasmediatorslinkingobesityandinsulin resistanceinperipheraltissues(suchastheliver).498,499 EVs facilitatecommunicationbetweenadipocytesandvariouscells withinadiposetissue.Togiveanexample,adipocyte-derivedEVs canchemotacticallyattractmonocytes,potentiallyleadingto adiposetissueinflammationinobeseinsulin-resistantanimalsand humans.500,501 Theformationofobesityandinsulinresistance correlateswithanincreasedinfiltrationofmacrophagesinto adiposetissue.502 Avastbodyofliteraturedescribesthe detrimentalroleofadiposetissuemacrophages(ATMs)in regulatingsystemicmetabolismthroughtheoverproductionof inflammatorycytokinesthatcanblockinsulinsignaling.503 ExosomesreleasedbyATMsplayapivotalroleinmodulating thefunctionalityofadiposetissueandinsulinsensitivity.504 PatientplasmaandurineenrichthesourcesofEVs,andstudies suggestthatEVmiRNAscanserveasdiagnostictoolsforpatients withmetabolic.472,477–479 andcardiovasculardiseases.505,506 The miRNA-155isoneofthemiRNAsoverexpressedinexosomes derivedfromobeseATMs.StudiesindicatethatmiRNA-155 regulatesadiposetissuehomeostasisbydirectlyinhibitingthe adipogenictranscriptionfactorsperoxisomeproliferator-activated receptor γ (PPARγ)andCCAAT/enhancer-bindingprotein β (CEBPβ).507 These findingsshedlightontheintricatesignaling networksbetweenadipocytes,stromalvascularcells,anddistant organsinhealthandmetabolicdiseases.

Developmentaldisorders

Indevelopmentalstudies,differentdevelopmentaltimepoints orsubpopulationscanbechosentostudythedynamicinterplay amongdiversecellularvarieties.Forexample,thereceptorligandinteractionsanalysisusedto ﬁ ne-mapmousehairfollicle developmentindicatedstronginteractionsbetweendifferent subgroupsatdifferentdevelopmentaltimepoints. 508 The enrichedreceptor-ligandpai rsofthesamecellsubgroups demonstratedstrongautocrinesignals,suggestingtheinvolvementofrobustintercellularcommu nicationinearlyhairfollicle development. 508 Inaddition,intercellularsignalingacross variouscelltypesisessentialfornervoussystemdevelopment, andligandengagementplaysapivotalroleinthesedevelopmentaldynamics.Dysfunctionofprefrontalcortexattributesto cognitivede ﬁ citsandmostneurodevelopmentaldisorders,so theintrinsicdevelopment-de pendentsignalsthatregulate neurongenerationandcircuitformationwasunveiled,which givesablueprintforcomprehendingthedevelopmentofthe humanprefrontalcortexduringtheearlyandmid-gestational periods. 509

Arecentstudyrevealedthat fivespecificligands(TGFβ2, NLGN1,TSLP,DKK1,andBMP4)havesynergisticcontributionson theprogressionofastrocytesinbothhumancerebralorganoids andprimaryfetaltissues.510 Moreover,thesynergisticimpactof theseligandspredominantlytargetsthemTORC1signalingpathway,leadingtothetranscriptomicsandmorphologicalcharacteristicsofastrocytedevelopment.Furthermore,reciprocalsignaling interactionsbetweenfetalgermcells(FGCs)andtheirgonadal nichecellswereobserved,showingthatthecellproliferationof FGCswaspromotedthroughBMPsignalingpathway.Then,BMP signalingpathwayregulatestheWntsignalingpathwayby coordinatingthechromatinaccessibilityofitsligandgenes, providingacomprehensiveroadmapforgermcelldevelopment invivophysiologically.511,512 Inaddition,theWnt/β-catenin pathwayplayscriticalrolesinembryonicdevelopmentandadult tissuehomeostasis.513 Furthermore,BMPsignalingpathwayplays aroleindevelopmentalstagedependenceandcell-type specificityinmalegermcells.514 Ontheotherhand,analysesof PI3KsignalingpathwayunmaskedtheoccurrenceofspecificCCCs duringthekidneydevelopmentofthehumanfetus,especiallythe interactionsbetweenreciprocalmesenchymeandepithelium

cells,whichmayhelptheappropriatecollectingductepithelial cellmorphogenesis.515

Otherapplications

Biologicaltransportsystem.Thebiologicaltransportsystemplays avitalroleinlivingorganismsandenablestheeffective transmissionandtransportationofnutrients,gases,andinformation,therebymaintainingthenormaloperationoflife.Molecular transportationincellsismainlyfacilitatedbyproteinchannelsand transporters.Regardingproteinchannels,theirselectivityand effectsonmoleculescanbefurtherunderstoodthroughthestudy ofthestructureandactivityofproteins.Asonekindofchannel proteinsthatcantransportglucoseandothermonosaccharides, SLC2A4 hasbeenfoundtobesigniﬁcantlydownregulatedinmost cancersanditshighmRNAexpressionissigniﬁcantlyrelatedto improvedprognosesinpatientswithbreastcancer.516

Fortransporters,themainpurposeofmostresearchistostudy theirselectivityandactivity.Forinstance, Glut1,theglucose transporterfoundinthecaterpillarinNamibianCanyon,hasbeen reportedtoincreaseduetogeneexpressionorproteinstabilization.517,518 Becausetheycanmaintaintheenergyneedsofvarious biochemicalproceduresintumorcells,itisimportantforusto understandhowglucoseintakeofspecificcelltypesaffectsthe behaviorofneighboringcellsinthesamemicroenvironment.519 In additiontointracellulartransportation,extracellulartransportation alsoplaysavitalrole.Extracellulartransportationincludesthe exchangeofsubstancesbetweenthematrixandextracellular fluids,theformationofextracellularmatricessuchascollagen fibers,andtheirroleintissuestructureandcelllifeactivities.The ECMiscomposedofaproteincalledcollagen,whichcansupport andprotectcells,andpromoteinteractionbetweencells.520 Atthe sametime,theECMcanalsoregulatephysiologicalprocessessuch assignaltransmissionandthereleaseofmediatedfactorsin cellularactivities.

Biologicaltransportsystemtechnologyleveragescellsignaling moleculestoregulatethetransportandreleasedrugsorother activesubstances.Thisapproachcanenhancetheeffectiveness andbioavailabilityofdrugs,offeringsigniﬁcantpotentialin diseasetreatment.Forinstance,cellsignalingmoleculescanbe harnessedtoinducecancercelldeath.Inaddition,ROS,produced duringoxidativemetabolism,serveascellsignalingmoleculesand areimplicatedinnumeroushumanpathologies.521 Theymaintain biologicalsystemhomeostasisthroughredoxreactionsanddrive cellularregulatorypathwaysthroughsubsequentsignaling.522 But accumulationofexcessiveROSpromotescellproliferationby triggeringthepathologicalalterationofnormalsignalingprocesses,leadingtothemalignanttransformationfromnormalcells. However,over-activatedROSlevelscouldinducecelldeathby inﬂictingdamageoncellularstructures.522 Consequently,therapeuticapproachesaimedatreducingexcessiveROStoavert earlytumorigenesisorenhancingROStoselectivelykillcancer cellsshowpotentialincancertreatment.

Predictionofdrugsideeffects.ThestudyofCCCnetworkscan assistinpredictingthesideeffectsofdrugs.Forinstance,certain drugsworkbyactivatingorinhibitingspeciﬁcsignalingpathways, whichmaybeinvolvedinvariousphysiologicalprocessesandresult inadversedrugreactions.Varioushormones,growthfactors,and cytokinesregulatecellproliferationanddifferentiation.These moleculesengagewithcellularreceptorsandinterfacewiththe cellnucleusthroughaseriesofintracellularsignaltransduction pathways.Sowhenkeycomponentsofthesepathwaysundergo alterationsduetooncogenemutationorover-expression,cancer cellsemergewithdisruptedcellsignalinganduncontrolledcellular growth.Thesekeycomponentsmutatedincancercellspresent viableselectivetargetsforinnovativeanticancertreatments, characterizedbytheirtumorspeciﬁcityandtolerabletoxicity.523

EXPERIMENTALMETHODSFORSTUDYINGCCC

VisualizingCCC

Imagingmethods

Electronmicroscopy:Inthe1930s,theGermanscientistErnst Ruskadiscoveredthatelectronscouldbefocusedundera magnetic field,leadingtotheinventionoftheworld’s first transmissionelectronmicroscope(EM).Currently,scientistshave inventedcryo-electronmicroscopy(cryo-EM)ontopoftransmissionEM,achieving “near-atomicresolution” ofbiologicalmolecules, finallyallowinghumanitytoglimpsehowbiological moleculesexecutetheirfunctions.Thebasicprincipleofcryo-EM technologyinvolvesplacingasolutionofbiologicalmacromoleculesonanEMgridtoformaverythinlayerofwater film,whichis thenrapidlyfrozentoliquidnitrogentemperatureusingcryotechniques.Thefreezingspeedissofastthatthewater filmdoes notformcrystalsbutinsteadformsavitreousicelayer.Biological macromoleculesareimmobilizedwithinthisthinlayerofice. Observingsuchfrozensamplesatlowtemperaturesundera transmissionelectronmicroscopeallowsustoobtainthestructure ofbiologicalmacromolecules.

Thenicotinicacetylcholinereceptor(nAChR)isatransmembraneproteinthatfacilitatesswiftcellularcommunicationunder theinfluenceofacetylcholine,anendogenousneurotransmitter. Asatypicaltransmembranemacromolecule,ithasextensive interactionswiththesurroundinglipidmicroenvironment.Recent cryo-EMstudieshaveunveiledthepresenceofphospholipidand cholesterolsiteswithinthelipid-exposedregionsofneuronaland electricorgannAChRs.524 These findingsareconsistentwith previousspectroscopyandaffinitylabelingstudies,which suggestedthatlipidmoleculescloselyinteractwiththetransmembranesegmentsofthereceptor.Forexample,electronspin resonance(ESR)studiesprovidedpreliminaryevidenceofmotionrestrictedlipidsincontactwithnAChRsinnativetorpedo membranes.525–527 InsubsequentESRexperimentsusingrecombinantnAChRs,directcontactbetweenthereceptorandadjacent orboundarylipidswasconfirmed.528 Theemergingdataoffers structuralevidencesupportingtheproposed “lipidsensor” functionoftheouterloopoftheM4transmembranedomain anditsregulatoryimpactonnAChRfunctionality.

Foranotherexample,thetransientreceptorpotentialvanilloid1 (TRPV1)channelisamultimodalreceptorthatcanrespondto variousstimuli,suchasheat,capsaicin,andprotons,makingita crucialpainsensorandaneffectivetargetforanesthetic drugs.529 530 Withtheemploymentofcryo-EM,thechannel structureofthemembraneproteinTRPV1wasresolvedatnearatomicresolution,alongwithitsstructureincomplexwith capsaicin.531 Asanextracellularchemicalsignal,capsaicinbinds tothemembraneproteinTRPV1locatedatthenerveendingson thetongue,openingachannelonthemembraneproteinthat allowsionsto ﬂowfromoutsidethecellmembranetotheinside. Thisionmovement,albeitminor,generatesacurrentthatis ultimatelytransmittedtoourbrainthroughnerve ﬁbers,enabling ustoexperiencethesensationofspiciness.

CCCplaysapivotalroleinthestructuringandfunctionalityof cellularnetworksandmulticellularsystems.532 Thisintricate networkiscoordinatedbysignalsemanatingfromthemicroenvironment,suchasparacrineorautocrineactionsofsoluble factorsorstimulimediatedbysubstratesfromtheECM.533,534 In addition,directCCChappensthroughstructuressuchasgap junctionsandtunnelingnanotubes(TNTs),535–537 whicharethin bridgesformedbythecytoskeletalactin filaments,capableof transferringcytoplasmandorganellesbetweenconnectedcells.538 Scanningelectronmicroscopy(SEM)isacrucialtoolfordirectly observingcell-to-cellTNTs.539 540 Todate,asignificantnumberof TNTshavebeenobservedinsinglecellsorthree-dimensional tumorcellaggregates.537,541,542 Mesenchymalstemcellshave beenextensivelystudiedduetotheiraccessibility,multipotency, andpotentialforanti-inflammatoryandpro-angiogeniceffects.

SEMwas ﬁrstemployedtoinvestigatealargepopulationofMSC’s spheroidsandrevealedthepresenceofTNTswithinhomotypic three-dimensionalclustersformedbyhumanMSCs.These observationswerefacilitatedthroughdirectvisualizationusing SEMandlaserscanningconfocalmicroscopy.543

Opticalmicroscopy:EMimagingrequires fixedcellsandspecial treatmentssuchasdehydrationandembedding,makingit unsuitableforlive-cellimaging,andwhethertheimagesobtained cantrulyreflectthestructuralinformationinlivingcellsisalso uncertain.Opticalmicroscopes,alongwithvarious fluorescence microscopyimagingtechniquesdevelopedlaterincombination with fluorescentlabeling,areoneoftheindispensablemeansfor studyinglifesciencesandbiomedicalissuesatthecellularlevel.In wide-fieldepifluorescencemicroscopy,spatialresolutioniseasily distortedduetodefocusblur,especiallywhen fluorescent moleculesaredistributedinthreedimensionsandformdensely packedstructures,asistypicalwithbiologicalsamples.Confocal microscopyusesapinholetocompletelyeliminateout-of-focus blur,achievingopticalsectioning.544 Two-photonmicroscopy utilizesthetwo-photonabsorptionprocesstoexcite fluorescent molecules,where fluorescenceoccursonlyatthefocalpointof theobjective,thusprovidinganopticalsectioningeffect.545 However,thespatialresolutionofsuchmicroscopytechniquesis stilllimitedbydiffraction.Therefore,advancementsinthelife sciencesurgentlycallforinnovationsthatcanunveilnanoscale moleculardynamicsandstructuralintricacieswithinlivingcells, surpassingthediffractionlimittoenhancetheresolutionofoptical microscopes.

Asthe firstfar-fieldmicroscopyimagingtechniquetobreakthe opticaldiffractionlimit,stimulatedemissiondepletion(STED) microscopyachievesathree-dimensionalresolutionof30–50nm throughtheuseofnonlineareffects.546–548 Duetoitshigh temporalresolutionandthree-dimensionaltomographiccapability,itrepresentsanimportantdirectioninthedevelopmentof opticalsuper-resolutiontechniques.Pellettetal. firstachieved live-celldual-colorSTEDimagingandusedimprovedSNAPfand CLIPflabelingtechniquestolabelEGFandEGFRforobserving theirinteractions.549 Furthermore,theuseofSTED-FCS(fluorescencecorrelationspectroscopy)combinedtechniquestostudy theinteractionsbetweenmembraneproteinsorlipidmolecules hasalsobecomeahottopicinmodernbiologicalresearch.550,551 Opticalactivationtechniques,includingstochasticoptical reconstructionmicroscopy(STORM),havesignificantlyimproved thespatialandtemporalresolutionavailableforexaminingthe physicalinteractionsbetweencells.24 STORMselectivelyactivates multiplephoto-switchable fluorescentgroupstodeterminethe lateralpositionofeach fluorescentsource,enablingthereconstructionofindividualimageswithnanoscaleresolution.552 The three-dimensionalextensionofSTORM,knownas3D-STORM, integratesenhancedaxialresolution,offeringavaluabletoolfor probingtheorganizationofproteinsatthecell–cellinterface withindensetissuesorenvironmentscharacterizedbynumerous uniformcellinteractions.553 Inbraintissue,thistechnique facilitatesthedetailedobservationoftheorganizationof scaffoldingproteinsandneurotransmitterreceptorswithin synapses.554

STEDandSTORMimagingtechniquesachievehighresolution; however,adrawbackistherequirementforintenseexcitation lightforillumination.Inaddition,the ﬂuorescentgroupsinthe specimenarequicklybleached,andthegeneratedfreeradicals havethepotentialtocausedamagetothespecimen.Therefore, theseimagingmodesaremoresuitablefor ﬁxedspecimensrather thanforobservingandstudyinglivebiologicalsamples.Consequently,anothermodeofachievingsuper-resolutionimaging throughalteringilluminationhasemerged.

Structuredilluminationmicroscopy(SIM)appliespatterned illumination ﬁeldsinsteadofconventionalwide-ﬁeldillumination,

improvingthespatialresolutionofopticalmicroscopyand providingbeneﬁtsforobservinglivecells.42 Inopticalmicroscopy, theobjectivelenshasalimitedabilitytocollecthigh-frequency informationfromthesample,resultinginthelossofsuchdetails duringimaging.SIMtechnologyaddressesthislimitationbyusing Moiréfringestotransferthesehigh-frequencydetails,which exceedthelens’scollectioncapacity,tothelow-frequencyrange. Thisenablesthemicroscopetocaptureinformationthatwas previouslyunattainable.Byapplyingspeciﬁcimagealgorithmsto processthiscombinedlow-frequencyandhigh-frequencydata, SIMproducessuper-resolutionimagesthatareapproximately twiceasdetailedasthoseobtainedthroughtraditionaloptical microscopy.42,43 Becauseofitsquickimagingspeed,minimal phototoxicity,andbroaddyecompatibility,SIMishighlyappropriateforprolongedmonitoringofdynamiceventsinlivingcells.

Fluorescenceresonanceenergytransfer.Fluorescenceresonance energytransfer(FRET)isthemechanismusedtodetect interactionsbetweentwobiomolecules,allowingfortheinference oftheirspatialproximity.Thisprocessinvolvesthetransferof energyfromanexciteddonor fluorophore(D)toacompatible acceptor(A)proteinor fluorophorethroughanon-radiative means.Thedonorabsorbsenergyatshorterwavelengths,while theacceptorabsorbsenergyatlongerwavelengths.555,556 This processonlyoccurswhenthetwomoleculesareinveryclose proximity,adistancethatisassociatedwiththeformationof complexesandconformationalchangesinvolvingmostbiomoleculesortheirconstituentdomains.557 Whenthedistanceisless than1nm,thedonorandacceptorcollide,andwhenthedistance isgreaterthan10nm,thedonoremitsphotons.Therefore,FRET onlyoccursinthenear field,withinarangeof1–10nm.558,559 Whentwocloselypositionedmoleculesare fluorescentsubstances,theobservableeffectsofFRETwillmanifestinthespectral propertiesofthese fluorescentdyes,includingalterationsin fluorescenceintensity, fluorescencelifetime,quantumefficiency, andanisotropy.560,561

FRETisexceptionallywell-suitedformeasuringawiderangeof dynamicmolecularevents,includingtheconformationalalterationsofmacromolecules,bothcisandtransbindingand/or assemblyofmacromolecules,aswellasthemodulationof physiologicaleventsacrossbothinvitroandinvivosettings.556 Traditionalopticalmicroscopesareconstrainedbylateraldiffractiontoaspatialresolutionof~250nm,ascalethatexceedsthe averagesizeofproteinmoleculesbyseveralordersofmagnitude withinarangeofafewnanometers.562 Thismakesitdifficultto predictwhethertwomoleculesareinteractinginanimage obtainedbytraditionalmicroscopy.Incontrast,utilizingFRET increasestheaccuracyofmolecularcolocalizationwithinthe diffractionlimit.Inprinciple,anyinstrumentcapableofrecording fluorescenceemissioncanbeusedtomeasureFRET,giventhe presenceofappropriate fluorophoresalongwithcorresponding filtersanddetectors.Therefore,earlyFRETexperimentswere primarilyconductedusing fluorescencespectroscopy, 563–565 whichgraduallyevolvedto flowcytometry564 566 567 andvarious microscopes,564,568–571 andlaterdevelopedintolaserscanning cytometry.572–574 FRETallowsresearcherstodirectlyobserve interactionsbetweenspecificproteinswithinlivingcells,whichis crucialforunderstandingintracellularsignalingnetworks.FRETis usedtostudysingle-moleculeinteractions,575 withinlivingcells,576 andevenacrossentiretissues.577

FRETprobeshavebeendevelopedtoprobevariousprocesses incellularsignaltransduction.578,579 Thesepowerfulapproaches allowforinvivoimagingacrosssystemsrangingfromCaenorhabditiseleganstotransgenicmousemodelsexpressingFRET probes.580–582 ImagingmethodsformeasuringFRETencompass epiﬂuorescenceandconfocalmicroscopyonexvivotissues,skin samples,orisolatedvesselsand/ortissuespecimens,extendingto multiphotonimagingwithinintacttissues.581–584 Inaddition,

lifetimeFRETmeasurementswereachievedbasedonmultiphoton imagingand ﬂuorescencethroughacranialwindowinmouse modelsofthenervoussystem.585 ThepotentialofutilizingFRETbasedprobesincombinationwithinvitrocellcultures,exvivo tissuepreparations,andinvivomodelsystemsforinvestigating cellularsignalingsystemsishighlycompelling.

Cellsurfacedetectionmethods.Insupportedplanarlipidbilayers (SLBs), fluorescentlylabeledproteinsareincorporatedintothe lipidbilayertofacilitateimagingofproteinmovementand organization,therebyenablingtrackingthroughouttheentireCCC process.586 SLBshaveevolvedintoaplatformforstudying molecularpatterns.587–589 UsingSLBtechniques,itispossibleto measurethetwo-dimensionalaffinityandkineticratesofcontact areas,therebyprovidingaquantitativebasisforunderstanding theinteractionswithincontactzones.590 Besides,GRASPisa proteincomplementationstrategythatfusestwononfluorescent fragmentsofGFPtointeractingpartnersonopposingcellsto detectCCCs.Whencellsareinclosecontactwitheachother,the splitproteinfragmentsassociateandreassembleintoGFP.45 GRASPhasbeenappliedtostudybothpre-andpostsynaptic interactions,enablingtheanalysisofconnectivityandthe distributionofinhibitoryandexcitatorysynapsesinmouse hippocampalneurons.591 Thisapproachhasbeenexpandedto includeothersplit fluorescentproteinfragments,suchasYFP (yellow)andCFP(cyan),allowingforsimultaneousimagingof multiplesynapticinteractionfactors.46

Anotherreportedstrategyformonitoringinteractingcellular partnersinvolvesachemo-geneticsystemthatutilizes fluorogenactivatingprotein(FAP)incombinationwithadyeactivatedby proximalanchoring(DAPA),whichiscomposedofmalachite greenandchloroalkane.592 FAPsserveasafusionproteintoolthat actsasa fluorescentmarkerbybindingtononfluorescentdyes knownas fluorogens.593 Malachitegreenisdisplayedoncells expressingHaloTagthroughattachmentwithchloroalkane,and whenitcomesintocontactwithadjacentcellsexpressingFAP,the contactbetweencellsisreportedthroughenhanced fluorescence. When fluorogensbindtoFAP,this fluorescentreadoutcantarget differentsubcellularlocations.594 andbeexpressedinvarious modelspecies.595–598 Furthermore,enzyme-basedamplification methodshavealsocontributedtoenhancingthevisualizationof CCCs.599,600

ChemicallytaggingCCCs

Contact-dependenttagging.Contact-dependentlabelingtechniquesnecessitatethephysicalinteractionbetweenanenzyme presentedonthesurfaceofonecellandareceptorsubstrateona neighboringcelltofacilitatecell-to-cellproximitylabeling.24 The labelingimmunepartnershipsbysortaggingintercellularcontacts (LIPSTIC)utilizesamodiﬁedsortaseenzymederivedfrom Staphylococcusaureus(SrtA)thatcanbefusedtoacellsurface ligand.Thisenzymetransfersabiotinylatedsubstratetoa pentaglycinereceptorpeptidepresentonamatchingreceptor ofadjacentcells,enablingtheidentiﬁcationofreceptor-ligand interactionswithinlivinganimalcells.LIPSTIChassuccessfully facilitatedthedirectbiotinylationofvariousligand–receptorpairs (LRPs)byexploitingphysicalinteractions.601 SoLIPSTICallowsfor thedirectmeasurementofdynamicCCCsbothinvitroandinvivo. ThroughtheapplicationofLIPSTIC,ithasbeenshownthatthe interactionsbetweendendriticcellsandCD4+ TcellsduringT-cell priminginvivothroughtwodistinctphases:aninitial,cognate stagemarkedbyCD40–CD40LinteractionsuniquetoTcellsand antigen-presentingdendriticcells,andasubsequent,non-cognate stagewheretheseinteractionsnolongerrequireprioractivation oftheT-cellreceptor. 601

Anothertechniquecalledenzyme-mediatedcellularproximity labeling(EXCELL),whichisbasedonasimilarsortaseenzyme mechanism,utilizesanenhancedformoftheStaphylococcus

aureustranspeptidasesortaseAenzyme(mgSrtA).602 Thisvariant hastheabilitytocovalentlytagarangeofcellsurfaceproteins thatcontainasingleglycineresidueattheirterminus.This techniqueenableshigh-resolutionimagingofCCCs,allowingfor in-depthexaminationofthemolecularcompositionandstructure atthecontactsites.Italsominimizesperturbationtothenatural stateofcells,ensuringthattheobservedinteractionsclosely resemblephysiologicalconditions.Byavoidingtheneedforpreinstallationofoligoglycine,EXCELLholdsthepotentialtodetect novelcellinteractions.602 Inparticular,ithasbeenusedtomonitor CCCsinlivingmicesincethesmallpentapeptide “LPETG” canbe easilyconjugatedwithothermolecules.601 Inshort,EXCELLcould becomeapowerfultoolfordetectinganddiscoveringCCCsin morecomplexinvivoenvironments.

Thenecessityofgeneticallyincorporatinglabelingenzymes mightposeasignificantobstacletothewidespreadapplicationof thesemethodologiesinthecomprehensivestudyofCCCs. Interaction-dependentfucosylation(FucoID)circumventsthis challengebyautonomouslyanchoringthelabelingenzyme Helicobacterpylori α1,3-fucosyltransferaseontothecellsurface.603 Theefficacyofimmunotherapiesaimedatbolsteringendogenous T-cellimmunityhingesupontheTcells’ capacitytoidentify tumor-specificantigens(TSAs).604 Inthequesttoexpedite advancementsincancerimmunotherapy,thedevelopmentofa computation-freemethodologythatenablestheswiftidentificationofTSA-reactiveTcells,andisstraightforwardtoimplement,is highlydesirable.FucoIDemergesasapivotalinnovation,capable ofidentifyingendogenoustumorantigen-specificTcellsthrough interaction-dependentfucosylationwithoutpriorknowledgeof TSAidentity.Employingthisapproachfacilitatestheisolationof TSA-reactiveCD4+,CD8+ Tcells,andTSA-suppressiveCD4+ Tcells withintumors.603 Thistechniqueexhibitswide-rangingutility acrossmultiplemousetumormodelscharacterizedbyobservable T-cellinfiltration,underscoringitssignificanttestingprospectsin clinicalscenarios.

Contact-independenttagging.Non-contacttechniques,incontrasttocontact-dependentlabelingmethods,generatehighly reactivelabelscapableofdiffusingbeyondthecatalyst’s immediatevicinity.Proximitylabelingprovidesamethodto capturetheimmediatebiochemicalenvironmentofproteins insitu,therebypreservingkeyspatialandtemporalcontexts.605 Whenintegratedwithmassspectrometry(MS)-basedproteomics, theseapproachesenabletheelucidationoftheproteomic landscapeofspatiallyrestrictedcell–cellinterfaces.Thisintegrationprovidescriticalinsightsintothemannerinwhichthe structuralorganizationofproteinsaffectsthefunctionalconsequencesofCCCs.24

APEXhasbeenusedtocapturetheentireorganellarproteome withhightemporalresolutionandhasbecomeanimportanttool forproximitylabeling.InthepresenceofH2O2,APEXconverts biotin-phenol(BP)labelsintoshort-lived(t1/2 ≈ 100 μs)reactive phenoxyradicals,markingneighboringproteinsontyrosineand otherelectron-richaminoacidsidechainsinthemitochondrial matrix.606 Forexample,combiningproximitylabelingwith quantitativeproteomicscancapturethelocationandtimingof GPCRfunctioninlivingcells.606

Thewell-knownproximitylabelingmethodBioIDutilizesaBirA ligasemutant(BirA*)tobiotinylateproximalproteins.607 Inthe presenceofATP,BirA*catalyzestheconversionofbiotininto activebiotin-AMP,whichthenreactswithnearbynucleophilic lysinesidechains.TheBioIDmethodisprimarilyusedtoidentify intracellularbindingpartners,605 includingthecytoplasmicregion ofcadherins.608–610 andotheradhesionproteins.611 BioIDfusedto theextracellulardomainofN-cadherin(Ncad)hasalsoidentiﬁed proteinssecretedbyratneurons.612 Bycombiningproximity labelingwithsingle-moleculebindinganalysis,previouslyundiscloseddirectconnectionshavebeenunveiledbetweenthe

extracellulardomainsofnumeroustransmembraneproteinsand E-cadherin(anessentialcell–celladhesionprotein).613 Asthe labelingefﬁciencywithBirA*wasfoundtobeslow(taking 18–24h),amorerapidsystemcalledTurboIDwascreated,capable ofcompletinglabelinginamere10min.614 TheTurboID techniquehasbeenemployedtoidentifyproteinsatepithelial celljunctionsbyfusingtheenzymewiththeextracellulardomain ofE-cadherin.613

Mechanicalforceanalysis

Althoughtheshapeoforganismsisencodedintheirgenomes, theinformationcodedbyDNAisnotenoughtoruletheultimate architectureoftissuesandorgans,norcanthecellexpression profilestellushowcomplexfunctionsareachieved.The developmentaltrajectoriesculminatinginthedefinitivemorphologyofvertebratesinvolvecontinuousfeedbackbetweendynamic mechanicalforcesalongwithcellgrowthandmovement. Mechanicalforcesareruledbycellsandintegratedintotissues throughmechanotransductionprocessesthataffectcellshape, proliferation,migration,andprogrammedcelldeath,collectively sculptingthe finalformoforganism.615 Thecoreofthese processesisprimarilythemyosinmotors,andthequasi-stable stateofcelltensionismaintainedbythesemyosincontraction forcemechanosensors,allowingcellstodefinetheshapeand tensionoforgans.615 Theinitialdiscoverythatcancercellscan growinsoftagarinananchorage-independentmanner,616,617 whilemostnoncancercellscannot,sparkedinterestintheroleof mechanotransductionatthecellularlevel.Redbloodcells exposedtoanionicandcationicdrugsundergodifferentchanges inintracellularandextracellularsurfacemembranetension, resultinginmodificationstocellmorphology.618 Thisobservation impliesthepresenceofacellularmechanismcapableofdetecting changesinmembranetension,whichwassubsequentlydemonstratedtobecrucialforcellspreadingandmigration.619

Cellmigrationplaysacrucialroleinmanyphysiologicaland pathologicalprocessessuchasmorphogenesis,620 woundhealing,621 andtumormetastasis.622 Inturn,migrationinvolvesa coordinatedseriesofevents,includingtheprotrusionof pseudopodia,formationofnewadhesions,developmentof tractionforces,andreleaseofoldadhesions.623 Toachieve appropriatephysiologicaloutcomes,cellmovementmustmaintainacertaindirectionandspeedinresponsetoenvironmental stimuli.Tractionforcemicroscopy(TFM)isatechniqueusedto measuretheforcesexertedbycellsontheirsubstrate.624–626 Itis basedontheprinciplethatcellsgeneratetractionforcesontheir attachedsubstrateduringmigration,extension,orcontraction. Onecaninferthemagnitudeanddirectionoftheforcesexerted bythecellsbyobservingtheminutedeformationsonthecellattachedsubstrate.However,TFMislimitedbypoorresolution, typicallyconfinedtodetectingforcesonamicrometerscale.627,628 Therefore,astrategyhasbeenproposedtoenhancetheoutputof TFMbyincreasingtheachievableheaddensityandtheaccuracy ofheadtracking.629–631 Thisinvolvescombininganalgorithmfor fluctuation-basedsuper-resolution(FBSR)imagingwithsoftwareenabledsuper-resolutionmicroscopy.Throughtheanalysisof fluorescencegroupintensity fluctuations,thisapproachallowsfor theresolutionofdenselypackedbeadsandsignificantlyimproves thetractionforceoutput.632

Thearrangementofintermolecularforcesinspacedictatesthe interplayamongmacromolecules,withbothlong-rangeand short-rangeinteractionsplayingpivotalrolesinthedynamic behaviorofbiologicalsystemsandtheirassemblies.633 Atomic forcemicroscopy(AFM)isahigh-resolutionscanningprobe microscopeusedtostudytheinteractionforcesbetweenobjects atthenanoscale.625,626,634,635 AFMenablesadirectmeasurement ofintercellularinteractionsbydelicatelycontactingtheprobewith thecellsurfaceandsubsequentlycapturingtheforcedisplacementcurveastheprobeinteractswiththecellsurface.

Themethodforexaminingthespatialdistributionofforceswithin avolumeusingAFMinvolvescollectingaseriesofforcecurveson thesurfaceandassemblingthemintoa “forcevolume” (FV).633 Radmacheretal.usedtheHertzmodeltoanalyzetheFVimages ofplateletsandconstructedthe ﬁrstcellmechanicalproperties mapbasedonAFM.636 Asimilarmethodologywasemployedto investigatethecontributionoftheactincytoskeletontothelocal mechanicalcharacteristicsofcardiomyocytes.637 andmacrophages.638 However,achievingatomicresolutionimaginghas longbeenchallengingwhennanoscalemanipulationunder differentenvironmentsbecomesroutine.Theinitialdeﬁciency wasduetothecontactwiththesampledullingtheatomictip, whichisessentialforsuccessfulatomicresolutionimaging.639

Withtheintroductionofnon-contactatomicforcemicroscopy (NC-AFM),thisissuewas finallyovercome.InNC-AFM,the cantileveroscillatesnearthesurfaceofthesamplewithout actually “touching” it,allowingthepreservationofthetip’satomic sharpnesswhilequantifyingthetip-sampledistanceusingthe changesinthecantilever’sresonancefrequencycausedby interactions.639 Thelatestprogressinhigh-speedatomicforce microscopy(HS-AFM)hasenabledtheexaminationofconformationaldynamicsinindividualunlabeledtransmembranechannels andtransporters.TheprogressinHS-AFMnowallowsnotonlyfor thedetectionoffasterdynamicsbutalsoprovidessub-molecular structuralinformationinrealspace,640–643 significantlyimproving temporalresolution.644 TheemergenceofHS-AFM,characterized byunprecedentedscanningrates,resultsfromablendofdiverse technologicaladvancements.Theseincludeenhancementsin cantileverbeams,samplestagescanners,cantileverbeam deflectiondetection,andfeedbacksystems.645 HS-AFMimaging hassucceededinapplyingtovariousbiologicalsystems,suchas molecularmotors,641 membrane-associatedproteins,646 macromolecularsystems,647 andprotein–DNAcomplexes.648

DOWNSTREAMANALYSISANDEXPERIMENTALVALIDATION

Researchonmoleculesinsignalingpathwaysthatplayakeyrole inregulatinggrowthanddevelopmenthasrevealedthatthe responseprocessisnottheresultofasinglepathway’sactionbut rathertheresultofcrosstalkbetweendifferentpathways.649 Accuratesignaltransductionrequirescrosstalkbetweenvarious pathways,furtherformingcomplexintracellularsignalingnetworks.Cellularsignaltransductionbeginsatthecellmembrane, propagatesthroughthecytoplasm,andultimatelyregulatesgene expressionpatternsdeepwithinthenucleus.Thisprocessis mediatedbyaseriesoftypicallyweakandtransient protein–proteininteractions,enablingcellstorapidlyadaptto changingenvironmentalconditions.650 Tofulﬁlltheircriticalroles incellularprocesses,theseproteinsinteractwitheachotherstably ortransiently,formingavastnetwork.651,652 Theapplicationof proteomicsandcellmanipulationtechniquesincellsignal transductionresearchprovidesimportantmeansforrevealing complexintracellularsignalingnetworksandidentifyingsignal moleculecomplexes.Theyalsoenableexplorationofthe molecularbasisofprotein–proteininteractions,discoveryofnew partnermolecules,andstudyofthecrosstalkbetweenknown pathwaysandthedynamicchangesincellsignaltransduction.

Co-immunoprecipitation

Co-immunoprecipitation(Co-IP)isoneofthestrongestmethods foridentifyingphysicalinteractionsbetweentwoormoreproteins invivo.653–655 Itisatechniquewhereantibodiesareusedto precipitateaspecificmolecule,andothermoleculesthat specificallybindtothatmoleculeareco-precipitatedalongwith it.Thistechniqueiscommonlyusedtoverifythespecificbinding betweenproteins.656 Co-IPisabletoidentifyproteininteractions involvedinthecellcommunicationprocess,includinginteractions betweenreceptorsandligands,signaltransductionmolecules,

andtheactivationofeffectorproteins.Theseinteractionsformthe basisofhowcellsrespondtoexternalsignalsandtriggerinternal responses.Proteininteractions,formingcomplexesofvarying sizes,exhibitspatiotemporaldependency.657 Theexecutionof speciﬁcproteinfunctionsstronglyreliesoncontactwiththe surfacesofneighboringproteins.Mostprocessesdemanddirect contactbetweenproteins,eitherinbinaryformoraspartoflarge complexesinvolvingmultipleproteins.658 Co-IPcanidentify proteincomplexesformedunderspeciﬁccellularstatesor conditions,therebyrevealingkeyparticipantsinthecellcommunicationprocess.Inmostcases,theseinvitrobindingassaysare combinedwithMS.ApreviousstudyutilizedtheCo-IP/MSmethod toidentifyBMPR-1Bprotein–proteininteractions(PPIs).Inaddition,thesignalpathwayofthetargetproteinwasanalyzed,and bioinformaticspredictionindicatedthatBMPR-1Binteractswith ovulation-promotingproteinsineweovaries.659 Asatransmembraneprotein,BMPR-1Bmediatessignaltransductionbetweenthe intracellularandextracellularcompartmentsbyparticipatingin vitalactivitiesandsubstanceexchange.660

Inaddition,Co-IPtechnologycanbeusedtoexploreunknown proteininteractions,therebydiscoveringnewsignalingmolecules andpathwaysinvolvedincellcommunication.ThroughCo-IPand GSTpull-downassays,Angiogenin(ANG)wasreportedforthe first timetointeractwithribonucleaseinhibitor(RI)bothendogenouslyandexogenously.661 UpregulatingANG,includingtheANG His37Alamutant,significantlydecreasedRIexpressionand activatedphosphorylationofkeydownstreamtargetmolecules ofthePI3K/AKT/mTORsignalingpathway.661–663 Thisdiscoveryled tothepromotionoftumorangiogenesis,tumorigenesis,and metastasisinvivo,highlightinganovelmechanismofANGin regulatingthePI3K/AKT/mTORsignalingpathwayviaRI.Therefore,PPIsplayacrucialroleinalmosteverycellularprocesssince theydictatethespecificityofsignaltransduction,controlthe strengthanddurationofsignals,andintegratevarioussignaling pathwaystoorchestrateintricatecellularresponses.664.Inturn, understandingPPIswillhelpelucidatethepathophysiologyand progressionofmanydiseases.665

Functionalexploitation

Althoughthepreviouslymentionedmethodsareadeptat identifyingproteinsincloseproximityandpotential ligand–receptorpairs,furtherapproachesarenecessarytoexplore andinterfereproteinfunctionsatthecell–cellinterface.Cell manipulationtechniquesthatinducetheloss,obtainment,or modiﬁcationofproteinfunctionalitiesofferadirectavenuefor investigatingligand–receptorinteractionsortheensuingsignaling pathways.Thisapproachhelpsingaininginsightsintothetypesof cellularinteractionsthattakeplace,theconsequencesofthese interactions,andhowtoleveragethembymanipulating transcriptionalprograms.24 Thesemethods,byalteringcell behavior,communicationmodes,orenvironmentalresponse capabilities,playacrucialroleinbothbasicresearchandclinical applications.

CRISPR-Cas9screeninghasbeenemployedtocomprehendthe functionalrolesofproteinsimplicatedintheevasion,recognition, andclearanceofcancercellsduringtheadaptiveimmune system.24 AlterationsinsomaticgenescanmodifythesusceptibilityofcancercellstoT-cell-basedimmunotherapy.Toidentify proteinsintumorcellsthatregulateand/oraresensitivetoT-cell effectorfunctions,adual-cell-typeCRISPR(2CT-CRISPR)screening methodwasdevisedforconductingloss-of-functionanalyses.666 Interferon-gamma(IFN-γ)drivenphosphorylationof JAK1stimulatestheJAK-STATsignalingcascadetoenhance antigenprocessingandpresentationintumors,therebyenhancingT-cellrecognitionandcytolysis.667 Utilizingaco-culture systemofIFN-γ signaling-deﬁcienttumorcellsandTcells,CRISPRCas9screeningidentiﬁedseveralgeneswithintheTNFsignaling pathwayascriticaltorenderingtumorcellsvulnerabletoT-cell-

mediatederadication,therebyunveilingpotentialtargetsfor alternativeimmunetherapeuticpathways.668

Activationofdownstreambiologicalprocessesmediatedbythe cellsurfacecanalsobeachievedthroughtheengineering expressionofreceptorsand/orligandsonthecellsurface. SyntheticNotch(synNotch)receptorsprovideextraordinary flexibilityinengineeredcells,allowingforthecustomizationof sensing/responsebehaviorsbasedonuser-specifiedextracellular signals.669–671 SynNotchreceptorsareengineeredtoincorporate thecoreregulatorydomainsoftheNotchcell–cellsignaling receptor,butwithsyntheticextracellularrecognitiondomains (suchassingle-chainantibodies)andsyntheticintracellular transcriptionaldomains.669,672 ThisNotchintracellulardomain actsasatranscriptionalregulator,operationalonlyafteritsrelease fromthemembraneandenableittoenterthecellnucleusto activategenespivotalforcell–cellsignalingduringdevelopmental processes.673 ThesesyntheticNotchreceptorsareversatile, functioningacrossvariouscelltypes,includingimmunecells andneurons.ThedeploymentofmultiplesyntheticNotch pathwaysisallowedwithinthesamecellandusedtodesign complexcombinatorialsensingcircuits.The flexibilityofsynthetic Notchreceptorsinengineeringnewcellbehaviorsmakesthema powerfultoolforconstructingtherapeuticcells,drivingthe formationofcomplexmulticellularpatterns,orregulatingor reportingcellbehaviorincomplexinvivoenvironments.669 NaturalT-cellresponseprogramslackcertaindesirablecharacteristics.674 Togiveanexample,evenwhenredirectedtoidentify tumors,Tcellshavelimitedabilitytoovercometheimmunosuppressivemicroenvironmentoftumors. 675 ButTcellsengineered withsyntheticNotchreceptorsexhibitrobustand finelytunable customizedfunctionalities.Inaddition,Tcellsequippedwith syntheticNotchcircuitscanpreciselyhomeinonsolidtumors, enablingthelocalizeddeliveryoftheirtailoredpotentpayloads withinthebody.670 (Fig. 4).

COMPUTATIONALMETHODSFORINFERRINGCCC

Usingsingle-cellomicsdata,variousbioinformaticsandcomputationalmethodshavebeendevelopedtodecipherbiological CCCs.676 (Table 2).Scientiﬁcresearchcommonlyadoptstwo principalapproaches:ligand–receptor(LR)signal-basedalgorithm andphysicallocation-orientedstrategy.7 Theavailabilityofsinglecelldata,particularlytranscriptomedata,hasledtothedevelopmentofplentyofcomputationaltoolsfordecipheringCCC(Fig. 5). Thesetoolsleveragediversemethodsforpredictingpotential intercellularcommunicationevents(CEs)basedonpriorknowledgeofCCCs.4 Variousmediatorsfacilitatethedevelopmentof toolsforCCCanalysis,includingCa2+ , 677 lipids,678 peptides,679 proteins,680 EVs,681 andelectricalsignals.Thesedevelopedtools forCCCanalysisutilizedifferentmedia,algorithms,anddatatypes toinferCCC,leadingtothediscoveryofdifferenttypesofCCCs basedondifferentprinciplesandresultinginvariousvisualizations (Fig. 6).

Single-celltranscriptome-basedtools

Single-celltranscriptometechnologyconductslarge-scaledetectionofgeneexpressioninasinglecellandaccuratelyrevealsthe activityoftranscriptionfactors(TFs)ofeachcell,whichprovides greatsupportforanin-depthunderstandingofcelldifferentiation, development,andmetabolism.Therapiddevelopmentofsinglecelltranscriptomicstechnology,suchasDrop-seq,inDrop,682,683 CITE-seq,684 10XGenomics,685 providingwithadeeperand comprehensiveunderstandinginmany ﬁeldsoflifesciences. Severalstrategieshavebeenemployedtoconstructcellular communicationnetworksbasedonLRIsusingsingle-celltranscriptomedata.7 Toolsanalyzingcellularcommunicationbasedon single-celltranscriptomedataareprimarilylimitedtointercellular communicationmediatedbyproteinligand–receptorcomplexes,

andtheiranalysisreliesongeneexpressionleveland ligand–receptordatabases.9 Thislimitationsigniﬁcantlyincreased false-positivepredictionsofCCC.686 Absenceofexpressioninany subunitimpedestheinferenceofinteractionbetweenligandand receptorandthesubsequentcommunication. 687 Socurrently developedcomputationalapproachescanbeclassiﬁedintofour types,dependentonthemathematicalframeworksforpinpointingLRIs,including(1)expressionpermutation-basedtools,(2) difference-assembly-basedtools,(3)network-basedtools,and(4) tensor-basedtools.5

Expressionpermutation-basedtools.Expressionpermutationbasedtoolsemployvariousmethodstocalculatecommunication scoresforeachLRPandevaluatethecomparativesigniﬁcancetoa nullmodelusingclusteringlabelarrangement,non-parametric testing,orempiricalmethods.ExamplesincludeCellPhoneDB,688 CellChat,686 ICELLNET,689 SingleCellSignalR,690 CellCall,691 and NATMI.692 Notably,CellPhoneDB,CellChat,andICELLNETconsider multisubunitcomplexesforligandsandreceptors.5

CellPhoneDBisattheforefrontofheteromermodeling, recognizingthatnumerousreceptorsandligandsoperate exclusivelyasheteromers.Itstandsoutforitscomprehensive cellularcommunicationligand–receptordatabase,whichincludes receptorsandligandsannotatedbypublicsourcesandspeciﬁc familiesofhand-selectedproteinsinvolvedincellcommunication. However,thedatabasedoesnotencompassallpotentialLRIsand neglectsothervitalsignalingcofactorswhichCellChatintegrates, suchassolubleagonists,antagonists,andbothstimulatoryand inhibitorymembrane-boundco-receptors.693

CellChatfacilitatestheanalysisofintercellularinteractionsand communicationnetworksbyprovidingcellinteractionnetwork diagramsandcommunicationpathwayanalysis.Ithasexpanded itscoveragetoinclude229signalingpathways,classiﬁedinto threecategories:contactbetweencells,receptorsintheECM,and signalingviasecretion,686 anotableexpansionfromtheapproximate900LRPsfeaturedinCellPhoneDB.CellPhoneDBinfersthe enrichedLRinterplaysamongthetwocellulargroupsrootedin pronouncedspeciﬁcity,whileCellChatemphasizesdifferential overexpressedligandsandreceptorstomeasuretheassociation betweenLRPsundertheprincipleofmassaction.

ICELLNETcalculatesanoverallCCCscorebysummingallLRI productionscoresacrosstwoclusters.Meanwhile,itdetermines interactionsthroughthemultiplicationofgeometricaverages fromexpressionsofbothligandsandreceptors.689 Notably,with theexceptioninasingularresearchwork,694 ICELLNETstandsas thesoledatabasethatclassiﬁespredictedinteractionsinto biologicalfamilies.689 DespiteICELLNEThavingfewerinteractions thanitscounterparts,itboastspreciseandintricatecytokine interactions,extendingtimelytoallchemokinesandcheckpoint interactions,therebyofferingdistinctresourcesforinvestigating intercellularcommunicationintheimmunesystem.Forexample, itcontains14cytokineinteractionsnotincludedinCellPhoneDB, forinstance,MIF/CXCR2andMIF/CXCR4.695

AnalogoustoCellPhoneDB,SingleCellSignalRintroducesa notionofinteractionscore.696 thatisdefinedasthefunctionof theaverageexpressionofligandsintypeAcellsandreceptorsin typeBcells.SingleCellSignalRreliesonanewcuratedLRdatabase andusesregularizedexpressionproductstodeducetheunderlyingLRIswithincellularnetworks.Althoughthefalse-positive resultsareabletobeavoidedthroughusingthepermutationtest thatutilizedinCellPhoneDB,highlyrepresentativecommunicationsinthedatasetmaynotbestatisticallysignificant.Tosolveit, adefinitivecut-offvalueforscoringscoreisofferedin SingleCellSignalR,capableofattainingasuitableerrordiscovery rategroundedinempiricalevidence.5

CellCallisatoolkitthatcandeducebothintercellularand intracellularcommunicationroutesbyamalgamatingcoupledLRIs andTFactivity.Distinguishedfromscoringmethodofcellular

Fig.4 RepresentativeexperimentalmethodsforstudyingCCC.Technologiestoexpandthemolecular-levelunderstandingofcell–cell interactionbiologyinclude a microscopyimaging, b chemicaltagging, c mechanoforce,andCo-IPanalysis,and d functionalexploitation

interactionsinSingleCellSignalRandCellPhoneDB,thealgorithm ofCellCallusestheexpressioninformationoftheRegBregulon whicharetargetgenesactivatedbytheco-expressedTF.696 Furthermore,CellCallutilizesanintegratedpathwayactivity analysistechniquetopinpointnotablyactivepathwaysin intercellulardialogamongdistinctcelltypes.However,CellCall focusesexclusivelyonthedownstreamgeneregulatorynetworks (GRNs)relatedtoLRIs.ItprimarilyfocusesonLRPscomprising protein-basedpartners,therebyneglectingnon-peptidicentities likelipids,smallmolecules,nucleicacidligands,andcarbohydrates.691 Inshort,CellCallandSingleCellSignalRcandetectalarge numberofcommunications,includingnonspeciﬁccommunications,butmaymisslow-intensitycommunications.690

NATMIcanbeusedtosummarizethecompletenetworkof communicationtodisplaythecommunicationintensityor specificitybetweeneachcelltypeandothercelltypeincomplex samples,soastoidentifyhighlycommunicativecellpairsor specificcommunities.692 ItusesconnectomeDB2020oruserdefinedLRPstoforecastandvisualizecellcommunicationnetwork betweencelltypesindatasets.

Theseexpressionpermutation-basedtools,asmentioned above,typicallysolvethelimitationthatmostCCCtoolsdonot considermultisubunitproteincomplexes.

Difference-assembly-basedtools.Severaldifference-assemblybasedtoolshavebeendeveloped,includingPyMINEr,697 iTALK,694 andCellTalker.698 PyMINErandiTALKaimedtoidentifythe differentialexpressiongenesbetweencellularclustersandused themascandidatesforthe finalLRpairinteractions.PyMINEr establishesgeneco-expressionnetworksascertainedthrough Spearmancorrelationandintegratesthemwithprotein–protein interactionnetworks.697 UnlikeSingleCellSignalR,whichdepends ongeneticsignaturesforconductingcell-typeidentification,690 PyMINEr’scharacteristicgenesignatureisnotprovided.Instead,its approachtodelineatingcelltypeshingesupontheenrichmentof subgroup-specificgenepathways.Actually,PyMINEridentifies alteredsignalingpathwaysbasedondifferentiallyexpressedpairs ofligandsandreceptors.ButiTALKcategorizesLRPsinto cytokines,growthfactors,immunecheckpoints,andothers, focusingsolelyoncommunicationbetweentumorcellsand normalcells.694 Incontrast,CellTalkeridentifiesunique

interactionsbetweenclustersbyusingdifferentiallyexpressed ligandsandreceptorswithinindividualclusters.698 Itassumes cellularcommunicationhingesontheuniformexpressionof ligandsandreceptorsamonginteractingcells.Inshort,CellTalker andiTALKemployslightlydifferentdownstreamanalysistechniquestoassemblethedeﬁnitiverosterofpivotalinteractingLRPs. However,thesemethodsmayoverlookcommoninteractions amongallgroupsthoughtheyexcelatidentifyingLRIswithinthe dataset’sbackground.

Network-basedanalysistools.Thenetworkapproachisutilizedby severaltools,leveraginggeneconnectivityproperties.Intercellular communicationencompassesintercellularsignaling,intracellular transmission,andsignalampliﬁcationthroughspeciﬁcsignaling pathways.Thesepathwaysoftenresultinactivitychangesof downstreamTFandGRNs.699,700 Variousapproacheshave consideredintracellularsignalingtotacklethesecomplexities, includingCCCExplorer,701 NicheNet,699 scMLnet,702 SoptSC,703 Scriabin,704 CytoTalk, 705 RNA-Magnet,706 andContactTracing. 707

CCCExplorerbuildsacomprehensivegraphdepictingvarious signalingpathwaysandcomputesastatisticakintoFisher’s methodbyemployingtheexpressionofligands,receptors,and downstreamTFstopinpointkeyinteractions.701 Itincorporates differentiallyexpressedgenesandPPInetworkstoanalyze downstreamtargetsandTFstodeterminesignaleventsofcell activationorinactivation.Becauseafunctionalunderstandingof CCCrequiresknowledgeabouttheeffectofligandonreceptor’s geneexpression,theexpressiondataofinteractingcellsisneeded toinfertheeffectofsender-cellligandsontheexpressionof receptorcell.

Toaddressthisproblem,acomputationalmethodcalled NicheNethasbeendevelopedbyintegratingdatafromvarious sources,includingligand–receptorrelationships,signalingpathways,andtranscriptionalregulatoryrelationships.Itcandirectly outputtheinter-relationshipsamongligands,receptors,and targetgenes.699 Sincethepriormodelofligand-targetregulation potentialmainlyreliesonpriornetworkinformationinsteadof expressionrelationshipsinspeciﬁccells,theconstructionof context-dependentmultilayer,intercellularandintracellularsignalingnetworksisneededtodeeplyunderstandCCCthrough single-cellgeneexpressionsfunctionally.702

Table2. ExistingbioinformatictoolsforinferringCCC

ExistingbioinformatictoolsforinferringCCC

IDToolFeatureAlgorithmLinkInputOutputVisualizationAvailableinURLRefs

https://github.com/Teichlab/ cellphonedb 688

Pythonand Web interface

Heatmap;Dot plots;Cluster combinations

L-RscRNA-seqUpregulatedand downregulated interactions;Listof moststatistically signi ﬁ cantL-Rr interactions

https://github.com/sqjin/ CellChat 686

RandWeb interface

AlluvialandCircos plots;Dotplots

L-RscRNA-seqLikelihoodofCCC betweenallclusters forallinteractions

R https://github.com/ soumelislab/ICELLNET 689

Barplots;Network visualization

L-RscRNA-seqIntergroup communication scores;matrixofCCC probabilities

https://github.com/SCA-IRCM 690

Circosplots,tables andgraph visualizationsof interactions betweenclusters

L-RscRNA-seqInteractionscoresfor eachLRIbetweenall clustersinthe dataset

R https://github.com/ ShellyCoder/cellcall 691

Circosplots; Sankeyplots; Bubbleplots; Ridgeplots,etc.

L-R-TFscRNA-seqIntracellularsignaling andathresholdfor intercellular communication scores

Python https://github.com/forrest- lab/NATMI/ 692

Heatmap; Network-graph; Circosplots

L-RscRNA-seqSummarizinghow strongly(or speci ﬁ cally)eachcell typeis communicatingto anothercelltype

https:// www.sciencescott.com/ pyminer 697

Pythonand standalone application

Network visualizationand Circosplots

ProteinscRNA-seqLikelihoodofCCCfor allinteractions;Listof gene-gene interactionnetworks foreachcellcluster

https://github.com/ Coolgenome/iTALK 694

Expression permutation

1CellPhoneDBAdatabaseofligands, receptors,andtheir interactions;Thesubunit architectureofligands andreceptors

Expression permutation

2CellChatIntercellularinteractions; Communicationnetworks; Cellinteractionnetwork diagramsand communicationpathway

Expression permutation

3ICELLNETSummingtheproductof allLRIscoresbetweentwo clusterstocomputean overallCCIscore

Expression permutation;A regularizedscoreto assessthecon ﬁ dence inpredicted ligand –receptor interactions

4SingleCellSignalRTheligand –receptor interactionsthatunderlie cellularnetworks;Anew curatedLRdatabaseanda novelregularizedscoreto performinferences

Expressionofligands/ receptorsand downstreamTF activities

5CellCallIdenti ﬁ ngthesigni ﬁ cantly activatedpathways involvedinintercellular crosstalkbetweencertain celltypes

Mean-expression weight;Speci ﬁ city weight;Cell- connectivity-summary- networkedgeweights

6NATMIInteractionsbetween clustersaremodeled, calculatedbytheproduct ofnormalizedligandand receptorexpressionsof thetwoclusters

Differentially expressedgenes

7PyMINErConstructinggeneco- expressionnetworks, whicharethenintegrated withprotein –protein interactionnetworks

CCInetworks; Circosplots;Box plots

L-RscRNA-seqUpregulatedand downregulated interactions;CCC probabilitiesformost signi ﬁ cantL-R interactions

Differentially expressedgenes

8iTALKTheexpressionof receptorsandligandsin eachcellsubpopulation; Onlyfocusonthe communicationbetween tumorcellsandnormal cells

Table2. continued ExistingbioinformatictoolsforinferringCCC

IDToolFeatureAlgorithmLinkInputOutputVisualizationAvailableinURLRefs

https://github.com/arc85/ celltalker

https://github.com/ methodistsmab/CCCExplorer 701

Standalone application

Circosplotsof differential interactions betweenclusters R

L-RscRNA-seqUpregulatedand downregulated interactionsbetween allclusters

Differentially expressedgenes

9CellTalkerDifferentiallyexpressed ligandsandreceptorsin eachclustertoidentify uniqueinteractions betweenclusters

https://github.com/saeyslab/ nichenetr 699

L-RscRNA-seqGraphvisualizations ofallinteractions Interactive directedgraphs

Priornetwork, statisticalinference (Fisher ’ sexacttest)and adirectedgraph

10CCCExplorerAgraphofallsignaling pathways;Usingligand, receptoranddownstream TFexpressiontoidentify signi ﬁ cantinteractions

703

Circosplotsof interactions betweencellsor clusters R

WeightingnetworkL-RscRNA-seqLigandinteraction scoresand expressingcelltypes forprovidedtarget pathway

11NicheNetDatabasesfromvarious sources,including ligand –receptor relationships,signaling pathwaysand transcriptionalregulatory relationships

R https://github.com/ SunXQlab/scMLnet

Networkdiagram; Violinplots; Heatmap

L-R-TFscRNA-seqTissue microenvironment- mediatedinter-/ intracellularsignaling mechanismsofACE2 regulation

Cell-typespeci ﬁ cgene expression,prior networkinformation andstatistical inference

12scMLnetFunctionalintercellular communications; intracellulargene regulatorynetworks

MATLAB/R https://github.com/ WangShuxiong/SoptSC https://github.com/ mkarikom/RSoptSC

Circosplotsof interactions betweencells

L-RscRNA-seqIndividualcellCCC probabilities,cell clusterCCC probabilities

Inferring communication networksbasedon cell-speci ﬁ cexpression ofligands,receptors, andtargetgenes

13SoptSCIndividualcellCCC probabilitiesare calculated;Integrates downstreamsignaling measurementsintoanLRI scoringfunction

https://github.com/BlishLab/ scriabin 704

https://github.com/ tanlabcode/CytoTalk 705

MATLAB/ Python/R

Dotplots;Bar plots;Boxplots R

NetworkanalysisL-RscRNA-seqCell –cellpairswith differenttotal communicative potentialand ﬁ nds modulesofco- expressed ligand –receptorpairs

14ScriabinComplexing communicativepathways; modelsofdownstream intracellularsignaling, anchor-baseddataset integratio,andgene network

http://git.embl.de/velten/ rnamagnet/ 706

Heatmap;Venn diagrams

L-RscRNA-seqIntegratedsignal transductiongene network

Prize-collectingSteiner forestalgorithm

15CytoTalkConstructsintegrated networkofintercellular andintracellulargene- geneinteractionsbased onmutualinformation

Heatmap;Scatter plots R

NetworkL-RscRNA-seqThesumof interaction probabilities; Averageinteraction scoresinalocal neighborhood

16RNA-MagnetIncorporatinginformation onsurfacereceptorswith lowmRNAexpression; identifyingthe enrichmentofsignaling interactions

Table2. continued ExistingbioinformatictoolsforinferringCCC

IDToolFeatureAlgorithmLinkInputOutputVisualizationAvailableinURLRefs

https://github.com/ LaughneyLab/ ContactTracing_Tutorial 707

https://github.com/rikenbit/ scTensor 715

HeatmapPython

NetworkL-RscRNA-seqInteractionsbetween cells

17ContactTracingAnalysisoftumor microenvironmentsin mouseandpatient

https://github.com/ zhengrongbin/MEBOCOST 716

Python

Manyoptionsfor interaction, expressionand pattern visualization R

TensordecompositionL-RscRNA-seqHTML ﬁ lewith summariesof clustering, decompositionand interaction components

18scTensorIdentifykeyLRIspresentin certaincelltypes; Interactionsmodeled usingtensor decomposition,whichare thenscored

737

https://github.com/zcang/ SpaOTsc

Barplots;Dot plots;Violinplots; Communication network

MetabolitesscRNA-seqCommunication scores,sensorsand eachmetabolite- sensorpartnerto characterizethe communication likelihood

Metabolitemediated intercellular communications

19MEBOCOSTIdentifyingcell –cell communicationsinwhich metabolites,aresecreted bysendercellsand traveledtointeractwith sensorproteinsofreceiver cells

https://github.com/ QSonggithub/spaCI 738

NotMentionedPython

L-RSTListofinferredligand andreceptor expressions;CCC matrixforagiven signalingpathway

Spatialcell –cell distanceandaverage enrichmentofgenes

20SpaOTscInferringthespatial distancebetweentwo cells;quantifyingthe con ﬁ denceofthe estimatedcell –cell distance

https://github.com/ BiomedicalMachineLearning/ stlearn 739

Python

Boxplots; Heatmap;Scatter plots;Network diagram;String plots;Spatialplots

L-RSTPredictingbothL –R interactionsandtheir upstreamregulators suchastranscription factors

Spatialrelationships; Network

21spaCISpatiallocationsandgene expressionpro ﬁ lesofcells toidentifytheactiveL –R signalingaxisacross neighboringcells

Python

Gene,SCTP, ClusterandPSTS visualization

L-RSTLigand –receptor expressionacross discretizedtissue

Python/R https://github.com/RubD/ Giotto 740

Heatmap;Dot plots

L-RSTUpregulatedand downregulated interactions;Listof mostsigni ﬁ cant ligand –receptor interactions

https://saezlab.github.io/ mistyR/ 741

Expression permutation

22stLearnSigni ﬁ cant ligand –receptorpairsare determinedon normalizedgene expressionwhichis normalizedacrossspatial location

Expression permutation

23GiottoGenerateanull distributionofLRIscores usingspatialinformation

Intrinsic (intraview),local nicheview (juxtaview),the broader,tissue view(paraview),or others R

L-RSTNetworkofsignaling geneinteractions withincellclusters andbetweencell clusters

Randomforest methods;Expression permutation

24MISTyInteractionsarecalculated byweightingthegene expressionsoflocalcell neighborhood

Table2. continued ExistingbioinformatictoolsforinferringCCC

IDToolFeatureAlgorithmLinkInputOutputVisualizationAvailableinURLRefs

https://github.com/ damienArnol/svca 742

ViolinplotsPython/R

L-RSTPredictinggenes withsigni ﬁ cant spatialvariation

Differentdimensions ofspatialvariation; Expression permutation

25SVCAAccountsforintrinsic effects,environmental effects,andcell –cell interactions

https://github.com/ ZJUFanLab/SpaTalk 743

Heatmap;Sankey plot;Diagramof theLRIfrom sendersto receiversinspace; LRTsignaling pathways

L-RSTInferringspatially resolvedcell –cell communicationsand downstreamsignal pathways

Cell-type decomposition;Spatial LRIenrichment

26SpaTalkIntegrating ligand –receptorproximity and ligand –receptor –target (LRT)co-expressionto modelandscoretheLRT signalingnetwork betweenspatially proximalcells

Python http://lewislab.ucsd.edu/ cell2cell/ 744

Barplots; Heatmap;Diagram oftheLRIfrom sendersto receiversinspace; LRTsignaling pathways

TensordecompositionL-RSTListofenrichedand depleted ligand –receptor interactions;Matrix ofcell –cell interactiondistances

27Tensor-Cell2CellModelinginteractions scoresandoptimizes Spearmancorrelation betweendistancesand interactionscores; Inferringcommunication distance

Multi-viewgraphPython https://github.com/lhc17/ HoloNet 745

NetworkL-RSTGeneratingtarget geneexpressionwith theCEnetworks; DecodingtheFCEs forspeci ﬁ c downstreamgenes

28HoloNetDecodingFCEsby integratingLRpairs,cell- typespatialdistribution anddownstreamgene expression

Python https://github.com/zcang/ COMMOT 746

Heatmap; Signaling pathways

L-RSTInferringCCCforall ligandandreceptor species;Visualizing spatialCCCatvarious scales;Analyzing downstreameffects

Collectiveoptimal transport

29COMMOTItaccountsforthe competitionbetween differentligandand receptorspeciesand spatialdistances,handles complexmolecular interactionsandspatial constraints

Circleplots; Heatmap;Chord diagram R https://github.com/Wei- BioMath/NeuronChat 747

NetworkL-RSTAweighteddirected graphcomposedof signi ﬁ cantlinks betweeninteracting cellgroups

30NeuronChatTheinference, visualizationandanalysis ofneural-speci ﬁ c communicationnetworks amongpre-de ﬁ nedcell groupsusingsingle-cell expressiondata

Theavailabilityofsingle-celltranscriptomedataandsingle-cellspatialtranscriptomedata,haveledtothedevelopmentofplentyofcomputatio naltoolsforreasoningaboutCCC.Thesetoolsleveragediverse methodsforpredictingpotentialintercellularcommunicationeventsbasedonpriorknowledgeofligand –receptorinteractions

Fig.5 Thetimelineofsingle-cellandspatialomicsandrelatedCCCsoftwares. a Timelineofthekeytechnologiesforsingle-cellandspatial omicswereretrospectivelysummarizedfrom2011tothepresentday.Cellnumbersreportedinrepresentativepublicationsbypublication date.AfulltablewithcorrespondingcellnumbersisavailableasSupplementaryTable1.SCTsingle-celltranscriptome,STspatial transcriptome,SCPsingle-cellproteomics,SPspatialproteomics. b Thehistoryofvariousbioinformaticsandcomputationalmethods developedtoinferbiologicalcell–cellcommunicationsbasedonsingle-cellomicsdata.SCTsingle-celltranscriptome,STspatialtranscriptome

Thus,anothertoolnamedscMLnethasbeendevelopedusing speciﬁctypeofcellgeneexpression,priornetworkinformation, andstatisticalinference.Thisapproachcannotonlymodel communicationsandGRNsamongcells,butalsoinferhow intracellulargeneexpressionisaffectedbythecellular interactions.708

DifferentfrommostmethodswhichhavetriedtopredictCCC betweenvariouscellularclusters,SoptSCenablestodecipherthe interactionsbetweenindividualcells.703 InSoptSC,individualcell CCCprobabilitiesarecalculatedusingnonlinearfunctionsinvolvingtheproductsofligandandreceptorexpressions,where targetgeneresponsescanbeweighed.However,itcouldnot automaticallydetectdisconnectedlineagesandinferbidirectional arrowsforcertaincellstatetransitions.703

SimilartoSoptSC,Scriabinisa flexibleandcomputationally effectiveapproachforanalyzingcommunicationpathwaysusing single-celllevelinformation.704 ItutilizescomprehensivedatabasesofcuratedLRIs,688 700 709 intracellularsignalingandanchor pointstoanalyzegenenetworks.710 Itshouldbenotedthatthis methodassumestheconsistentcredibilityofLRPswithinexpertly curatedprotein–proteininteractionrepositories.Downstream signalinganalysesinScriabinaredependentonNicheNet’smatrix ofligand-targetactivities,potentiallyinfluencedbythespecific celltypesandstimulationconditionsemployedinitscreation.In addition,NicheNet’sdatabaselackscapabilitiesforanalyzing inhibitorysignaling,leadingScriabintoprimarilyreturnCCCedges thatareanticipatedtoactivatesignals.704

Inaddition,CytoTalkinitiallybuildsacomprehensivenetwork containingbothintracellularandintercellularcommunications. ComparedwithNicheNetandSoptSC,thedifferentialexpression ofdownstreampathwaygenesismoresigniﬁcantfromCytoTalk prediction.Unlikethepreviousmethodsusingknownpathway

annotations,699,703 CytoTalkisabletoconstructsignaltransductionpathwaysfromscratchandcomparethemindifferenttissues orconditions,representingasigniﬁcantimprovementover existingalgorithms.705

Moreover,RNA-Magnetutilizesfuzzylogicfortheidentiﬁcation ofactiveligandsandreceptorsincellularcommunication.5 This methodforecastspotentialphysicalinteractionsamongindividual cellsandchosenattractorgroupsbyintegratingtheexpression patternsofcellsurfacereceptorswiththeircorrespondingsurfaceexpressedmRNA.688,711,712 RNA-Magnetassignsscorestoindicate thedegreeofattractionforeachcell,alongwithadirection showingtheattractorgrouptowhichthecellisprimarilydrawn.It hasbeenreportedthattheRNA-Magnetalgorithmcanaccurately inferthethree-dimensionalorganizationofbonemarrowfromthe expressiondataofsingle-cellgenes.713 However,RNA-Magnet mayhavelimitationswithonlyheterodimerreceptorinformation forintegrinsinearlyversionandtheinstallationprocessmay involvemultipledependencieswhichmaybechallengingfor someusers.706

Furthermore,ContactTracingrepresentsaninnovativesystemic methodtoforecasttheimpactofcondition-dependentcellular interactionswithinTME.707 ThismethodanalyzesTMEalongwith varyinglevelsofchromosomalinstabilitybyutilizingtheinherent variabilityofscRNA-seqdatatoinfercellresponsesto ligand–receptor-mediatedinteractions,independentofpreviously existingdownstreamtargetgeneknowledge.707

Theadvantageofthesenetwork-basedmethodsliesintheir utilizationofligandandreceptorexpressionlevelstocalculate interactionscoreandalteredexpressionofdownstreamsignaling targets.However,suchapproacheshavelimitationsinaddressing signalcrosstalk,whichmayresultintheoccurrenceoffalsepositiveornegativeoutcomes,especiallyincaseswhere

Fig.6 CCCnetworksinferredfromsingle-cellomics.Intercellularcommunicationnetworkscanbeinferredthroughvarioussingle-cellmultiomicstechniquesandmethods.(1)CCCofsingle-celltranscriptome:geneexpressionmatricesofdifferentcelltypesareobtainedby performingsingle-cellRNA-seq,andthenclusteringanalysisiscarriedouttoinfercommunicationnetworksofvariouscelltypes.CCCof single-cellproteomics:asingle-cellsuspensionismadeaftercollectingsamplessuchasliver,pancreas,lungandmousebrainwhichlabeling withconjugatedantibodiestaggedwithmetalisotopes.Thencell–cellcommunicationofdifferentcelltypesisinferredthroughmass spectrometry ﬂowcytometryandclusteringanalysis.(2)CCCofspatialproteomics:tissuesarepreparedonslidesfollowedbylabelingof conjugatedantibodiestaggedwithmetalisotopesandlaserablation,thenproteinexpressionmapandCCCnetworkisobtainedbyanalysis ofionmassspectrometry.CCCofsingle-cellspatialtranscriptome:bycombiningscRNA-seqwithspatiallocalization,geneexpressionmapof variouscelltypesisobtainedtoinferCCCsindifferentspatiallocations

intracellularpathwaysaremodulatedthroughposttranslational modiﬁcationsinsteadoftranscriptionalregulation,asobservedin certaincytokinesignalingpathways.714

Tensor-basedanalysistools.Toolsbasedontensoranalysis constitutethegroupwiththehighestmathematicalcomplexity. Forinstance,scTensorisaninnovativeapproachforderiving representativetriadicrelationships,encompassingligandexpression,receptorexpression,andassociatedLRPs.Oneofits attractivefeaturesisthatLRreferenceisavailableformany organisms.ThisapproachutilizesTuckerdecompositionona third-ordertensortopinpointkeyligand–receptorinteractions (LRIs)thatarespeciﬁctoparticularcelltypes.715 ThescTensor utilizesapotentialLRPdatabaseautomaticallygeneratedby interactionsfromSTRINGandannotationsfromSwissprot (secreted/membrance),andrevealedasigniﬁcantquantityof presumedLRpairs.TheCCCnetworkisconstructedasadirected hypergraphwithmultipleedgetypesrepresentingdifferentLRPs. Tensordecompositionisusedtomodeltheseinteractionsand calculatetheirscores. 715 Althoughthesetoolscancapture communicationpathwaysinvolvingallcellpairssimultaneously andextractrelationshipsbetweendifferentCEs,interpreting fractionsfromtensordecompositionmightnotbeasstraightforwardasothertools.

Otherprinciplesandstrategies.UnliketoolsthatuseLRPsas mediators,MEBOCOSTisanalgorithmbasedoncomputational

methodologydesignedtoinferthedynamicsofmetabolite-driven intercellularcommunicationsquantitativelyusingscRNA-Seq data.716 Byconsideringtheexpressionofenzymeproduction, dataregardingthesecretionofmetaboliteshasbeenincorporated inthetransmissionstudy,andit’spossibletodeducethesynthesis ofparticularmetabolitesfromtranscriptomedata.717–719 This algorithmdetectsinteractionsbetweencellswheresendercells secretemetaboliteslikelipids,whichthenengagewiththesensor proteinsinreceivercells.MEBOCOSTidentiﬁescellsemittingand receivinganextracellularmetabolite,contingentontheirrespectiveenzymeandsensorexpressionlevels,therebyidentifying communicationsbetweencellsinvolvingmetabolitesensors.The MEBOCOSTalgorithmaccountsforboththesynthesisand utilizationreactionsofmetabolites.Itsdesignensurescompatibilitywithestablishedalgorithmsthatascertaintheexistenceof single-cellmetabolitevia ﬂuxbalanceanalysis,suchasscFEAand COMPASS.Nevertheless,thefrequentlynonlinearcorrelation betweenmetabolitequantitiesandRNAcontentsofmetabolic enzymesposesachallengethatthisalgorithmcannotprovide quantitativecalculationsofmetaboliteabundance.716

Whilehigh-throughputscRNA-seqmethodsdescribecell populationsheterogeneity,720 theylacktheabilitytooffer phenotypicinformation,suchascellsurfaceproteinlevels.684 Meanwhile,thetargetedmethodformeasuringexpressed proteinsinasinglecellislimitedinscaleandlimitedproﬁling methodisachievablefordetectingaplentifulofgenesand proteinsinparallel.721,722 Fluorescent-labeledantibodiestargeting

cellsurfaceproteinsserveasreliableindicatorsofcellularactivity andfunction.723 ThemethodknownasCITE-sequsing oligonucleotide-labeledantibodiesthroughsequencingaddresses thislimitationbyutilizingsequencing-basedstrategythat simultaneouslyquantifyingtranscriptomeandcellsurfaceprotein insingle-celllevel.CITE-seq.684 notonlydescribescellular transcriptomesandepitopesindexingbutalsoiscompatiblewith existingsingle-cellanalysisapproaches.Comparedtoseparate transcriptomemeasurements,multimodaldataanalysisthrough CITE-seqprovidesdetailedcellularphenotypefeatures.

ItisnoteworthythatsinceCCCserveasthedownstreamofall dataanalyses,settingthresholdsforLRIscanimpactthe interpretationandexplanatorypowerofCCCresults.Alower thresholdmayleadtotheidentiﬁcationofmoreinteractions, includingthosewithlowerexpressionlevelsorfrequencies,aiding inthediscoveryofpotentialnovelLRPs.However,thismayalso introducenoise,includingfalse-positiveresults.5,9 Conversely,a higherthresholdmayreducefalsepositivesbutcouldalsoresult inmissingsometrueLRIs.Therefore,whensettingthethreshold forLRIs,abalanceneedstobestruckbasedonthespeciﬁc researchobjectivesandcharacteristicsofthedatausedtoachieve themostaccurateandinterpretableCCCresults.

Spatialtranscriptome-basedtools Typically,cellularinteractionsareconfinedtorestrictedareas, whichisnotcapturedbyscRNA-seq.5 Inordertominimize incorrectexclusioninCCCanalysis,integratingthecell’smedium spatialpositionisessential.724 Spatialtranscriptometechnology enablestranscriptomeprofilingfromcellsindifferentlocationson tissuesections,facilitatingtheanalysisofgeneexpression characteristicsatdiversespatialpositionswithintissues.mRNA servesasthefunctionalcopyofactivegenes,andtheirlocalization withinlivingtissuesisoftenrelatedtotheregulationofcelland tissuegrowthanddevelopment.Previously,theanalysisof multiplemRNAssimultaneouslyrequiredthecrushingofcells, makingitimpossibletounderstandthelocalizationofmRNA withincells.FluorescentinsituRNAsequencing(FISSEQ)can revealenvironmentallyspecifictranscriptswhilepreservingthe tissuearchitecturenecessaryforRNAlocalization.725 Thistechniqueisapplicabletotissuesectionsandwholeembryos,isnot overlylimitedbyopticalresolution,andcanreducenoisesignals insingle-moleculedetection.726 Inaddition,itenableslarge-scale paralleldetectionofgeneticelements,assistingresearchersin analyzingcellphenotypes,generegulation,andinsituenvironments.Currently,the10XGenomicsVisium.727,728 technology standsasamainstreamcommercialspatialtranscriptiontechnology;however,itsdetectionresolutionremainsbelowthetrue single-celllevel.Conversely,the10XGenomicsXenium.729 technologysignificantlyenhancesspatialresolutionbyinsitu fluorescentimaging,capturingRNAexpressionsatsingle-cellor subcellularlevels.Thistechnologyswiftlydetectstheinsitu expressionlevelofnumeroustargetsonfreshfrozen(FF)or formalin-fixedparaffin-embedding(FFPE)tissuesections.By employingexistingorcustomizedprobepanelsandtargets,this methodachievessubcellularresolution,offeringinsightsintocell structureandfunction.Asanotherimage-basedspatialapproach, MERFISHfacilitatesthedetectionandmeasurementofamultitude ofRNAtypes,rangingfromhundredstothousands,atthesinglecelllevel.730 Itemploysspecific fluorescentlabelingstrategiesto simultaneouslydetectmultipleRNAmolecules.Notably,MERFISH demonstratesfault-tolerantcapabilities,accuratelyidentifying RNAspeciesdespiteminor fluorescentlabelingerrors.Xiaowei Zhuang’steamatHarvardUniversitysuccessfullyemployed MERFISHtechnologytorecognizeover100neuronalandnonneuronalcellpopulationsinthehumanbrainwithhigh-resolution images.731 Currently,fault-tolerant fluorescenceinsituhybridizationtechniqueslikeMERFISH730 andseqFISH+732 areprimary hybridization-basedinsitutranscriptomicmethods.ST

technologiescancreate “atlases” withspatialinformation,revealingwhichcellsconstituteeachtissueandhowtheyareorganized andcommunicate.733 However,theimbalancebetweenresolution,genecapture,and ﬁeldofviewincurrentmethodshinders theconstructionofatlaseswith “higherspatialresolution” and “broadertranscriptomecoverage” 734 TheStereo-seqtechnique activelyaddressesthesechallenges,andiscapableofanalyzing genesandimagingsimultaneously.735,736 Thistechnologyallows forultra-highprecisionanalysisofgeneandcellchangesover timeandspaceduringthedevelopmentalprocessesoflife, achievingacomprehensivespatiotemporalmolecularatlasoflife.

Sothespatialtranscriptomeiscrucialinlocatingand distinguishingtheactivegenefunctionexpressedindistinct tissueareas,offeringkeyinsightsfordiagnosticandtherapeutic purposes.Creatingtoolsforsingle-cellSTanalysistoclarify regulatoryprocessescontrollingcellstatechangesholdgreat signiﬁcanceforresearchin ﬁeldssuchascancerpathogenesis, neuroscience,developmentalbiology,andothers.Therefore, manytoolssuchasSpaOTsc,737 spaCI,738 stLearn,739 Giotto,740 MISTy,741 SVCA,742 SpaTalk,743 Tensor-Cell2Cell,744 HoloNet,745 COMMOT,746 andNeuronChat747 havebeendeveloped.

Non-spatialsingle-cellmethodsfrequentlyyieldconsiderable falsepositives,asCCCoccurswithinconﬁnedspatialranges unmeasuredinsuchdatasets.Thus,SpaOTscwasdevelopedto inferthespatialdistanceoftwocellsbycontrastingtheir predictedspatialdistributions,thenprovideausefullinkage betweenthemandquantifythereliabilityoftheestimated distance.737 Asanetworkapproach,optimaltransportationisused inSpaOTsctomodelintercellularcommunication.However, computationalchallengesariseasdatasetsexpandbeyond manageablesizes.Inaddition,thisapproachdoesnotaccount forpotentialtimedelaysinCCC.Owingtofrequentsignal dropoutsandnoisesignalsinsingle-cellSTdata,anothernetworkbasedtoolspaCIhasbeenproposedusinganadaptivegraph modelwithattention-basedmechanisms.Itcombinesthe neighboringcells’ spatialpositionandexpressionproﬁlesto determinetheactiveLRsignalingaxis.Moreimportantly,spaCI allowsdetectionofupstreamTFsthatmediatestheLRsignaling axis,andenhancescomprehensionofthepotentialmolecular mechanismofintercellularcrosstalkwhichnetwork-basedmethodsinsingle-celltranscriptomeareblindto.

MethodssuchasSpaOTscandspaCIhavenotcombinedspatial cell-typedistributionandLRinteractionto findhotspotsthatmay havehighCCCactivities.So,atoolbasedonexpression permutation,calledstLearn,wasdevelopedtoautomaticallyscan areaswithhighcell-typedensitiesandco-expressedLRPs, suggestingahighlyinteractivearea.732,748,749 Similarly,Giotto, MISTyandSVCAcaninfertheinteractioninthelocalcellnicheby establishingthestatisticalsignificanceoftheautomatically recognizedcell-typedistributioninneighborhood.62,740,749–752 Giottoincorporatesspatialexpressioninformationwiththe possibilityofcellinteractionsbycreatinganulldistributionof LRIscorestorecognizekeyinteractions.Itanalyzesandisolates interactionsbetweennearbycellclustersbasedontheconstructionofspatialnetworksfromspatialtranscriptomics.Incontrast, MISTyisanexplainableframeworkforanalyzinghighlymultiplexedspatialdatawithoutrequiringcell-typeannotation.This methodidentifiescrucialmarkergenesinparticularregions throughrandomforestalgorithmsandcalculateinteractionsby applyingweightstogeneexpressionsinlocalcellularenvironments.SimilartoGiottoandMISTy,anothercomputational frameworkSVCAwasdevelopedtoquantifyspatialvariationin differentdimensionsbyanalyzingtheinteractionsbetween markerswithindifferentspatialcontexts.

However,GiottoandSpaOTscarelimitedtoinferringCCC betweensingle-cellSTdataratherthanthespot-basedSTdata andbetweenpairedcelltypesratherthanpairedcells. 743 Itstill lacksmethodscapableofinferri ngandvisualizingspatially

resolvedCCCatsingle-cellresolutionthroughSTdata.The emergenceofSpaTalkenablesstatisticalanalysisandvisualizationofspatiallyproximalLRIs,formingadynamicCCCnetwork. 753 Byincorporatingspatialinformation,SpaTalkdisplays enrichedLRIsamongspatiallypr oximalco-expressedcellpairs atsingle-cellresolution,providinganinformativemethodfor analyzingandvisualizingLRIsandtheirmediatedCCCfrom differentperspectives. 743 Thisoffersapowerfultoolfor resolvingkeyCCCsinnormalphysiologyandpathological processesatspatialsingle-cellre solution.Inaddition,TensorCell2Cellisanunsuperviseda pproachbasedontensordecompositionandunravelscontext-speci fi cCCCbyanalyzingvarious cellstages,states,orlocationsconcurrently. 744 Inshort,these methodsfacilitatestheintegrati onofspatiallocation,structural characteristics,andexpressi onpatternstoaddresssigni fi cant biologicalquestionsincludingcell-typeidenti fi cationand intercellularcommunications. 739

AlthoughSVCAandTensor-Cell2Cellwasbuilttocharacterize thedependenciesofsender-receivercellaswellastherelated phenotypes,amethodforsystematicallydecodingfunctionalCEs wasstilllacking.ConsideringonlyfunctionalbutnotirrelevantCEs involvingspeciﬁcbiologicalprocessescanhelptobetterunderstandtheroleofintercellularcommunicationinshapingcertain cellphenotypesandformulatepossiblediseaseinterventions.745 ThenHoloNetwasdevelopedtocharacterizecommunication landscapeandidentifybothcelltypesservingasmainsenderand LRPsservingascoremediatorsofthespeciﬁcdownstreamgenein functionalCEs.745

Multipleligandscanbindtomultiplereceptors,thereby generatingcompetition,aubiquitousandcrucialbiophysical processamongmultiplemolecularspecies.746 However,current methodsexamineCCConlocalandindependentcellpairs, focusingoninformationbetweencellsornearindividualcells. Thus,collectiveorglobalinformationinCCC,suchascompetition betweencells,isoverlooked.Toaddressthisissue,COMMOTwas developedbytakingintoaccountthecompetitionbetween differentligandandreceptorspeciesaswellasthespatialdistance betweencells.746 Besides,brainfunctiondependsonsignal transmissionbetweenavastnumberofneuronsandnonneuronalcells.Theconnectome theconnectivetissueofneural connections issubjecttotranscriptionalregulation.754,755 Emergingspatialtranscriptomicsmethods,66,732 besidesmeasuring geneexpressionwithincells,alsomeasurethespatiallocationof neuronalcells,providingarichresourcefordissectingneuronal heterogeneity.However,thesemethodsarenotsuitablefor characterizingcommunicationbetweenneurons,asneuronscan extendaxonsanddendritesoverlongdistancestoformsynapses andprimarilycommunicatethroughneurotransmittersignals.756–758 ThedevelopmentofNeuronChattookintoaccount neurotransmittersignalingandsystem-levelneuron-speciﬁccellto-cellcommunicationnetworks,incorporatingtheprocessof neuralsignaltransmissiontoinferintercellularcommunication. ThismakesNeuronChatdistinctfromexistingmethodsfor inferringintercellularcommunicationthatdonotaccountfor neuronalactivity.747

However,thelimitedabilityofinsituhybridizationtechnology andtheapplicabilityofNGSsolelytohomogenizedtissuesfailto fullycapturethecomplexityofahumanTME.759 Toovercomethis challenge,GeoMxDSPspatialmulti-omicstechnology71 and CosMxSMIsingle-cellspaceinsituimagingtechnology.760 are developedforspatialanalysisofmultipletargets,whichrealize thedirectevaluationofcompletetissuemicroenvironmentand localdrugeffectsinsituofpatients’ tumortissues.761 Furthermore,thesemethodscapableofsimultaneouslydetectingmRNA andproteinbybindingoligomerantibodies,762 offeracomprehensiveviewofthefulltranscriptome,successfullyappliedin spatialgeneexpressionstudiesacrossvariousorgansand tissues.763

Single-cellproteomicsandspatialproteomics Focusingonunderstandingthequalitativeandquantitative aspectsofproteincompositionwithinsinglecells,proteomic analysisatthesingle-celllevelandspatialproteomicstudiesare emerging.Thisanalysisunveilsdifferencesintheproteome betweenindividualcells,providingadetailedmolecularmapof proteins.Thisinformationaidsincomprehendingcellularvariationsinphenotypeandfunction.Intherealmofcellcommunication,single-cellproteomicsoffersin-depthinsightsintohowcells interactthroughspeciﬁcproteinsandsignalingpathways.These interactionsinvolvethe “secretlanguage” composedofsignals likecytokinesandmembraneproteins,whichconnectcellsto ensuretheefﬁcientfunctioningoflife,acoreaspectofcell communicationresearch.

Single-cellproteomicstechnologies,suchasmasscytometry, enablethesimultaneousanalysisof50parametersatthesinglecelllevel,encompassingproteins,nucleicacids,andsmall molecules,allachievedwithahighsignal-to-noiseratio.CyTOF,764 whichstandsforCytometrybyTimeofFlight,employsmass cytometrytoquantifylabeledtargetsonboththesurfaceand interiorofindividualcells.ThistechnologyenablesthesimultaneousquantificationofmultiplecellularcomponentsbyemployingadetectorbasedoninductivelycoupledplasmaMS(ICP-MS). TheprincipalbenefitofCyTOFliesinitscapacitytoleverage immunolabelingtoquantifyproteins,carbohydrates,orlipids withinacell.Thisinnovativetechnologyhasrevolutionized discoveryandclinicalresearchbyallowingresearchersto simultaneouslyinterrogateover50markersonmillionsof individualcells.Furthermore,CyTOFcombinesMSand flow cytometryprinciples,enablingsingle-cellproteinexpression analysis,whichiscrucialforadvancingstudiesinbothsteadystateandpathologicalprocesses.Mostimportantly,MS flow cytometryallowshigh-throughputandhigh-resolutiondetection ofmultipleparametersinasinglecell,makingitinvaluablefor studyingCCC.Thistechnologycannotonlydetectreceptorsand ligandsonthecellsurfacebutalsoidentifysignalingmolecules withinthecell.Onesignificantadvantageisthatit’snot constrainedbyoverlapping fluorescencespectra,allowingthe simultaneousdetectionofmoreparameters.Thiscomprehensive informationprovidesdeeperinsightsintothemechanisms underlyingcellcommunication.

Spatialproteomicsexploresthespatialdistributionandfunction ofproteinswithincells,consideringthateukaryoticcellsarehighly compartmentalized,anddifferentbiologicalprocessesoccurin distinctcellularcompartments.Aprotein’sfunctiondepends closelyonitssub-localizationwithinthecell,asdifferent compartmentsprovidevaryingchemicalenvironments,suchas pHandredoxconditions.Proteinsarefunctionalmoleculesofall cellularfunctionsandprocesses.Thus,thespatialexpressionof proteinsisessentialfordeterminingtheirpreciselocationsand rolesintissues.Proteinscanchangedependingoncell-type,cycle stage,diseasestate,andtreatmentmethods.Consequently,spatial proteomicsservesasaneffectiveapproachforexamining alterationsinspatialexpressionpatternofproteinsassociated withdiseases,offeringnewperspectivesforbiomarkerdiscovery andtherapeuticdevelopment.Recently,the ﬁeldofspatial proteomicshasachievedsigniﬁcantadvancementsintheaspects ofmicroenvironmentanddiseasedevelopment,mechanismsand drugtargets,organstructuralheterogeneity,andtissueororgan spatialmapping.

Traditionalproteomictechniquesprimarilyfocusondetecting proteinexpressionlevelsincellortissuelysates,lackingessential spatiallocationinformation.Spatialproteomicstechnologieshave emergedtoaddressthelimitation.Nevertheless,thesetechnologiesformthegroundworkfortwoessentialspatialimaging techniques,imagingMScytography(IMC)andmultipleionbeam imaging(MIBI).763 IMCintegratingimmunocytochemistryand immunohistochemistrytechniqueswithhigh-resolutionlaser

ablationintoCyTOFMS ﬂowcytometry.765 Itcomplements existingimagingmethods,delineatescellsubgroupsandintercellularinteractions,andaccentuatestumorheterogeneity. Similarly,MIBIemployssecondaryionMSforimagingantibodies taggedwithmetalisotopesandanalyzessamplesmarkedwithas manyas100differentmetalisotope-labeledantibodies.766 This techniqueiscompatiblewithconventionalFFPEtissueslices,a prevalentspecimentypeinclinicalrepositoriesworldwide.767 The emergingMS.768 technologyforidentifyingandquantifying proteinscannotonlymeasuretheabundanceofproteinsand PTMsinindividualcells,butalsomeasuretheircomplexesand subcellularlocalization.

However,thesemethodsbasedonionMSencounterlimitations concerningtheavailabilityofsufficientpuremetals.Ontheother hand,traditional fluorescenceimmunohistochemistrytechnology faceslimitationsinachievingsingle-cellanalysisduetooptical constraintsanddifficultyinimagingmorethansevenbiomarkers inasample.Incontrast,CellDIVEcircumventsthishurdlethrough multipleroundsofstaining,enablingtheimagingdetectionof60 biomarkersinasinglesamplebydirectlabelingwith fluorescent dyes.69 Similarly,anothersingle-cellproteomicanalysisplatform CODEXenablestoofferintricatedetailsregardingprotein distributionin2Dspace.62,769 Thefundamentaldesignprinciple underlyingCODEXinvolvesmarkingspecificoligonucleotide “Barcode” onindividualantibodies,770–772 insteadofdirect labelingwith fluorescentdyesusedinCellDIVE.69 The fluorescent dyenecessaryforimagingselectivelybindstothecomplementary oligonucleotidesequenceofthe “Barcode”.Insummary,these innovativeapproachesallowustosurpasslimitationsassociated withthenumberofvisiblespectral fluorescenceimaging channels,facilitatingthesimultaneousdetectionandanalysisof 50ormoreproteinindicators.Meanwhile,pathologicalanalysis softwarefacilitatesthesemi-quantitativeanalysisofdiverse biomarkermoleculesineachcell.

Single-cellmultiomictools

Thecontinuousadvancementofsingle-cellomicstechnologyhas equippeduswithapotenttooltoexplorecellinteractionand communication.Techniquesincludingsingle-celltranscriptomics andproteomics,offerdetailedobservationsofgeneexpression andproteinsynthesisinsinglecells,respectively.Thesetechnologieshavebroadapplicationsinbiologicalandmedicalresearch, offeringfreshinsightsandpossibilitiesfordeepeningourunderstandingofcellinteractionandcommunication.

Single-cellsequencingisanadvancedbiotechnologythat facilitatesdetaileddecodingofgeneexpressionandgenetic alterationforeachcellinatissuesample.Forexample,singlecellDNAsequencingpermitsaccurateDNAmappingforeach uniquecell.Goingbeyondthis,themoreintricatesingle-cell multi-omicssequencing,thisistosaysingle-cellmultimodal omicsanalysis,facilitatesthesimultaneousacquisitionofvarious dimensionsofomicsdatafromani ndividualcell.Practically, single-cellmulti-omicsanalysisef ﬁ cientlyrecordsdifferent featuresoftheidenticalcellinmultipletissuesamples, encompassingDNA,RNA,epigeneticregulations,andprotein patterns.Thispowerfulapproac hhasfoundextensiveapplicationinsystematicallyunravelingtheintricateinteraction mechanismsofcriticalcomponentsandpathwayswithincells, contributingsigni ﬁ cantlytoourunderstandingofcomplex cellularprocesses. 773

Single-cellmulti-omicsresearchintegratesdiversetechniques fromvariousdisciplinestoscrutinizethevariabilityamong differentcells.Simultaneously,thisintegrationenablesacomprehensiveandquantitativeanalysisofthemulti-dimensionaldata associatedwithdistinctcells,exploringtheirpotentialbiological signiﬁcance.Nonetheless,itconfrontschallengessuchasintricate technicalcomplexities,substantialdatavolumes,andmultiple dimensionsofdata,necessitatingongoingdevelopmentand

optimizationofanalyticalmethodsandresearchmodelsinthe realmofmulti-omicstechnology.

Besides,thesingle-celltranscriptomeoffersthecapacityfor large-scalesimultaneousanalysisofthousandsofmolecular attributeswithinasinglecell,uncoveringpivotalgenesassociated withdistinctcelltypesandhighlightingdisparitiesamongvarious cellcategories.However,theunderlyingmechanismsgoverning thesedifferentialexpressionsremainlargelyunclear.Inthis context,single-cellmulti-omicsemergesasapotenttoolcapable ofelucidatingtheinternalinterplaybetweengeneexpressionand epigeneticregulationwithinthesamecell.Itprovidesameansto establishthedirectconnectionsbetweencandidateregulatory elementsandtheirtargetgenes,allowingforthedeﬁnitionof regulatoryelementsandcellstatesspeciﬁctouniquecelltypes. Thisapproachaidsinelucidatingtherootcausesofgene expressiondisparitiesandunveilstheregulatorynetworkunderpinninggeneslinkedtotumorsanddiseases,alongwiththe mechanismsgoverningthem.

Finally,considerablestrideshavebeentakeninrefining methodsandapplyingsingle-cellandspatialmulti-omicstechnologies.Sometoolshaveincorporatedmulti-omicsinformation ininferringCCC,preciselymappingsingle-celldataintospatial transcriptomicsdata.Forinstance,theupdatedversionof CellPhoneDBv5significantlyimprovesthedatabaseandcomputationalmethodstoinfer,prioritize,andvisualizeCCC,utilizing othersingle-cellmodalitiessuchasspatialinformationorTF activity.774 Apartfromthis,CellChatv2nowenablestheinference ofCCCfrommultiplespatiallyresolvedtranscriptomicsdatasetsas well.ByoptimizingtheCCCalgorithmthroughtheintegrationof multi-omicsdata,theaccuracyofcell-typeclassificationcanbe improved,therebyenhancingthecalculationresultsofcell proportionsandlog2values.Thesetechniquesempowerthe explorationofthemolecularhierarchy,bridgingthegapfromthe genometothephenotypewithinasinglecell.Theyofferinsights intothedynamicinterplaybetweengeneregulationfrom epigenomeandgeneexpressionfromtranscriptomeorproteome acrossvariousbiologicalprocesses,includingdevelopment,aging, anddiseases.Inaddition,thesetechnologiesfacilitatethestudyof theimpactofgeneticvariationsacquiredbyindividualcellson theiruniquefunctionalandphenotypiccharacteristics,alongwith theirinfluenceonsurroundingtissuefunctionsandother factors.775 Withongoingadvancementsinsingle-celltechnology, wecanlookforwardtodeepeningourunderstandingofthe intricatenetworkofinteractionsbetweencells.Inturn,thisoffers novelstrategiesandpathwaysfordiseasepreventionand treatment.SincethemethodforinferringCCCpossessesunique advantagesandconstraints,theutilizationofthesemethodologiesnecessitatesawarenessoftheirstrengthsandweaknesses, andthecarefulchoiceofanalyticalparameters.5 Whilemethodologicalandtechnicalchallengespersist,thereareabundant opportunitiesforenhancingourcomprehensionofcellular interactions.Lookingahead,wecananticipatefurtherbreakthroughsintherealmsofbiologyandmedicinethrough continuedadvancementsinsingle-cellresearch.

InvestigatingmechanismsunderlyingCCCremainsaprominent areaofexplorationinphysiologyandthebroaderlifesciences. Althoughwehavemaderemarkableprogress,therearestillmany challengesinunderstandingsingle-cellcommunicationand interaction,whichrequirefurtherresearchandexplorationto bettersolvecurrentproblemsinbiomedicalresearch.Research hasrevealedthattheintricaciesofsignaltransductionpathways areexceedinglycomplex,involvingproteininteractionsandthe expressionprocessesofassociatedgenes.However,complex interconnectionsexistamongvarioussignaltransductionpathways,forminganintricatedialogbetweensignalsandevenan entiresignalnetworksystem.Despitesigniﬁcantprogressin recentyears,facilitatedbyvarious “omics” approaches,exploring signaltransductionmechanismsrequiresfurtherin-depthresearch

andexamination.Therefore,developingcutting-edgetoolsfor decipheringcellularinteractionsandtheirintegrationwithmultiomicsapproachesispivotalforadvancingthetreatmentof diversediseasesandtheprogressofthemedical ﬁeld.

CHALLENGESANDPERSPECTIVES

CCCisafundamentalmechanismformulticellularorganismsto adapttointernalandexternalenvironmental fluctuations,andto preservehomeostasis.ThroughCCC,biochemicalandphysical signalsaredispatchedandreceivedbetweencells,influencingcell phenotypesandfunctions.704 However,thepresentresearch challengeisthatcurrentapproachesfordecipheringintercellular communicationfromscRNA-seqdatapredominantlyanalyzeat theclassificationofcellsubtypesorclusterlevel,oftenoverlooking informationinindividualcells.

Thechallengesinclude:

(1)Dataparsingcomplexity:WhilescRNA-seqtechnologyoffers thecapabilitytodissectintricatemulticellularnichesat single-cellresolution,itisessentialtorecognizethatCCC doesnotoperateatapopulationlevelbuttranspiresat single-cellscale.Hence,thedevelopmentofnovelCCC inferencemethodsisimperative.Thesemethodsshould examinesingle-celldynamicsandtheirinterplays,and capitalizeonthefullspectrumofinformationencapsulated withinscRNA-seqdata.704

(2)Complexexperimentaldesign:Traditionalapproachesto investigatingintercellularinteractionsofteninvolveexpensiveequipmentandintricateprocedures.Moreover,these methodsexhibitlimited ﬂexibilityandareoftenincompatiblewithotheranalyticalprocesses.776

Besidesthis,thecurrentapplicabilityofCCCismorewidespread inthecell–cellinteractionsunderphysiologicalconditions. However,duringpathologicalorpost-treatmentprocesses,celltypetransitionsoccur,impactingtheaccuracyofanalysis.To improveaccuracy,thefollowingmethodscanbeconsidered:

(1)Useofmulti-omicsapproaches:Combiningsingle-cell transcriptomics,proteomics,andmetabolomicscancomprehensivelyanalyzechangesincell–cellinteractions, reducinganalysisbiasescausedbycell-typetransitions.

(2)Developmentofprecisecellmarkers:Developingnewcell markersthatcanmoreaccuratelydistinguishdifferentcell typesandmonitorchangesincellstatesmore ﬁnely.

(3)Conductingvalidationexperiments:Basedontheanalysis results,performvalidationexperimentstoconﬁrmthe impactofcell-typetransitionsontheresultsandfurther validatetheaccuracyoftheanalysis.

(4)Integrationwithclinicaldata:Integratingexperimentaldata withclinicaldataforanalysiscanbetterunderstandtherole ofcell-typetransitionsindiseasedevelopmentandtreatmentprocesses,improvingtheaccuracyandcredibilityof theanalysis.

AsOMICStechnologiesundergorapidadvancement, researchintocellularcommunicationnetworkshasalsomade substantialprogress.Thisresearchunveilsthefoundational structureandfunctionsofcel lcommunicationnetworksand laystheexperimentalgroundworkforapplicationinvarious related ﬁ elds.

Inthefuture,researchintocellcommunicationnetworksis poisedtoattaingreaterdepth,withspeciﬁcprospectsincluding: (1)Researchbasedonmulti-omicscellcommunicationnetwork. (2)Explorationofthedynamicchangesinthestructureand functionalityofcellcommunicationnetworks.(3)In-depth investigationofpivotalcomponentsandinteractionswithin

signalingpathways.(4)Researchfocusedondrug-targeted therapyandthepredictionofsideeffects.(5)Utilizationof microﬂuidicsystemshasemergedaspracticaltoolsforresearchingcell–cellandcell-ECMcommunications.Microﬂuidicsystems offeradvantagessuchaslowreagentconsumption,precise managementofreagents,highthroughput,andseamless integrationoffunctionalcomponents.Theyfacilitatecomprehensivestudiesofcellularinteractionsatpopulationandsingle-cell levels.776 (6)Thedevelopmentofnovelmulti-omicsanalysis methodsforinferringCCC.

Insummary,thestructuralanalysisandapplicationofCCC networkshavehugesigniﬁcance,anditsresearchholdssubstantialpotentialforaddressingamultitudeofchallengesinlife sciences.

ACKNOWLEDGEMENTS

ThisstudywassupportedbyfundingfromtheNationalNaturalScienceFoundation ofChina[Y.S.,No.82300433;F.M.,No.32170656],BeijingNovaProgram[F.M., Z211100002121039],BeijingMunicipalNaturalScienceFoundation[Y.S.,No. 7224348]andKeyClinicalProjectsofPekingUniversityThirdHospital[Y.S.,No. BYSYZD2023047].WeacknowledgetheuseofBioRender(www.biorender.com)for creating ﬁguresinthisreviewpaper.

AUTHORCONTRIBUTIONS

Conceptualization:F.M.andJ.Q.;writingandediting:J.S.,Y.S.,Z.Z.,X.H.,andJ.F.; supervision:F.M.andJ.Q.Figuredrawing:J.S.,Y.S.,andF.M.Allauthorshavereadand approvedthearticle.

ADDITIONALINFORMATION

Supplementaryinformation Theonlineversioncontainssupplementarymaterial availableat https://doi.org/10.1038/s41392-024-01888-z

Competinginterests: Theauthorsdeclarenocompetinginterests.

REFERENCES

1.Bonnans,C.,Chou,J.&Werb,Z.Remodellingtheextracellularmatrixindevelopmentanddisease. Nat.Rev.Mol.CellBiol. 15,786–801(2014).

2.Rouault,H.&Hakim,V.Differentcellfatesfromcell-cellinteractions:core architecturesoftwo-cellbistablenetworks. Biophys.J. 102,417–426(2012).

3.Zhou,X.etal.Circuitdesignfeaturesofastabletwo-cellsystem. Cell 172, 744–757e717(2018).

4.Dimitrov,D.etal.Comparisonofmethodsandresourcesforcell-cellcommunicationinferencefromsingle-cellRNA-Seqdata. Nat.Commun. 13,3224(2022).

5.Armingol,E.,Ofﬁcer,A.,Harismendy,O.&Lewis,N.E.Decipheringcell-cell interactionsandcommunicationfromgeneexpression. Nat.Rev.Genet. 22, 71–88(2021).

6.Singer,S.J.Intercellularcommunicationandcell-celladhesion. Science 255, 1671–1677(1992).

7.Shao,X.,Lu,X.,Liao,J.,Chen,H.&Fan,X.Newavenuesforsystematically inferringcell-cellcommunication:throughsingle-celltranscriptomicsdata. ProteinCell 11,866–880(2020).

8.Wang,X.,Song,W.,Kawazoe,N.&Chen,G.Theosteogenicdifferentiationof mesenchymalstemcellsbycontrolledcell-cellinteractiononmicropatterned surfaces. J.Biomed.Mater.Res.A 101,3388–3395(2013).

9.Ramilowski,J.A.etal.Adraftnetworkofligand-receptor-mediatedmulticellular signallinginhuman. Nat.Commun. 6,7866(2015).

10.Sonnen,K.F.&Janda,C.Y.Signallingdynamicsinembryonicdevelopment. BiochemJ. 478,4045–4070(2021).

11.Alon,U.Networkmotifs:theoryandexperimentalapproaches. Nat.Rev.Genet. 8,450–461(2007).

12.Lodish,H.F. MolecularCellBiology (Macmillan,2008).

13.Uzman,A.In MolecularBiologyoftheCell (4thed.):(edsAlberts,B.,Johnson,A., Lewis,J.,Raff,M.,Roberts,K.,andWalter,P.)212–219(JohnWiley&SonsInc., 2003).

14.Kholodenko,B.N.Cell-signallingdynamicsintimeandspace. Nat.Rev.Mol.Cell Biol. 7,165–176(2006).

15.Nusse,R.&Clevers,H.Wnt/beta-cateninsignaling,disease,andemerging therapeuticmodalities. Cell 169,985–999(2017).

16.Skoda,A.M.etal.TheroleoftheHedgehogsignalingpathwayincancer:a comprehensivereview. Bosn.J.BasicMed.Sci. 18,8–20(2018).

17.Zhou,B.etal.Notchsignalingpathway:architecture,disease,andtherapeutics. SignalTransduct.TargetTher. 7,95(2022).

18.Song,D.,Yang,D.,Powell,C.A.&Wang,X.Cell-cellcommunication:oldmystery andnewopportunity. CellBiol.Toxicol. 35,89–93(2019).

19.Tang,F.etal.mRNA-Seqwhole-transcriptomeanalysisofasinglecell. Nat. Methods 6,377–382(2009).

20.Li,X.etal.Single-cellRNAsequencingrevealsapro-invasivecancer-associated ﬁbroblastsubgroupassociatedwithpoorclinicaloutcomesinpatientswith gastriccancer. Theranostics 12,620(2022).

21.Belardi,B.,Son,S.,Felce,J.H.,Dustin,M.L.&Fletcher,D.A.Cell–cellinterfacesas specializedcompartmentsdirectingcellfunction. Nat.Rev.Mol.CellBiol. 21, 750–764(2020).

22.Darvin,P.,Toor,S.M.,SasidharanNair,V.&Elkord,E.Immunecheckpoint inhibitors:recentprogressandpotentialbiomarkers. Exp.Mol.Med. 50,1–11 (2018).

23.Raﬁq,S.,Hackett,C.S.&Brentjens,R.J.Engineeringstrategiestoovercomethe currentroadblocksinCARTcelltherapy. Nat.Rev.Clin.Oncol. 17,147–167(2020).

24.Bechtel,T.J.,Reyes-Robles,T.,Fadeyi,O.O.&Oslund,R.C.Strategiesfor monitoringcell-cellinteractions. Nat.Chem.Biol. 17,641–652(2021).

25.Arneson,D.etal.Singlecellmolecularalterationsrevealtargetcellsand pathwaysofconcussivebraininjury. Nat.Commun. 9,3894(2018).

26.Oh,E.-Y.etal.Extensiverewiringofepithelial-stromalco-expressionnetworksin breastcancer. GenomeBiol. 16,1–22(2015).

27.Han,X.etal.Mappingthemousecellatlasbymicrowell-seq. Cell 172, 1091–1107.e1017(2018).

28.Kramer,A.,Green,J.,Pollard,J.Jr.&Tugendreich,S.Causalanalysisapproaches inIngenuityPathwayAnalysis. Bioinformatics 30,523–530(2014).

29.Pires-daSilva,A.&Sommer,R.J.Theevolutionofsignallingpathwaysinanimal development. Nat.Rev.Genet. 4,39–49(2003).

30.Levi-Montalcini,R.&Hamburger,V.Selectivegrowthstimulatingeffectsof mousesarcomaonthesensoryandsympatheticnervoussystemofthechick embryo. J.Exp.Zool. 116,321–361(1951).

31.Kahn,C.R.,Neville,D.M.Jr&Roth,J.Insulin-receptorinteractionintheobesehyperglycemicmouse:amodelofinsulinresistance. J.Biol.Chem. 248,244–250 (1973).

32.Gromova,A.&LaSpada,A.R.Harmonylost:cell-cellcommunicationatthe neuromuscularjunctioninmotorneurondisease. TrendsNeurosci. 43,709–724 (2020).

33.Reyes-Ruiz,J.M.etal.Theregulationof ﬂavivirusinfectionbyhijacking exosome-mediatedcell-cellcommunication:newinsightsonvirus-hostinteractions. Viruses 12,765(2020).

34.Schwager,S.C.,Taufalele,P.V.&Reinhart-King,C.A.Cell-cellmechanical communicationincancer. CellMol.Bioeng. 12,1–14(2019).

35.Toda,S.,Frankel,N.W.&Lim,W.A.Engineeringcell-cellcommunicationnetworks:programmingmulticellularbehaviors. Curr.Opin.Chem.Biol. 52,31–38 (2019).

36.Moscona,A.&Moscona,H.Thedissociationandaggregationofcellsfromorgan rudimentsoftheearlychickembryo. J.Anat. 86,287(1952).

37.Steinberg,M.S.&Gilbert,S.F.TownesandHoltfreter(1955):directedmovementsandselectiveadhesionofembryonicamphibiancells. J.Exp.Zool.A Comp.Exp.Biol. 301,701–706(2004).

38.Prasher,D.C.,Eckenrode,V.K.,Ward,W.W.,Prendergast,F.G.&Cormier,M.J. Primarystructureofthe Aequoreavictoria green- ﬂuorescentprotein. Gene 111, 229–233(1992).

39.Chalﬁe,M.,Tu,Y.,Euskirchen,G.,Ward,W.W.&Prasher,D.C.Green ﬂuorescent proteinasamarkerforgeneexpression. Science 263,802–805(1994).

40.Betzig,E.&Trautman,J.K.Near-ﬁeldoptics:microscopy,spectroscopy,and surfacemodiﬁcationbeyondthediffractionlimit. Science 257,189–195(1992).

41.Willig,K.I.,Rizzoli,S.O.,Westphal,V.,Jahn,R.&Hell,S.W.STEDmicroscopy revealsthatsynaptotagminremainsclusteredaftersynapticvesicleexocytosis. Nature 440,935–939(2006).

42.Gustafsson,M.G.Surpassingthelateralresolutionlimitbyafactoroftwousing structuredilluminationmicroscopy. J.Microsc 198,82–87(2000).

43.Gustafsson,M.G.Nonlinearstructured-illuminationmicroscopy:wide-ﬁeld ﬂuorescenceimagingwiththeoreticallyunlimitedresolution. Proc.Natl.Acad. Sci.USA 102,13081–13086(2005).

44.Hofmann,M.,Eggeling,C.,Jakobs,S.&Hell,S.W.Breakingthediffractionbarrier in ﬂuorescencemicroscopyatlowlightintensitiesbyusingreversiblyphotoswitchableproteins. Proc.Natl.Acad.Sci.USA 102,17565–17569(2005).

45.Feinberg,E.H.etal.GFPReconstitutionAcrossSynapticPartners(GRASP) deﬁnescellcontactsandsynapsesinlivingnervoussystems. Neuron 57, 353–363(2008).

46.Macpherson,L.J.etal.Dynamiclabellingofneuralconnectionsinmultiple coloursbytrans-synaptic ﬂuorescencecomplementation. Nat.Commun. 6, 10024(2015).

47.Betzig,E.etal.Imagingintracellular ﬂuorescentproteinsatnanometerresolution. Science 313,1642–1645(2006).

48.Cloonan,N.&Grimmond,S.M.Transcriptomecontentanddynamicsatsinglenucleotideresolution. GenomeBiol. 9,234(2008).

49.Mardis,E.R.Theimpactofnext-generationsequencingtechnologyongenetics. TrendsGenet. 24,133–141(2008).

50.Schuster,S.C.Next-generationsequencingtransformstoday’sbiology. Nat. Methods 5,16–18(2008).

51.Wang,Z.,Gerstein,M.&Snyder,M.RNA-Seq:arevolutionarytoolfortranscriptomics. Nat.Rev.Genet. 10,57–63(2009).

52.Wold,B.&Myers,R.M.Sequencecensusmethodsforfunctionalgenomics. Nat. Methods 5,19–21(2008).

53.Cloonan,N.etal.Stemcelltranscriptomeproﬁlingviamassive-scalemRNA sequencing. Nat.Methods 5,613–619(2008).

54.Li,H.etal.Determinationoftagdensityrequiredfordigitaltranscriptome analysis:applicationtoanandrogen-sensitiveprostatecancermodel. Proc.Natl. Acad.Sci.USA 105,20179–20184(2008).

55.Marioni,J.C.,Mason,C.E.,Mane,S.M.,Stephens,M.&Gilad,Y.RNA-seq:an assessmentoftechnicalreproducibilityandcomparisonwithgeneexpression arrays. GenomeRes. 18,1509–1517(2008).

56.Mortazavi,A.,Williams,B.A.,McCue,K.,Schaeffer,L.&Wold,B.Mappingand quantifyingmammaliantranscriptomesbyRNA-Seq. Nat.Methods 5,621–628 (2008).

57.Pan,Q.,Shai,O.,Lee,L.J.,Frey,B.J.&Blencowe,B.J.Deepsurveyingof alternativesplicingcomplexityinthehumantranscriptomebyhigh-throughput sequencing. Nat.Genet. 40,1413–1415(2008).

58.Sultan,M.etal.Aglobalviewofgeneactivityandalternativesplicingbydeep sequencingofthehumantranscriptome. Science 321,956–960(2008).

59.Wang,E.T.etal.Alternativeisoformregulationinhumantissuetranscriptomes. Nature 456,470–476(2008).

60.Macosko,E.Z.etal.Highlyparallelgenome-wideexpressionproﬁlingofindividualcellsusingnanoliterdroplets. Cell 161,1202–1214(2015).

61.Klein,A.M.etal.Dropletbarcodingforsingle-celltranscriptomicsappliedto embryonicstemcells. Cell 161,1187–1201(2015).

62.Goltsev,Y.etal.DeepproﬁlingofmousesplenicarchitecturewithCODEX multiplexedimaging. Cell 174,968–981.e915(2018).

63.Merritt,C.R.etal.MultiplexdigitalspatialproﬁlingofproteinsandRNAin ﬁxed tissue. Nat.Biotechnol. 38,586–599(2020).

64.Chen,K.H.,Boettiger,A.N.,Mofﬁtt,J.R.,Wang,S.&Zhuang,X.RNAimaging. Spatiallyresolved,highlymultiplexedRNAproﬁlinginsinglecells. Science 348, aaa6090(2015).

65.Choi,J.etal.Spatialorganizationofthemouseretinaatsinglecellresolutionby MERFISH. Nat.Commun. 14,4929(2023).

66.Zhang,M.etal.Spatiallyresolvedcellatlasofthemouseprimarymotorcortex byMERFISH. Nature 598,137–143(2021).

67.Chu,X.etal.MultiplemicrovascularalterationsinpancreaticisletsandneuroendocrinetumorsofaMen1mousemodel. Am.J.Pathol. 182,2355–2367 (2013).

68.Wu,J.etal.Validationofmultipleximmunoﬂuorescenceanddigitalimage analysisforprogrammeddeath-ligand1expressionandimmunecellassessmentinnon-smallcelllungcancer:comparisonwithconventionalimmunohistochemistry. J.Clin.Pathol. 75,452–458(2022).

69.Uttam,S.etal.Spatialdomainanalysispredictsriskofcolorectalcancerrecurrenceandinfersassociatedtumormicroenvironmentnetworks. Nat.Commun. 11,3515(2020).

70.Kennedy-Darling,J.etal.Highlymultiplexedtissueimagingusingrepeated oligonucleotideexchangereaction. Eur.J.Immunol. 51,1262–1277(2021).

71.Zollinger,D.R.,Lingle,S.E.,Sorg,K.,Beechem,J.M.&Merritt,C.R.GeoMx™ RNA assay:highmultiplex,digital,spatialanalysisofRNAinFFPEtissue. MethodsMol. Biol. 2148,331–345(2020).

72.Adams,J.C.AJP-cellthemeon “cellsignaling:proteins,pathwaysand mechanisms” . Am.J.Physiol.CellPhysiol. 308,C197(2015).

73.Zeisberg,M.&Kalluri,R.Cellularmechanismsoftissue fibrosis.1.Commonand organ-specificmechanismsassociatedwithtissue fibrosis. Am.J.Physiol.Cell Physiol. 304,C216–C225(2013).

74.Drosten,M.,Lechuga,C.G.&Barbacid,M.GeneticanalysisofRasgenesin epidermaldevelopmentandtumorigenesis. SmallGTPases 4,236–241(2013).

75.Sun,C.etal.ReversibleandadaptiveresistancetoBRAF(V600E)inhibitionin melanoma. Nature 508,118–122(2014).

76.Masland,R.H.Neuronalcelltypes. Curr.Biol. 14,R497–R500(2004).

77.Hyman,S.E.Neurotransmitters. Curr.Biol. 15,R154–R158(2005).

78.O’Dell,T.J.,Hawkins,R.D.,Kandel,E.R.&Arancio,O.Testsoftherolesoftwo diffusiblesubstancesinlong-termpotentiation:evidencefornitricoxideasa possibleearlyretrogrademessenger. Proc.Natl.Acad.Sci.USA 88,11285–11289 (1991).

79.Stevens,C.F.&Wang,Y.Reversaloflong-termpotentiationbyinhibitorsof haemoxygenase. Nature 364,147–149(1993).

80.Hiller-Sturmhofel,S.&Bartke,A.Theendocrinesystem:anoverview. Alcohol HealthRes.World 22,153–164(1998).

81.Akdis,M.etal.Interleukins,from1to37,andinterferon-gamma:receptors, functions,androlesindiseases. J.AllergyClin.Immunol. 127,701–721(2011). e701-770.

82.O’Shea,J.J.&Murray,P.J.Cytokinesignalingmodulesininﬂammatory responses. Immunity 28,477–487(2008).

83.O’Shea,J.J.,Holland,S.M.&Staudt,L.M.JAKsandSTATsinimmunity, immunodeﬁciency,andcancer. NewEngl.J.Med. 368,161–170(2013).

84.Lovinger,D.M.Communicationnetworksinthebrain:neurons,receptors, neurotransmitters,andalcohol. AlcoholResHealth 31,196–214(2008).

85.Kimura,H.Hydrogensulﬁde:itsproduction,releaseandfunctions. AminoAcids 41,113–121(2011).

86.Starka,L.&Duskova,M.Whatisahormone? Physiol.Res. 69,S183–S185(2020).

87.Idriss,H.T.&Naismith,J.H.TNFalphaandtheTNFreceptorsuperfamily: structure-functionrelationship(s). MicroscRes.Tech. 50,184–195(2000).

88.Wang,X.&Zhao,X.Y.TranscriptionfactorsassociatedwithIL-15cytokinesignalingduringNKcelldevelopment. Front.Immunol. 12,610789(2021).

89.Goodman,L.S.&Gilman,A. ThePharmacologicalBasisofTherapeutics (The Macmillan,1955).

90.Kandel,E.R.etal. PrinciplesofNeuralScience.Vol.4(McGraw-HillNewYork, 2000).

91.Saxton,R.A.,Glassman,C.R.&Garcia,K.C.Emergingprinciplesofcytokine pharmacologyandtherapeutics. Nat.Rev.DrugDiscov. 22,21–37(2023).

92.Wang,X.,Lupardus,P.,Laporte,S.L.&Garcia,K.C.Structuralbiologyofshared cytokinereceptors. Annu.Rev.Immunol. 27,29–60(2009).

93.Wells,J.A.&deVos,A.M.Hematopoieticreceptorcomplexes. Annu.Rev.Biochem. 65,609–634(1996).

94.Rosenbaum,D.M.,Rasmussen,S.G.&Kobilka,B.K.Thestructureandfunction ofG-protein-coupledreceptors. Nature 459,356–363(2009).

95.Boulanger,M.J.&Garcia,K.C.Sharedcytokinesignalingreceptors:structural insightsfromthegp130system. Adv.ProteinChem. 68,107–146(2004).

96.Rall,T.W.&Sutherland,E.W.FormationofaCyclicAdenineRibonucleotideBy TissueParticles. J.Biol.Chem. 232,1065–1076(1958).

97.Gao,T.etal.cAMP-dependentregulationofcardiacL-typeCa2+ channels requiresmembranetargetingofPKAandphosphorylationofchannelsubunits. Neuron 19,185–196(1997).

98.Mikala,G.etal.cAMP-dependentphosphorylationsitesandmacroscopic activityofrecombinantcardiacL-typecalciumchannels. Mol.CellBiochem. 185, 95–109(1998).

99.Yan,X.etal.Adenylylcyclase/cAMP-PKA-mediatedphosphorylationofbasal L-typeCa2+ channelsinmouseembryonicventricularmyocytes. CellCalcium 50,433–443(2011).

100.Bahar,E.,Kim,H.&Yoon,H.ERStress-mediatedSignaling:ActionPotentialand Ca(2+)askeyplayers. Int.J.Mol.Sci. 17,1558(2016).

101.Horigane,S.-i,Ozawa,Y.,Yamada,H.&Takemoto-Kimura,S.Calciumsignalling:a keyregulatorofneuronalmigration. J.Biochem 165,401–409(2019).

102.Santulli,G.&Marks,A.R.Essentialrolesofintracellularcalciumreleasechannels inmuscle,brain,metabolism,andaging. Curr.Mol.Pharm. 8,206–222(2015).

103.Markevich,N.etal.SignalprocessingattheRascircuit:whatshapesRasactivationpatterns? Syst.Biol. 1,104–113(2004).

104.Wee,P.&Wang,Z.Epidermalgrowthfactorreceptorcellproliferationsignaling pathways. Cancers 9,52(2017).

105.Cuadrado,A.&Nebreda,A.R.Mechanismsandfunctionsofp38MAPKsignalling. BiochemJ. 429,403–417(2010).

106.Mann,G.,Riddell,M.C.&Adegoke,O.A.Effectsofacutemusclecontractionon thekeymoleculesininsulinandAktsignalinginskeletalmuscleinhealthandin insulinresistantstates. Diabetology 3,423–446(2022).

107.Merz,K.E.&Thurmond,D.C.Roleofskeletalmuscleininsulinresistanceand glucoseuptake. Compr.Physiol. 10,785–809(2011).

108.Sharma,M.&Dey,C.S.AKTISOFORMS-AS160-GLUT4:thedeﬁningaxisofinsulin resistance. Rev.Endocr.Metab.Disord. 22,973–986(2021).

109.Liu,T.-Y.etal.Irisininhibitshepaticgluconeogenesisandincreasesglycogen synthesisviathePI3K/Aktpathwayintype2diabeticmiceandhepatocytes. Clin.Sci. 129,839–850(2015).

110.Li,M.etal.AnessentialroleoftheNF-kappaB/Toll-likereceptorpathwayin inductionofinﬂammatoryandtissue-repairgeneexpressionbynecroticcells. J. Immunol. 166,7128–7135(2001).

111.Wu,Y.-d&Zhou,B.TNF-α/NF-κB/Snailpathwayincancercellmigrationand invasion. Br.J.Cancer 102,639–644(2010).

112.Gupta,M.K.,Mohan,M.L.&Prasad,S.V.N.Gprotein-coupledreceptor resensitizationparadigms. Int.Rev.CellMol.Biol. 339,63–91(2018).

113.Rajagopal,S.&Shenoy,S.K.GPCRdesensitization:acuteandprolongedphases. CellSignal 41,9–16(2018).

114.Cherry,J.A.&Pho,V.CharacterizationofcAMPdegradationbyphosphodiesterasesintheaccessoryolfactorysystem. Chem.Senses 27,643–652(2002).

115.Wahlang,B.,McClain,C.,Barve,S.&Gobejishvili,L.RoleofcAMPandphosphodiesterasesignalinginliverhealthanddisease. CellSignal 49,105–115 (2018).

116.Jeffrey,K.L.,Camps,M.,Rommel,C.&Mackay,C.R.Targetingdual-speciﬁcity phosphatases:manipulatingMAPkinasesignallingandimmuneresponses. Nat. Rev.DrugDiscov. 6,391–403(2007).

117.Fajardo,A.M.,Piazza,G.A.&Tinsley,H.N.Theroleofcyclicnucleotidesignaling pathwaysincancer:targetsforpreventionandtreatment. Cancers 6,436–458 (2014).

118.Finlay,D.B.,Duffull,S.B.&Glass,M.100yearsofmodellingligand-receptor bindingandresponse:afocusonGPCRs. Br.J.Pharm. 177,1472–1484(2020).

119.Hauser,A.S.,Attwood,M.M.,Rask-Andersen,M.,Schioth,H.B.&Gloriam,D.E. TrendsinGPCRdrugdiscovery:newagents,targetsandindications. Nat.Rev. DrugDiscov. 16,829–842(2017).

120.Vassart,G.&Costagliola,S.Gprotein-coupledreceptors:mutationsandendocrinediseases. Nat.Rev.Endocrinol. 7,362–372(2011).

121.Zhang,H.,Kong,Q.,Wang,J.,Jiang,Y.&Hua,H.ComplexrolesofcAMP-PKACREBsignalingincancer. Exp.Hematol.Oncol. 9,32(2020).

122.Berridge,M.J.Inositoltrisphosphateandcalciumsignallingmechanisms. BiochimBiophys.Acta 1793,933–940(2009).

123.Berridge,M.J.Neuronalcalciumsignaling. Neuron 21,13–26(1998).

124.Greengard,P.Calcium/calmodulin-dependentproteinkinaseIIincreasesglutamateandnoradrenalinereleasefromsynaptosomes. Nature 343,647–651 (1990).

125.Suizu,F.etal.CharacterizationofCa2+/calmodulin-dependentproteinkinaseI asamyosinIIregulatorylightchainkinaseinvitroandinvivo. BiochemJ. 367, 335–345(2002).

126.Hubbard,S.R.Juxtamembraneautoinhibitioninreceptortyrosinekinases. Nat. Rev.Mol.CellBiol. 5,464–471(2004).

127.Hubbard,S.R.&Miller,W.T.Receptortyrosinekinases:mechanismsofactivationandsignaling. Curr.Opin.CellBiol. 19,117–123(2007).

128.Schlessinger,J.Cellsignalingbyreceptortyrosinekinases. Cell 103,211–225 (2000).

129.Gammeltoft,S.Molecularstructureofreceptortyrosinekinases.In Textbookof ReceptorPharmacology ,(edsbyForeman,J.C.&Johansen,T.)131(CRCPress, 2003).

130.Hess-Stumpp,H.,Haberey,M.&Thierauch,K.H.PTK787/ZK222584,atyrosine kinaseinhibitorofallknownVEGFreceptors,repressestumorgrowthwithhigh efﬁcacy. Chembiochem 6,550–557(2005).

131.Katz,M.,Amit,I.&Yarden,Y.RegulationofMAPKsbygrowthfactorsand receptortyrosinekinases. Biochim.Biophys.Acta 1773,1161–1176(2007).

132.Gravandi,M.M.etal.TherapeutictargetingofRas/Raf/MAPKpathwaybynatural products:asystematicandmechanisticapproachforneurodegeneration. Phytomedicine 115,154821(2023).

133.SudheshDev,S.,ZainalAbidin,S.A.,Farghadani,R.,Othman,I.&Naidu,R. Receptortyrosinekinasesandtheirsignalingpathwaysastherapeutictargetsof curcuminincancer. Front.Pharm. 12,772510(2021).

134.Cuesta,C.,Arévalo-Alameda,C.&Castellano,E.TheimportanceofbeingPI3Kin theRASsignalingnetwork. Genes 12,1094(2021).

135.He,Y.etal.TargetingPI3K/Aktsignaltransductionforcancertherapy. Signal Transduct.TargetTher. 6,425(2021).

136.Guo,D.etal.ARac-cGMPsignalingpathway. Cell 128,341–355(2007).

137.Hofmann,F.ThecGMPsystem:componentsandfunction. Biol.Chem. 401, 447–469(2020).

138.Rybalkin,S.D.,Yan,C.,Bornfeldt,K.E.&Beavo,J.A.CyclicGMPphosphodiesterasesandregulationofsmoothmusclefunction. Circ.Res. 93,280–291(2003).

139.Ignarro,L.J.,Buga,G.M.,Wood,K.S.,Byrns,R.E.&Chaudhuri,G.Endotheliumderivedrelaxingfactorproducedandreleasedfromarteryandveinisnitric oxide. Proc.Natl.Acad.Sci.USA 84,9265–9269(1987).

140.Liu,T.,Schroeder,H.,Power,G.G.&Blood,A.B.Aphysiologicallyrelevantrole forNOstoredinvascularsmoothmusclecells:AnoveltheoryofvascularNO signaling. RedoxBiol. 53,102327(2022).

141.Rawlings,J.S.,Rosler,K.M.&Harrison,D.A.TheJAK/STATsignalingpathway. J. CellSci. 117,1281–1283(2004).

142.Roberts,A.B.TGF-β signalingfromreceptorstothenucleus. MicrobesInfect. 1, 1265–1273(1999).

143.Wrana,J.L.TGF-β receptorsandsignallingmechanisms. Min.ElectrolyteMetab. 24,120–130(1998).

144.Dontu,G.etal.RoleofNotchsignalingincell-fatedeterminationofhuman mammarystem/progenitorcells. BreastCancerRes. 6,1–11(2004).

145.Liu,J.,Sato,C.,Cerletti,M.&Wagers,A.Notchsignalingintheregulationofstem cellself-renewalanddifferentiation. Curr.Top.Dev.Biol. 92,367–409(2010).

146.Sanz-Ezquerro,J.J.,Münsterberg,A.E.&Stricker,S.Editorial:signalingpathways inembryonicdevelopment. Front.CellDev.Biol. 5,76(2017).

147.Kopan,R.&Ilagan,M.X.ThecanonicalNotchsignalingpathway:unfoldingthe activationmechanism. Cell 137,216–233(2009).

148.Bray,S.J.Notchsignalling:asimplepathwaybecomescomplex. Nat.Rev.Mol. CellBiol. 7,678–689(2006).

149.Kovall,R.A.StructuresofCSL,NotchandMastermindproteins:piecingtogether anactivetranscriptioncomplex. Curr.Opin.Struct.Biol. 17,117–127(2007).

150.Sigafoos,A.N.,Paradise,B.D.&Fernandez-Zapico,M.E.Hedgehog/GLIsignaling pathway:transduction,regulation,andimplicationsfordisease. Cancers 13, 3410(2021).

151.Liu,M.,Su,Y.,Peng,J.&Zhu,A.J.Proteinmodiﬁcationsinhedgehogsignaling: crosstalkandfeedbackregulationconferdivergenthedgehogsignaling activity. Bioessays 43,2100153(2021).

152.Rijsewijk,F.etal.TheDrosophilahomologyofthemousemammaryoncogene int-1isidenticaltothesegmentpolaritygenewingless. Cell 50,649–657(1987).

153.Croce,J.C.&McClay,D.R.EvolutionoftheWntpathways. MethodsMol.Biol. 469,3–18(2008).

154.Hayden,M.,West,A.&Ghosh,S.NF-κBandtheimmuneresponse. Oncogene 25, 6758–6780(2006).

155.Piva,R.,Belardo,G.&Santoro,M.G.NF-κB:astress-regulatedswitchforcell survival. Antioxid.RedoxSignal 8,478–486(2006).

156.Wang,S.,Liu,Z.,Wang,L.&Zhang,X.NF-κBsignalingpathway,inﬂammation andcolorectalcancer. CellMol.Immunol. 6,327–334(2009).

157.Moynagh,P.N.TheNF-kappaBpathway. J.CellSci. 118,4589–4592(2005).

158.Karin,M.,Cao,Y.,Greten,F.R.&Li,Z.-W.NF-κBincancer:frominnocent bystandertomajorculprit. Nat.Rev.Cancer 2,301–310(2002).

159.Yamamoto,Y.&Gaynor,R.B.TherapeuticpotentialofinhibitionoftheNF-κB pathwayinthetreatmentofinﬂammationandcancer. J.Clin.Investig. 107, 135–142(2001).

160.Burne,M.J.etal.IL-1andTNFindependentpathwaysmediateICAM-1/VCAM-1 up-regulationinischemiareperfusioninjury. J.Leukoc.Biol. 70,192–198(2001).

161.Mantovani,A.,Bussolino,F.&Introna,M.Cytokineregulationofendothelialcell function:frommolecularleveltothebedside. Immunol.Today 18,231–240 (1997).

162.Zhang,Y.,Liu,H.,Tang,W.,Qiu,Q.&Peng,J.ResveratrolpreventsTNF-alphainducedVCAM-1andICAM-1upregulationinendothelialprogenitorcellsvia reductionofNF-kappaBactivation. J.Int.Med.Res. 48,300060520945131(2020).

163.Panés,J.,Perry,M.&Granger,D.N.Leukocyte-endothelialcelladhesion:avenuesfortherapeuticintervention. Br.J.Pharm. 126,537(1999).

164.Singh,V.,Kaur,R.,Kumari,P.,Pasricha,C.&Singh,R.ICAM-1andVCAM-1: gatekeepersinvariousinﬂammatoryandcardiovasculardisorders. Clin.Chim. Acta 548,117487(2023).

165.Ulbrich,H.,Eriksson,E.E.&Lindbom,L.Leukocyteandendothelialcelladhesion moleculesastargetsfortherapeuticinterventionsininﬂammatorydisease. TrendsPharm.Sci. 24,640–647(2003).

166.Bohm,M.Catecholaminerefractorinessandtheirmechanismsincardiocirculatoryshockandchronicheartfailure. Thorac.CardiovascSurg. 46,270–275(1998).

167.Jiang,X.etal.AnovelEST-derivedRNAiscreenrevealsacriticalroleforfarnesyl diphosphatesynthaseinbeta2-adrenergicreceptorinternalizationanddownregulation. FASEBJ. 26,1995–2007(2012).

168.Kim,D.,Cho,S.,Woo,J.A.&Liggett,S.B.ACREB-mediatedincreaseinmiRNA let-7fduringprolongedbeta-agonistexposure:anovelmechanismofbeta(2)adrenergicreceptordown-regulationinairwaysmoothmuscle. FASEBJ. 32, 3680–3688(2018).

169.Kim,K.&Chung,K.Y.ManyfacesoftheGPCR-arrestininteraction. Arch.Pharm. Res. 43,890–899(2020).

170.Lefkowitz,R.J.Gprotein-coupledreceptors.III.Newrolesforreceptorkinases andbeta-arrestinsinreceptorsignalinganddesensitization. J.Biol.Chem. 273, 18677–18680(1998).

171.Pierce,K.L.&Lefkowitz,R.J.Classicalandnewrolesofbeta-arrestinsinthe regulationofG-protein-coupledreceptors. Nat.Rev.Neurosci. 2,727–733(2001).

172.Barisione,G.,Barofﬁo,M.,Crimi,E.&Brusasco,V.Beta-adrenergicagonists. Pharmaceuticals 3,1016–1044(2010).

173.Tomás,E.etal.Hyperglycemiaandinsulinresistance:possiblemechanisms. Ann. N.YAcad.Sci. 967,43–51(2002).

174.Costin,B.N.&Miles,M.F.Molecularandneurologicresponsestochronic alcoholuse. Handb.Clin.Neurol. 125,157–171(2014).

175.Hillbom,M.,Pieninkeroinen,I.&Leone,M.Seizuresinalcohol-dependent patients:epidemiology,pathophysiologyandmanagement. CNSDrugs 17, 1013–1030(2003).

176.Imam,I.Alcoholandthecentralnervoussystem. Br.J.Hosp.Med. 71,635–639 (2010).

177.Wu,J.,Tang,H.,Chen,S.&Cao,L.Mechanismsandpharmacotherapyfor ethanol-responsivemovementdisorders. Front.Neurol. 11,892(2020).

178.Barnett-Norris,J.,Lynch,D.&Reggio,P.H.Lipids,lipidraftsandcaveolae:their importanceforGPCRsignalingandtheircentralitytotheendocannabinoid system. LifeSci. 77,1625–1639(2005).

179.Chini,B.&Parenti,M.G-proteincoupledreceptorsinlipidraftsandcaveolae: how,whenandwhydotheygothere? J.Mol.Endocrinol. 32,325–338(2004).

180.Chiu,Y.-H.,Zhao,M.&Chen,Z.J.UbiquitininNF-κBsignaling. Chem.Rev. 109, 1549–1560(2009).

181.Walther,D.J.etal.Synthesisofserotoninbyasecondtryptophanhydroxylase isoform. Science 299,76–76(2003).

182.Koritnik,B., Črne-Finderle,N.&Sketelj,J.Acetylcholinesteraseintheneuromuscularjunction. Chem.Biol.Interact. 119,301–308(1999).

183.Chowdhury,R.,Chowdhury,S.,Roychoudhury,P.,Mandal,C.&Chaudhuri,K. Arsenicinducedapoptosisinmalignantmelanomacellsisenhancedby menadionethroughROSgeneration,p38signalingandp53activation. Apoptosis 14,108–123(2009).

184.Liu,P.,Ma,G.,Wang,Y.,Wang,L.&Li,P.Therapeuticeffectsoftraditional Chinesemedicineongoutynephropathy:BasedonNF-κBsignalingpathways. Biomed.Pharmacother. 158,114199(2023).

185.Ebied,A.M.,Elmariah,H.&Cooper-DeHoff,R.M.Newdrugsapprovedin2021. Am.J.Med. 135,836–839(2022).

186.Ebied,A.M.,Elmariah,H.&Cooper-DeHoff,R.M.Newdrugsapprovedin2022. Am.J.Med. 136,545–550(2023).

187.Sweet,B.V.,Schwemm,A.K.&Parsons,D.M.ReviewoftheprocessesforFDA oversightofdrugs,medicaldevices,andcombinationproducts. J.ManagCare Pharm. 17,40–50(2011).

188.Wang,Y.-T.,Yang,P.-C.,Zhang,Y.-F.&Sun,J.-F.Synthesisandclinicalapplication ofnewdrugsapprovedbyFDAin2023. Eur.J.Med.Chem. 265,116124(2024).

189.Tewari,D.,Patni,P.,Bishayee,A.,Sah,A.N.&Bishayee,A.Naturalproducts targetingthePI3K-Akt-mTORsignalingpathwayincancer:anoveltherapeutic strategy. SeminCancerBiol. 80,1–17(2022).

190.Asati,V.,Mahapatra,D.K.&Bharti,S.K.PI3K/Akt/mTORandRas/Raf/MEK/ERK signalingpathwaysinhibitorsasanticanceragents:Structuralandpharmacologicalperspectives. Eur.J.Med.Chem. 109,314–341(2016).

191.King,D.,Yeomanson,D.&Bryant,H.E.PI3Kingthelock:targetingthePI3K/Akt/ mTORpathwayasanoveltherapeuticstrategyinneuroblastoma. J.Pediatr. Hematol.Oncol. 37,245–251(2015).

192.Braun,T.P.,Eide,C.A.&Druker,B.J.ResponseandresistancetoBCR-ABL1targetedtherapies. CancerCell 37,530–542(2020).

193.Hua,H.etal.TargetingmTORforcancertherapy. J.Hematol.Oncol. 12,71 (2019).

194.Lee,Y.T.,Tan,Y.J.&Oon,C.E.Moleculartargetedtherapy:treatingcancerwith speciﬁcity. Eur.J.Pharm. 834,188–196(2018).

195.Lim,Z.-F.&Ma,P.C.Emerginginsightsoftumorheterogeneityanddrug resistancemechanismsinlungcancertargetedtherapy. J.Hematol.Oncol. 12, 134(2019).

196.Lui,G.Y.L.,Grandori,C.&Kemp,C.J.CDK12:anemergingtherapeutictargetfor cancer. J.Clin.Pathol. 71,957–962(2018).

197.Qin,S.etal.Recentadvancesonanti-angiogenesisreceptortyrosinekinase inhibitorsincancertherapy. J.Hematol.Oncol. 12,27(2019).

198.Chen,M.etal.CRISPR-Cas9forcancertherapy:opportunitiesandchallenges. CancerLett. 447,48–55(2019).

199.Wang,S.W.etal.CurrentapplicationsandfutureperspectiveofCRISPR/Cas9 geneeditingincancer. Mol.Cancer 21,57(2022).

200.Sever,R.&Brugge,J.S.Signaltransductionincancer. ColdSpringHarb.Perspect. Med. 5,a006098(2015).

201.Hanahan,D.&Coussens,L.M.Accessoriestothecrime:functionsofcells recruitedtothetumormicroenvironment. CancerCell 21,309–322(2012).

202.Calorini,L.&Bianchini,F.Environmentalcontrolofinvasivenessandmetastatic disseminationoftumorcells:theroleoftumorcell-hostcellinteractions. Cell Commun.Signal 8,1–10(2010).

203.Calvo,F.&Sahai,E.Cellcommunicationnetworksincancerinvasion. Curr.Opin. CellBiol. 23,621–629(2011).

204.Chung,A.S.,Lee,J.&Ferrara,N.Targetingthetumourvasculature:insightsfrom physiologicalangiogenesis. Nat.Rev.Cancer 10,505–514(2010).

205.Chen,Z.etal.Single-cellRNAsequencinghighlightstheroleofinﬂammatory cancer-associated ﬁbroblastsinbladderurothelialcarcinoma. Nat.Commun. 11, 5077(2020).

206.Li,Y.etal.Single-celllandscaperevealsactivecellsubtypesandtheirinteraction inthetumormicroenvironmentofgastriccancer. Theranostics 12,3818–3833 (2022).

207.Kaminska,K.etal.Theroleofthecell-cellinteractionsincancerprogression. J. CellMol.Med. 19,283–296(2015).

208.Crawford,Y.etal.PDGF-Cmediatestheangiogenicandtumorigenicproperties of ﬁbroblastsassociatedwithtumorsrefractorytoanti-VEGFtreatment. Cancer Cell 15,21–34(2009).

209.Koshida,Y.,Kuranami,M.&Watanabe,M.Interactionbetweenstromal ﬁbroblastsandcolorectalcancercellsintheexpressionofvascularendothelial growthfactor. J.Surg.Res. 134,270–277(2006).

210.Cristofanilli,M.,Charnsangavej,C.&Hortobagyi,G.N.Angiogenesismodulation incancerresearch:novelclinicalapproaches. Nat.Rev.DrugDiscov. 1,415–426 (2002).

211.Buczek,M.,Escudier,B.,Bartnik,E.,Szczylik,C.&Czarnecka,A.Resistanceto tyrosinekinaseinhibitorsinclearcellrenalcellcarcinoma:fromthepatient’s bedtomolecularmechanisms. BiochimBiophys.Acta 1845,31–41(2014).

212.Li,X.etal.RevascularizationofischemictissuesbyPDGF-CCviaeffectson endothelialcellsandtheirprogenitors. J.Clin.Investig. 115,118–127(2005).

213.Fearon,E.R.&Vogelstein,B.Ageneticmodelforcolorectaltumorigenesis. Cell 61,759–767(1990).

214.Sjoblom,T.etal.Theconsensuscodingsequencesofhumanbreastandcolorectalcancers. Science 314,268–274(2006).

215.Laplante,M.&Sabatini,D.M.mTORSignaling. ColdSpringHarb.Perspect.Biol. 4, a011593(2012).

216.Yang,L.,Li,A.,Wang,Y.&Zhang,Y.Intratumoralmicrobiota:rolesincancer initiation,developmentandtherapeuticefﬁcacy. SignalTransduct.TargetTher. 8, 35(2023).

217.Nejman,D.etal.Thehumantumormicrobiomeiscomposedoftumor type–speciﬁcintracellularbacteria. Science 368,973–980(2020).

218.Siegel,R.L.,Miller,K.D.&Jemal,A.Cancerstatistics,2020. CACancerJ.Clin. 70, 7–30(2020).

219.Jiang,Y.,Chen,M.,Nie,H.&Yuan,Y.PD-1andPD-L1incancerimmunotherapy: clinicalimplicationsandfutureconsiderations. Hum.VaccinImmunother. 15, 1111–1122(2019).

220.Seliger,B.BasisofPD1/PD-L1therapies. J.Clin.Med. 8,2168(2019).

221.Dantoing,E.,Piton,N.,Salaun,M.,Thiberville,L.&Guisier,F.Anti-PD1/PD-L1 Immunotherapyfornon-smallcelllungcancerwithactionableoncogenicdriver mutations. Int.J.Mol.Sci. 22,6288(2021).

222.Hosseini,A.,Gharibi,T.,Maroﬁ,F.,Babaloo,Z.&Baradaran,B.CTLA-4:from mechanismtoautoimmunetherapy. Int.Immunopharmacol. 80,106221(2020).

223.Hossen,M.M.etal.CurrentunderstandingofCTLA-4:frommechanismto autoimmunediseases. Front.Immunol. 14,1198365(2023).

224.Lingel,H.&Brunner-Weinzierl,M.C.CTLA-4(CD152):aversatilereceptorfor immune-basedtherapy. Semin.Immunol. 42,101298(2019).

225.Rowshanravan,B.,Halliday,N.&Sansom,D.M.CTLA-4:amovingtargetin immunotherapy. Blood 131,58–67(2018).

226.Li,Y.etal.IGSF8isaninnateimmunecheckpointandcancerimmunotherapy target. Cell S0092-8674,00355–00356(2024).

227.Clarke,M.F.Clinicalandtherapeuticimplicationsofcancerstemcells. NewEngl. J.Med. 380,2237–2245(2019).

228.Luo,Y.T.etal.Theviablecirculatingtumorcellswithcancerstemcellsfeature, whereisthewayout? J.Exp.Clin.CancerRes. 37,38(2018).

229.Visvader,J.E.&Lindeman,G.J.Cancerstemcellsinsolidtumours:accumulating evidenceandunresolvedquestions. Nat.Rev.Cancer 8,755–768(2008).

230.Hardavella,G.,George,R.&Sethi,T.Lungcancerstemcells-characteristics, phenotype. Transl.LungCancerRes. 5,272–279(2016).

231.Lapidot,T.etal.AcellinitiatinghumanacutemyeloidleukaemiaaftertransplantationintoSCIDmice. Nature 367,645–648(1994).

232.Bonnet,D.&Dick,J.E.Humanacutemyeloidleukemiaisorganizedasahierarchythatoriginatesfromaprimitivehematopoieticcell. Nat.Med. 3,730–737 (1997).

233.Das,P.K.etal.Plasticityofcancerstemcell:originandroleindiseaseprogressionandtherapyresistance. StemCellRev.Rep. 16,397–412(2020).

234.Lin,L.etal.STAT3isnecessaryforproliferationandsurvivalincoloncancerinitiatingcells. CancerRes. 71,7226–7237(2011).

235.Merchant,A.A.&Matsui,W.TargetingHedgehog-acancerstemcellpathway. Clin.CancerRes. 16,3130–3140(2010).

236.Zhou,Y.etal.Wntsignalingpathwayincancerimmunotherapy. CancerLett. 525,84–96(2022).

237.Jiang,B.H.&Liu,L.Z.PI3K/PTENsignalinginangiogenesisandtumorigenesis. Adv.CancerRes. 102,19–65(2009).

238.Kroon,P.etal.JAK-STATblockadeinhibitstumorinitiationandclonogenic recoveryofprostatecancerstem-likecells. CancerRes. 73,5288–5298(2013).

239.Liu,M.etal.ThecanonicalNF-kappaBpathwaygovernsmammarytumorigenesisintransgenicmiceandtumorstemcellexpansion. CancerRes. 70, 10464–10473(2010).

240.Chen,K.,Huang,Y.H.&Chen,J.L.Understandingandtargetingcancerstemcells: therapeuticimplicationsandchallenges. ActaPharm.Sin. 34,732–740(2013).

241.Duran‐Corbera,A.etal.APhotoswitchableligandtargetingthe β1‐adrenoceptorenableslight‐controlofthecardiacrhythm. Angew.Chem.Int.Ed.Engl. 61,e202203449(2022).

242.Song,Y.etal.Heterodimerizationwith5-HT2BRisindispensablefor β2ARmediatedcardioprotection. Circ.Res. 128,262–277(2021).

243.Parinandi,N.L.&Hund,T.J. CardiovascularSignalinginHealthandDisease [Internet] .(Cham(CH):Springer;2022).

244.Bers,D.M.CaMKIIinhibitioninheartfailuremakesjumptohuman. Circ.Res. 107,1044–1046(2010).

245.Sossalla,S.etal.InhibitionofelevatedCa2+/calmodulin-dependentprotein kinaseIIimprovescontractilityinhumanfailingmyocardium. Circ.Res 107, 1150–1161(2010).

246.Anderson,M.E.,Brown,J.H.&Bers,D.M.CaMKIIinmyocardialhypertrophyand heartfailure. J.Mol.CellCardiol. 51,468–473(2011).

247.Lehman,S.J.etal.Chroniccalmodulin-kinaseIIactivationdrivesdiseaseprogressioninmutation-speciﬁchypertrophiccardiomyopathy. Circulation 139, 1517–1529(2019).

248.Ljubojevic-Holzer,S.etal.CaMKIIdeltaCdrivesearlyadaptiveCa(2+)change andlateeccentriccardiachypertrophy. Circ.Res. 127,1159–1178(2020).

249.Voigt,N.etal.EnhancedsarcoplasmicreticulumCa2+ leakandincreasedNa +-Ca2+ exchangerfunctionunderliedelayedafterdepolarizationsinpatients withchronicatrial ﬁbrillation. Circulation 125,2059–2070(2012).

250.Wagner,S.,Maier,L.S.&Bers,D.M.Roleofsodiumandcalciumdysregulationin tachyarrhythmiasinsuddencardiacdeath. Circ.Res. 116,1956–1970(2015).

251.Ling,H.etal.Ca2+/Calmodulin-dependentproteinkinaseIIdeltamediates myocardialischemia/reperfusioninjurythroughnuclearfactor-kappaB. Circ.Res. 112,935–944(2013).

252.Weinreuter,M.etal.CaMKinaseIImediatesmaladaptivepost-infarctremodelingandpro-inﬂammatorychemoattractantsignalingbutnotacutemyocardialischemia/reperfusioninjury. EMBOMol.Med. 6,1231–1245(2014).

253.Erickson,J.R.etal.DiabetichyperglycaemiaactivatesCaMKIIandarrhythmias byO-linkedglycosylation. Nature 502,372–376(2013).

254.Hegyi,B.etal.CaMKIIserine280O-GlcNAcylationlinksdiabetichyperglycemia toproarrhythmia. Circ.Res. 129,98–113(2021).

255.Lu,S.etal.Hyperglycemiaacutelyincreasescytosolicreactiveoxygenspecies viaO-linkedGlcNAcylationandCaMKIIactivationinmouseventricularmyocytes. Circ.Res. 126,e80–e96(2020).

256.Mesubi,O.O.etal.OxidizedCaMKIIandO-GlcNAcylationcauseincreasedatrial ﬁbrillationindiabeticmicebydistinctmechanisms. J.Clin.Investig. 131,e95747 (2021).

257.Sun,J.,Steenbergen,C.&Murphy,E.S-nitrosylation:NO-relatedredoxsignaling toprotectagainstoxidativestress. Antioxid.RedoxSignal 8,1693–1705(2006).

258.Falk,E.,Shah,P.K.&Fuster,V.Coronaryplaquedisruption. Circulation 92, 657–671(1995).

259.Libby,P.,Ridker,P.M.&Hansson,G.K.Progressandchallengesintranslating thebiologyofatherosclerosis. Nature 473,317–325(2011).

260.Lusis,A.J.Atherosclerosis. Nature 407,233–241(2000).

261.Geovanini,G.R.&Libby,P.Atherosclerosisandinﬂammation:overviewand updates. Clin.Sci. 132,1243–1252(2018).

262.Welsh,P.,Grassia,G.,Botha,S.,Sattar,N.&Mafﬁa,P.Targetinginﬂammationto reducecardiovasculardiseaserisk:arealisticclinicalprospect? Br.J.Pharm. 174, 3898–3913(2017).

263.Libby,P.,Ridker,P.M.&Maseri,A.Inﬂammationandatherosclerosis. Circulation 105,1135–1143(2002).

264.Dai,G.etal.Distinctendothelialphenotypesevokedbyarterialwaveforms derivedfromatherosclerosis-susceptibleand-resistantregionsofhumanvasculature. Proc.Natl.Acad.Sci.USA 101,14871–14876(2004).

265.Owens,G.K.,Kumar,M.S.&Wamhoff,B.R.Molecularregulationofvascular smoothmusclecelldifferentiationindevelopmentanddisease. Physiol.Rev. 84, 767–801(2004).

266.Mozos,I.etal.Inﬂammatorymarkersforarterialstiffnessincardiovasculardiseases. Front.Immunol. 8,1058(2017).

267.Ouimet,M.,Barrett,T.J.&Fisher,E.A.HDLandreversecholesteroltransport: basicmechanismsandtheirrolesinvascularhealthanddisease. Circ.Res. 124, 1505–1518(2019).

268.Schwartz,G.G.etal.Effectsofdalcetrapibinpatientswitharecentacute coronarysyndrome. NewEngl.J.Med. 367,2089–2099(2012).

269.Update,A.S.Heartdiseaseandstrokestatistics–2017update. Circulation 135, e146–e603(2017).

270.Bailey,A.&Mohiuddin,S.S.Biochemistry,highdensitylipoprotein.In: StatPearls [Internet] .TreasureIsland(FL):StatPearlsPublishing;(2024).

271.Bobryshev,Y.V.,Killingsworth,M.C.&Orekhov,A.N.Increasedsheddingof microvesiclesfromintimalsmoothmusclecellsinathero-proneareasofthe humanaorta:implicationsforunderstandingoftheprediseasestage. Pathobiology 80,24–31(2012).

272.Leroyer,A.S.etal.Cellularoriginsandthrombogenicactivityofmicroparticles isolatedfromhumanatheroscleroticplaques. J.Am.Coll.Cardiol. 49,772–777 (2007).

273.Perrotta,I.&Aquila,S.Exosomesinhumanatherosclerosis:anultrastructural analysisstudy. Ultrastruct.Pathol. 40,101–106(2016).

274.Niu,C.etal.Macrophagefoamcell–derivedextracellularvesiclespromote vascularsmoothmusclecellmigrationandadhesion. J.Am.HeartAssoc. 5, e004099(2016).

275.Nguyen,M.-A.etal.Extracellularvesiclessecretedbyatherogenicmacrophages transfermicroRNAtoinhibitcellmigration. ArteriosclerThromb.Vasc.Biol. 38, 49–63(2018).

276.Zheng,B.etal.Exosome-mediatedmiR-155transferfromsmoothmusclecells toendothelialcellsinducesendothelialinjuryandpromotesatherosclerosis. Mol.Ther. 25,1279–1294(2017).

277.Hergenreider,E.etal.Atheroprotectivecommunicationbetweenendothelial cellsandsmoothmusclecellsthroughmiRNAs. Nat.CellBiol. 14,249–256 (2012).

278.Givvimani,S.Synergismbetweenarrhythmiaandhyperhomo-cysteinemiain structuralheartdisease. Int.J.Physiol.Pathophysiol.Pharm. 3,107–119(2011).

279.Leuschner,F.&Nahrendorf,M.Novelfunctionsofmacrophagesintheheart: insightsintoelectricalconduction,stress,anddiastolicdysfunction. Eur.HeartJ. 41,989–994(2020).

280.Narmoneva,D.A.,Vukmirovic,R.,Davis,M.E.,Kamm,R.D.&Lee,R.T.Endothelialcellspromotecardiacmyocytesurvivalandspatialreorganization: implicationsforcardiacregeneration. Circulation 110,962–968(2004).

281.Zhou,X.,Smith,Q.R.&Liu,X.Brainpenetratingpeptidesandpeptide–drug conjugatestoovercometheblood–brainbarrierandtargetCNSdiseases. Wiley Interdiscip.Rev.Nanomed.Nanobiotechnol. 13,e1695(2021).

282.Verkhratsky,A.&Nedergaard,M.Physiologyofastroglia. Physiol.Rev. 98, 239–389(2018).

283.Campanella,C.etal.Heatshockprotein60levelsintissueandcirculating exosomesinhumanlargebowelcancerbeforeandafterablativesurgery. Cancer 121,3230–3239(2015).

284.Campanella,C.etal.HeatshockproteinsinAlzheimer’sdisease:roleandtargeting. Int.J.Mol.Sci. 19,2603(2018).

285.MarinoGammazza,A.etal.Alzheimer’sdiseaseandmolecularchaperones: currentknowledgeandthefutureofchaperonotherapy. Curr.Pharm.Des. 22, 4040–4049(2016).

286.Quek,C.&Hill,A.F.Theroleofextracellularvesiclesinneurodegenerative diseases. BiochemBiophys.Res.Commun. 483,1178–1186(2017).

287.Korkut,C.etal.Regulationofpostsynapticretrogradesignalingbypresynaptic exosomerelease. Neuron 77,1039–1046(2013).

288.Gross,J.C.,Chaudhary,V.,Bartscherer,K.&Boutros,M.ActiveWntproteinsare secretedonexosomes. Nat.CellBiol. 14,1036–1045(2012).

289.Sheldon,H.etal.NewmechanismforNotchsignalingtoendotheliumata distancebyDelta-like4incorporationintoexosomes. Blood 116,2385–2394 (2010).

290.Antonucci,F.etal.Microvesiclesreleasedfrommicrogliastimulatesynaptic activityviaenhancedsphingolipidmetabolism. EMBOJ. 31,1231–1240(2012).

291.Frühbeis,C.etal.Neurotransmitter-triggeredtransferofexosomesmediates oligodendrocyte–neuroncommunication. PLoSBiol. 11,e1001604(2013).

292.Wang,S.etal.SynapsinIisanoligomannose-carryingglycoprotein,actsasan oligomannose-bindinglectin,andpromotesneuriteoutgrowthandneuronal survivalwhenreleasedviaglia-derivedexosomes. J.Neurosci. 31,7275–7290 (2011).

293.Arantes,R.M.&Andrews,N.W.AroleforsynaptotagminVII-regulatedexocytosisoflysosomesinneuriteoutgrowthfromprimarysympatheticneurons. J. Neurosci. 26,4630–4637(2006).

294.Ghidoni,R.etal.CystatinCisreleasedinassociationwithexosomes:anewtool ofneuronalcommunicationwhichisunbalancedinAlzheimer’sdisease. Neurobiol.Aging 32,1435–1442(2011).

295.Putz,U.etal.Nedd4family-interactingprotein1(Ndﬁp1)isrequiredforthe exosomalsecretionofNedd4familyproteins. J.Biol.Chem. 283,32621–32627 (2008).

296.Bakhti,M.,Winter,C.&Simons,M.Inhibitionofmyelinmembranesheathformationbyoligodendrocyte-derivedexosome-likevesicles. J.Biol.Chem. 286, 787–796(2011).

297.Fauré,J.etal.Exosomesarereleasedbyculturedcorticalneurones. Mol.Cell Neurosci. 31,642–648(2006).

298.Marzesco,A.-M.etal.Releaseofextracellularmembraneparticlescarryingthe stemcellmarkerprominin-1(CD133)fromneuralprogenitorsandotherepithelialcells. J.CellSci. 118,2849–2858(2005).

299.Porro,C.,Trotta,T.&Panaro,M.A.Microvesiclesinthebrain:Biomarker,messengerormediator? J.Neuroimmunol. 288,70–78(2015).

300.CarusoBavisotto,C.etal.Extracellularvesicle-mediatedcell(-)cellcommunicationinthenervoussystem:focusonneurologicaldiseases. Int.J.Mol.Sci 20, 434(2019).

301.Chen,C.C.etal.Elucidationofexosomemigrationacrosstheblood–brain barriermodelinvitro. CellMol.Bioeng. 9,509–529(2016).

302.Zhuang,X.etal.Treatmentofbraininﬂammatorydiseasesbydeliveringexosomeencapsulatedanti-inﬂammatorydrugsfromthenasalregiontothebrain. Mol.Ther. 19,1769–1779(2011).

303.CarusoBavisotto,C.etal.ExosomalchaperonesandmiRNAsingliomagenesis: State-of-artandtheranosticsperspectives. Int.J.Mol.Sci. 19,2626(2018).

304.Fiandaca,M.S.etal.IdentiﬁcationofpreclinicalAlzheimer’sdiseasebyaproﬁle ofpathogenicproteinsinneurallyderivedbloodexosomes:acase‐control study. AlzheimersDement. 11,600–607.e601(2015).

305.VanNiel,G.,Porto-Carreiro,I.,Simoes,S.&Raposo,G.Exosomes:acommon pathwayforaspecializedfunction. J.Biochem. 140,13–21(2006).

306.Emmanouilidou,E.etal.Cell-produced α-synucleinissecretedinacalciumdependentmannerbyexosomesandimpactsneuronalsurvival. J.Neurosci. 30, 6838–6851(2010).

307.Reddy,A.P.,Ravichandran,J.&Carkaci-Salli,N.Neuralregenerationtherapiesfor Alzheimer’sandParkinson’sdisease-relateddisorders. BiochimBiophys.Acta Mol.BasisDis. 1866,165506(2020).

308.Shi,M.etal.Plasmaexosomal α-synucleinislikelyCNS-derivedandincreasedin Parkinson’sdisease. ActaNeuropathol. 128,639–650(2014).

309.Zhu,B.,Yin,D.,Zhao,H.&Zhang,L.TheimmunologyofParkinson’sdisease. SeminImmunopathol. 44,659–672(2022).

310.Gao,P.,Li,X.,Du,X.,Liu,S.&Xu,Y.Diagnosticandtherapeuticpotentialof exosomesinneurodegenerativediseases. Front.AgingNeurosci. 13,790863 (2021).

311.Gupta,A.&Pulliam,L.Exosomesasmediatorsofneuroinﬂammation. J.Neuroinﬂammation 11,68(2014).

312.Gustafsson,G.etal.Secretionanduptakeofalpha-synucleinviaextracellular vesiclesinculturedcells. CellMol.Neurobiol. 38,1539–1550(2018).

313.Harischandra,D.S.etal.Environmentalneurotoxicantmanganeseregulates exosome-mediatedextracellularmiRNAsincellculturemodelofParkinson’s disease:relevanceto α-synucleinmisfoldinginmetalneurotoxicity. Neurotoxicology 64,267–277(2018).

314.Huang,Y.etal.Parkinson’sdiseasederivedexosomesaggravateneuropathologyinSNCA*A53Tmice. Ann.Neurol. 92,230–245(2022).

315.Ibáñez,F.,Montesinos,J.,Ureña-Peralta,J.R.,Guerri,C.&Pascual,M.TLR4 participatesinthetransmissionofethanol-inducedneuroinﬂammationvia astrocyte-derivedextracellularvesicles. J.Neuroinﬂammation 16,1–14(2019).

316.Li,D.etal.EffectofregulatorynetworkofexosomesandmicroRNAsonneurodegenerativediseases. Chin.Med.J. 131,2216–2225(2018).

317.Pinnell,J.R.,Cui,M.&Tieu,K.ExosomesinParkinsondisease. J.Neurochem. 157, 413–428(2021).

318.Nila,I.S.etal.Identiﬁcationofexosomalbiomarkersanditsoptimalisolation anddetectionmethodforthediagnosisofParkinson’sdisease:asystematic reviewandmeta-analysis. AgeingRes.Rev. 82,101764(2022).

319.Kluge,A.etal.Detectionofneuron-derivedpathological α-synucleininblood. Brain 145,3058–3071(2022).

320.Shi,M.etal.CNStauefﬂuxviaexosomesislikelyincreasedinParkinson’s diseasebutnotinAlzheimer’sdisease. AlzheimersDement. 12,1125–1131 (2016).

321.Bhattacharyya,P.,Biswas,A.&Biswas,S.C.Brain-enrichedmiR-128:reducedin exosomesfromParkinson’spatientplasma,improvessynapticintegrity,and prevents6-OHDAmediatedneuronalapoptosis. Front.CellNeurosci. 16, 1037903(2022).

322.Cao,X.-Y.etal.MicroRNAbiomarkersofParkinson’sdiseaseinserumexosomelikemicrovesicles. Neurosci.Lett. 644,94–99(2017).

323.Yao,Y.,Qu,M.,Li,G.,Zhang,F.&Rui,H.CirculatingexosomalmiRNAsas diagnosticbiomarkersinParkinson’sdisease. Eur.Rev.Med.Pharm.Sci. 22, 5278–5283(2018).

324.Fraser,K.B.,Moehle,M.S.,Alcalay,R.N.,West,A.B.&Consortium,L.C.Urinary LRRK2phosphorylationpredictsparkinsonianphenotypesinG2019SLRRK2 carriers. Neurology 86,994–999(2016).

325.Wang,S.,Kojima,K.,Mobley,J.A.&West,A.B.Proteomicanalysisofurinary extracellularvesiclesrevealbiomarkersforneurologicdisease. EBioMedicine 45, 351–361(2019).

326.Majbour,N.etal.Disease-associatedalpha-synucleinaggregatesasbiomarkers ofParkinsondiseaseclinicalstage. Neurology 99,e2417–e2427(2022).

327.Arega,Y.&Shao,Y.Heartfailureandlate-onsetAlzheimer’sdisease:aMendelianrandomizationstudy. Front.Genet. 13,1015674(2022).

328.Zheng,T.etal.Plasmaexosomesspreadandclusteraroundbeta-amyloid plaquesinananimalmodelofAlzheimer’sdisease. Front.AgingNeurosci. 9,12 (2017).

329.Chen,X.etal.M1microglia-derivedexosomespromoteactivationofresting microgliaandampliﬁesproangiogeniceffectsthroughIrf1/miR-155-5p/Socs1 axisintheretina. Int.J.Biol.Sci. 19,1791–1812(2023).

330.Li,N.,Shu,J.,Yang,X.,Wei,W.&Yan,A.ExosomesderivedfromM2microglia cellsattenuatesneuronalimpairmentandmitochondrialdysfunctioninAlzheimer’sdiseasethroughthePINK1/Parkinpathway. Front.CellNeurosci. 16, 874102(2022).

331.Wang,Y.etal.Circulatingextracellularvesicle-containingmicroRNAsreveal potentialpathogenesisofAlzheimer’sdisease. Front.CellNeurosci. 16,955511 (2022).

332.Ding,L.etal.ExosomesMediateAPPDysregulationviaAPP-miR-185-5pAxis. Front.CellDev.Biol. 10,793388(2022).

333.Duggan,M.R.,Lu,A.,Foster,T.C.,Wimmer,M.&Parikh,V.Exosomesinagerelatedcognitivedecline:mechanisticinsightsandimprovingoutcomes. Front. AgingNeurosci. 14,834775(2022).

334.You,Y.&Ikezu,T.Emergingrolesofextracellularvesiclesinneurodegenerative disorders. Neurobiol.Dis. 130,104512(2019).

335.Wu,Q.etal.Implicationsofexosomesderivedfromcholesterol-accumulated astrocytesinAlzheimer’sdiseasepathology. Dis.ModelMech. 14,dmm048929 (2021).

336.Asai,H.etal.Depletionofmicrogliaandinhibitionofexosomesynthesishalttau propagation. Nat.Neurosci. 18,1584–1593(2015).

337.Kang,S.S.,Ahn,E.H.&Ye,K.Delta-secretasecleavageofTaumediatesits pathologyandpropagationinAlzheimer’sdisease. Exp.Mol.Med. 52, 1275–1287(2020).

338.Song,L.,Wells,E.A.&Robinson,A.S.Criticalmolecularandcellularcontributors toTaupathology. Biomedicines 9,190(2021).

339.DeLeo,A.M.&Ikezu,T.ExtracellularvesiclebiologyinAlzheimer’sdiseaseand relatedtauopathy. J.NeuroimmunePharm. 13,292–308(2017).

340.Vella,L.J.,Hill,A.F.&Cheng,L.Focusonextracellularvesicles:exosomesand theirroleinproteintrafﬁckingandbiomarkerpotentialinAlzheimer’sand Parkinson’sdisease. Int.J.Mol.Sci. 17,173(2016).

341.Grey,M.etal.Accelerationofalpha-synucleinaggregationbyexosomes. J.Biol. Chem. 290,2969–2982(2015).

342.Reilly,P.etal.Novelhumanneuronaltaumodelexhibitingneuroﬁbrillarytanglesandtranscellularpropagation. Neurobiol.Dis. 106,222–234(2017).

343.Sun,R.etal.Changesinthemorphology,number,andpathologicalprotein levelsofplasmaexosomesmayhelpdiagnoseAlzheimer’sdisease. J.Alzheimers Dis. 73,909–917(2020).

344.Zou,Y.,Mu,D.,Gao,J.&Qiu,L.Reviewontherolesofspeciﬁccell-derived exosomesinAlzheimer’sdisease. Front.Neurosci. 16,936760(2022).

345.Su,H.etal.Characterizationofbrain-derivedextracellularvesiclelipidsinAlzheimer’sdisease. J.Extracell.Vesicles 10,e12089(2021).

346.Gamez-Valero,A.etal.ExploratorystudyonmicroRNAproﬁlesfromplasmaderivedextracellularvesiclesinAlzheimer’sdiseaseanddementiawithLewy bodies. Transl.Neurodegener. 8,31(2019).

347.Liu,W.L.etal.Emergingbloodexosome-basedbiomarkersforpreclinicaland clinicalAlzheimer’sdisease:ameta-analysisandsystematicreview. Neural Regen.Res. 17,2381–2390(2022).

348.Zhang,B.etal.PlasmaexosomalmiRNAsinpersonswithandwithoutAlzheimer disease:alteredexpressionandprospectsforbiomarkers. PLoSONE 10, e0139233(2015).

349.Forsythe,P.Mastcellsinneuroimmuneinteractions. TrendsNeurosci. 42,43–55 (2019).

350.Skaper,S.D.,Facci,L.,Zusso,M.&Giusti,P.Aninﬂammation-centricviewof neurologicaldisease:beyondtheneuron. Front.CellNeurosci. 12,72(2018).

351.Piguet,F.etal.Thechallengeofgenetherapyforneurologicaldiseases:strategiesandtoolstoachieveefﬁcientdeliverytothecentralnervoussystem. Hum.GeneTher. 32,349–374(2021).

352.Samal,J.,Rebelo,A.L.&Pandit,A.Awindowintothebrain:toolstoassesspreclinicalefﬁcacyofbiomaterials-basedtherapiesoncentralnervoussystem disorders. Adv.DrugDeliv.Rev. 148,68–145(2019).

353.Terstappen,G.C.,Meyer,A.H.,Bell,R.D.&Zhang,W.Strategiesfordelivering therapeuticsacrosstheblood–brainbarrier. Nat.Rev.DrugDiscov. 20,362–383 (2021).

354.Wang,H.etal.Curcumin-primedexosomespotentlyamelioratecognitive functioninADmicebyinhibitinghyperphosphorylationoftheTauprotein throughtheAKT/GSK-3β pathway. Nanoscale 11,7481–7496(2019).

355.Bang,O.Y.&Kim,J.-E.Stemcell-derivedextracellularvesicletherapyforacute braininsultsandneurodegenerativediseases. BMBRep. 55,20(2022).

356.Cone,A.S.etal.Mesenchymalstemcell-derivedextracellularvesiclesameliorate Alzheimer’sdisease-likephenotypesinapreclinicalmousemodel. Theranostics 11,8129–8142(2021).

357.Ma,X.etal.ADSCs-derivedextracellularvesiclesalleviateneuronaldamage, promoteneurogenesisandrescuememorylossinmicewithAlzheimer’sdisease. J.ControlRelease 327,688–702(2020).

358.Narbute,K.etal.Intranasaladministrationofextracellularvesiclesderivedfrom humanteethstemcellsimprovesmotorsymptomsandnormalizestyrosine hydroxylaseexpressioninthesubstantianigraandstriatumofthe6hydroxydopamine-treatedrats. StemCellsTransl.Med. 8,490–499(2019).

359.Alvarez-Erviti,L.etal.DeliveryofsiRNAtothemousebrainbysystemicinjection oftargetedexosomes. Nat.Biotechnol. 29,341–345(2011).

360.deAbreu,R.C.etal.Nativeandbioengineeredextracellularvesiclesforcardiovasculartherapeutics. Nat.Rev.Cardiol. 17,685–697(2020).

361.Greening,D.W.,Xu,R.,Ale,A.,Hagemeyer,C.E.&Chen,W. SeminCancerBiol. 90,73–100(2023).

362.Haney,M.J.etal.ExosomesasdrugdeliveryvehiclesforParkinson’sdisease therapy. J.ControlRelease 207,18–30(2015).

363.Kim,M.,Lee,Y.&Lee,M.Hypoxia-speciﬁcanti-RAGEexosomesfornose-to-brain deliveryofanti-miR-181aoligonucleotideinanischemicstrokemodel. Nanoscale 13,14166–14178(2021).

364.LaBarbera,L.,Mauri,E.,D’Amelio,M.&Gori,M.Functionalizationstrategiesof polymericnanoparticlesfordrugdeliveryinAlzheimer’sdisease:currenttrends andfutureperspectives. Front.Neurosci. 16,939855(2022).

365.Qu,M.etal.Dopamine-loadedbloodexosomestargetedtobrainforbetter treatmentofParkinson’sdisease. J.ControlRelease 287,156–166(2018).

366.Weng,S.etal.Theroleofexosomesasmediatorsofneuroinﬂammationinthe pathogenesisandtreatmentofAlzheimer’sdisease. Front.AgingNeurosci. 14, 899944(2022).

367.Yang,J.etal.Exosome-mediateddeliveryofantisenseoligonucleotidestargeting α-synucleinamelioratesthepathologyinamousemodelofParkinson’s disease. Neurobiol.Dis. 148,105218(2021).

368.O’Shea,J.J.,Kontzias,A.,Yamaoka,K.,Tanaka,Y.&Laurence,A.Januskinase inhibitorsinautoimmunediseases. Ann.Rheum.Dis. 72,ii111–ii115(2013).

369.Yamaoka,K.Januskinaseinhibitorsforrheumatoidarthritis. Curr.Opin.Chem. Biol. 32,29–33(2016).

370.Banerjee,S.,Biehl,A.,Gadina,M.,Hasni,S.&Schwartz,D.M.JAK–STATsignaling asatargetforinﬂammatoryandautoimmunediseases:currentandfuture prospects. Drugs 77,521–546(2017).

371.Lai,P.S.etal.ASTATinhibitorpatentreview:progresssince2011. ExpertOpin. Ther.Pat. 25,1397–1421(2015).

372.Kotyla,P.J.,Engelmann,M.,Giemza-Stoklosa,J.,Wnuk,B.&Islam,M.A. ThromboembolicadversedrugreactionsinJanuskinase(JAK)Inhibitors:does theinhibitorspeciﬁcityplayarole? Int.J.Mol.Sci. 22,2449(2021).

373.Lanier,L.L.Theoriginandfunctionsofnaturalkillercells. Clin.Immunol. 95, S14–S18(2000).

374.Lanier,L.L.Naturalkillercellreceptorsignaling. Curr.Opin.Immunol. 15, 308–314(2003).

375.Ljunggren,H.-G.&Kärre,K.Insearchofthe ‘missingself’:MHCmoleculesand NKcellrecognition. Immunol.Today 11,237–244(1990).

376.Seaman,W.E.NaturalkillercellsandnaturalkillerTcells. ArthritisRheum. 43, 1204–1217(2000).

377.Vivier,E.etal.Innateoradaptiveimmunity?Theexampleofnaturalkillercells. science 331,44–49(2011).

378.Vivier,E.,Tomasello,E.,Baratin,M.,Walzer,T.&Ugolini,S.Functionsofnatural killercells. Nat.Immunol. 9,503–510(2008).

379.Zhang,C.,Zhang,J.&Tian,Z.Theregulatoryeffectofnaturalkillercells:do “NKregcells” exist. CellMol.Immunol. 3,241–254(2006).

380.Perricone,R.,Perricone,C.,DeCarolis,C.&Shoenfeld,Y.NKcellsinautoimmunity:atwo-edg’dweaponoftheimmunesystem. Autoimmun.Rev. 7, 384–390(2008).

381.Tian,Z.,Gershwin,M.E.&Zhang,C.RegulatoryNKcellsinautoimmunedisease. J.Autoimmun. 39,206–215(2012).

382.Baxter,A.G.&Smyth,M.J.TheroleofNKcellsinautoimmunedisease. Autoimmunity 35,1–14(2002).

383.Perussia,B.,Chen,Y.&Loza,M.J.PeripheralNKcellphenotypes:multiple changingoffacesofanadapting,developingcell. Mol.Immunol. 42,385–395 (2005).

384.Zhuang,L.etal.ActivityofIL-12/15/18primednaturalkillercellsagainst hepatocellularcarcinoma. Hepatol.Int. 13,75–83(2019).

385.Lan,Y.etal.SimultaneoustargetingofTGF-beta/PD-L1synergizeswithradiotherapybyreprogrammingthetumormicroenvironmenttoovercomeimmune evasion. CancerCell 39,1388–1403e1310(2021).

386.Pizzirani,C.etal.StimulationofP2receptorscausesreleaseofIL-1β–loaded microvesiclesfromhumandendriticcells. Blood 109,3856–3864(2007).

387.Admyre,C.,Johansson,S.M.,Paulie,S.&Gabrielsson,S.DirectexosomestimulationofperipheralhumanTcellsdetectedbyELISPOT. Eur.J.Immunol. 36, 1772–1781(2006).

388.Skokos,D.etal.Mastcell-derivedexosomesinducephenotypicandfunctional maturationofdendriticcellsandelicitspeciﬁcimmuneresponsesinvivo. J. Immunol. 170,3037–3045(2003).

389.Buzas,E.I.,György,B.,Nagy,G.,Falus,A.&Gay,S.Emergingroleofextracellular vesiclesininﬂammatorydiseases. Nat.Rev.Rheumatol. 10,356–364(2014).

390.Clayton,A.etal.Analysisofantigenpresentingcellderivedexosomes,basedon immuno-magneticisolationand ﬂowcytometry. J.Immunol.Methods 247, 163–174(2001).

391.Andre,F.etal.Exosomesaspotentcell-freepeptide-basedvaccine.I.Dendritic cell-derivedexosomestransferfunctionalMHCclassI/peptidecomplexesto dendriticcells. J.Immunol. 172,2126–2136(2004).

392.Mallegol,J.etal.T84-intestinalepithelialexosomesbearMHCclassII/peptide complexespotentiatingantigenpresentationbydendriticcells. Gastroenterology 132,1866–1876(2007).

393.Montecalvo,A.etal.Exosomesasashort-rangemechanismtospreadalloantigenbetweendendriticcellsduringTcellallorecognition. J.Immunol. 180, 3081–3090(2008).

394.Morelli,A.E.etal.Endocytosis,intracellularsorting,andprocessingofexosomes bydendriticcells. Blood 104,3257–3266(2004).

395.Théry,C.etal.IndirectactivationofnaïveCD4+ Tcellsbydendriticcell–derived exosomes. Nat.Immunol. 3,1156–1162(2002).

396.Vincent‐Schneider,H.etal.ExosomesbearingHLA‐DR1moleculesneeddendritic cellstoefﬁcientlystimulatespeciﬁcTcells. Int.Immunol. 14,713–722(2002).

397.Segura,E.etal.ICAM-1onexosomesfrommaturedendriticcellsiscriticalfor efﬁcientnaiveT-cellpriming. Blood 106,216–223(2005).

398.Turpin,D.etal.Roleofextracellularvesiclesinautoimmunediseases. Autoimmun.Rev. 15,174–183(2016).

399.Zhou,H.,Fan,E.K.&Fan,J.Cell–cellinteractionmechanismsinacutelung injury. Shock 55,167–176(2021).

400.Moldoveanu,B.etal.Inﬂammatorymechanismsinthelung. J.Inﬂamm.Res. 2, 1–11(2009).

401.Lee,H.,Abston,E.,Zhang,D.,Rai,A.&Jin,Y.Extracellularvesicle:anemerging mediatorofintercellularcrosstalkinlunginﬂammationandinjury. Front. Immunol. 9,924(2018).

402.Linkermann,A.,Stockwell,B.R.,Krautwald,S.&Anders,H.-J.Regulatedcell deathandinﬂammation:anauto-ampliﬁcationloopcausesorganfailure. Nat. Rev.Immunol. 14,759–767(2014).

403.Lomas-Neira,J.etal.Neutrophil-endothelialinteractionsmediateangiopoietin2-associatedpulmonaryendothelialcelldysfunctioninindirectacutelunginjury inmice. Am.J.Respir.CellMol.Biol. 50,193–200(2014).

404.Silva,M.T.Neutrophilsandmacrophagesworkinconcertasinducersand effectorsofadaptiveimmunityagainstextracellularandintracellularmicrobial pathogens. J.Leukoc.Biol. 87,805–813(2010).

405.Abraham,E.Neutrophilsandacutelunginjury. Crit.CareMed. 31,S195–S199 (2003).

406.Fan,J.,Frey,R.S.&Malik,A.B.TLR4signalinginducesTLR2expressionin endothelialcellsvianeutrophilNADPHoxidase. J.Clin.Investig. 112,1234–1243 (2003).

407.Fan,J.,Frey,R.S.,Rahman,A.&Malik,A.B.RoleofneutrophilNADPHoxidasein themechanismoftumornecrosisfactor-alpha-inducedNF-kappaBactivation andintercellularadhesionmolecule-1expressioninendothelialcells. J.Biol. Chem. 277,3404–3411(2002).

408.Standiford,T.J.&Ward,P.A.Therapeutictargetingofacutelunginjuryand acuterespiratorydistresssyndrome. Transl.Res. 167,183–191(2016).

409.Agache,I.&Akdis,C.A.Precisionmedicineandphenotypes,endotypes,genotypes,regiotypes,andtheratypesofallergicdiseases. J.Clin.Investig. 129, 1493–1503(2019).

410.Maison,N.etal.T2-highasthmaphenotypesacrosslifespan. Eur.Respir.J. 60, 2102288(2022).

411.Wenzel,S.E.Asthmaphenotypes:theevolutionfromclinicaltomolecular approaches. Nat.Med. 18,716–725(2012).

412.Li-Weber,M.&Krammer,P.H.RegulationofIL4geneexpressionbyTcellsand therapeuticperspectives. Nat.Rev.Immunol. 3,534–543(2003).

413.Olin,J.T.&Wechsler,M.E.Asthma:pathogenesisandnoveldrugsfortreatment. BMJ 349,g5517–g5517(2014).

414.Xie,H.&He,S.H.Rolesofhistamineanditsreceptorsinallergicandinﬂammatoryboweldiseases. WorldJ.Gastroenterol. 11,2851–2857(2005).

415.Ax,E.etal.T2andT17cytokinesalterthecargoandfunctionofairway epithelium-derivedextracellularvesicles. Respir.Res. 21,155(2020).

416.Bartel,S.etal.HumanairwayepithelialextracellularvesiclemiRNAsignatureis altereduponasthmadevelopment. Allergy 75,346–356(2020).

417.Bourdonnay,E.etal.TranscellulardeliveryofvesicularSOCSproteinsfrom macrophagestoepithelialcellsbluntsinﬂammatorysignaling. J.Exp.Med. 212, 729–742(2015).

418.Chahar,H.S.,Corsello,T.,Kudlicki,A.S.,Komaravelli,N.&Casola,A.Respiratory syncytialvirusinfectionchangescargocompositionofexosomereleasedfrom airwayepithelialcells. Sci.Rep. 8,387(2018).

419.Draijer,C.etal.Residentalveolarmacrophage-derivedvesicularSOCS3dampensallergicairwayinﬂammation. FASEBJ. 34,4718–4731(2020).

420.Fujita,Y.,Kosaka,N.,Araya,J.,Kuwano,K.&Ochiya,T.Extracellularvesiclesin lungmicroenvironmentandpathogenesis. TrendsMol.Med. 21,533–542(2015).

421.Gupta,R.etal.Intercellularcommunicationbetweenairwayepithelialcellsis mediatedbyexosome-likevesicles. Am.J.Respir.CellMol.Biol. 60,209–220 (2019).

422.Huang,L.etal.Exosomesfromthymicstromallymphopoietin-activateddendriticcellspromoteTh2differentiationthroughtheOX40ligand. Pathobiology 86,111–117(2019).

423.Lee,H.,Zhang,D.,Laskin,D.L.&Jin,Y.Functionalevidenceofpulmonary extracellularvesiclesininfectiousandnoninfectiouslunginﬂammation. J. Immunol. 201,1500–1509(2018).

424.Lee,H.,Zhang,D.,Zhu,Z.,DelaCruz,C.S.&Jin,Y.Epithelialcell-derived microvesiclesactivatemacrophagesandpromoteinﬂammationvia microvesicle-containingmicroRNAs. Sci.Rep. 6,35250(2016).

425.Mills,J.T.etal.Airwayepithelialcellsgeneratepro-inﬂammatorytenascin-Cand smallextracellularvesiclesinresponsetoTLR3stimuliandrhinovirusinfection. Front.Immunol. 10,1987(2019).

426.Mwase,C.etal.Mechanicalcompressionofhumanairwayepithelialcells inducesreleaseofextracellularvesiclescontainingtenascinC. Cells 11,256 (2022).

427.Jette,C.A.etal.Cryo-EMstructuresofHIV-1trimerboundtoCD4-mimetics BNM-III-170andM48U1adoptaCD4-boundopenconformation. Nat.Commun. 12,1950(2021).

428.Kreger,J.etal.Quantifyingthedynamicsofviralrecombinationduringfree virusandcell-to-celltransmissioninHIV-1infection. VirusEvol. 7,veab026 (2021).

429.Lambert,G.S.&Upadhyay,C.HIV-1envelopeglycosylationandthesignal peptide. Vaccines 9,176(2021).

430.Mielke,D.etal.ADCC-mediatingnon-neutralizingantibodiescanexertimmune pressureinearlyHIV-1infection. PLoSPathog. 17,e1010046(2021).

431.Vezina,D.etal.StabilizingtheHIV-1envelopeglycoproteinstate2Aconformation. J.Virol. 95,e01620–20(2021).

432.Wang,L.etal.Areplication-competentHIVclonecarryingGFP-Envrevealsrapid EnvrecyclingattheHIV-1Tcellvirologicalsynapse. Viruses 14,38(2021).

433.Zhang,S.etal.Dualpathwaysofhumanimmunodeﬁciencyvirustype1 envelopeglycoproteintrafﬁckingmodulatetheselectiveexclusionofuncleaved oligomersfromvirions. J.Virol. 95,e01369–20(2021).

434.Dufrancais,O.etal.Cellularandmolecularactorsofmyeloidcellfusion: podosomesandtunnelingnanotubescallthetune. CellMol.LifeSci. 78, 6087–6104(2021).

435.Ljubojevic,N.,Henderson,J.M.&Zurzolo,C.Thewaysofactin:whytunneling nanotubesareuniquecellprotrusions. TrendsCellBiol. 31,130–142(2021).

436.Rilla,K.Diverseplasmamembraneprotrusionsactasplatformsforextracellular vesicleshedding. J.Extracell.Vesicles 10,e12148(2021).

437.Schiff,A.E.etal.Tcell-tropicHIVefﬁcientlyinfectsalveolarmacrophages throughcontactwithinfectedCD4+ Tcells. Sci.Rep. 11,3890(2021).

438.Zurzolo,C.Tunnelingnanotubes:reshapingconnectivity. Curr.Opin.CellBiol. 71, 139–147(2021).

439.Gao,Z.etal.Anovelmetabolism-relatedgenesignatureforpredictingthe prognosisofHBV-infectedhepatocellularcarcinoma. J.Oncol. 2022,2391265 (2022).

440.Gao,Z.etal.Interleukin-33mediatesbothimmune-relatedandnon-immunerelatedinhibitoryeffectsagainsthepatitisBvirus. Antivir.Res. 206,105404 (2022).

441.Peneau,C.etal.HepatitisBvirusintegrationspromotelocalanddistant oncogenicdriveralterationsinhepatocellularcarcinoma. Gut 71,616–626 (2022).

442.Wu,Q.etal.PresenceofintacthepatitisBvirionsinexosomes. CellMol.Gastroenterol.Hepatol. 15,237–259(2023).

443.Guo,C.etal.Single-cellanalysisoftwosevereCOVID-19patientsrevealsa monocyte-associatedandtocilizumab-respondingcytokinestorm. Nat.Commun. 11,3924(2020).

444.Hoffmann,M.etal.SARS-CoV-2cellentrydependsonACE2andTMPRSS2andis blockedbyaclinicallyprovenproteaseinhibitor. Cell 181,271–280e278(2020).

445.Braga,L.etal.DrugsthatinhibitTMEM16proteinsblockSARS-CoV-2spikeinducedsyncytia. Nature 594,88–93(2021).

446.Davies,N.G.etal.EstimatedtransmissibilityandimpactofSARS-CoV-2lineage B.1.1.7inEngland. Science 372,eabg3055(2021).

447.Hoffmann,M.etal.SARS-CoV-2variantsB.1.351andP.1escapefromneutralizingantibodies. Cell 184,2384–2393(2021).

448.Johnson,B.A.etal.LossoffurincleavagesiteattenuatesSARS-CoV-2pathogenesis. Nature 591,293–299(2021).

449.Korn,E.D.&Olivecrona,T.Discussionpaper:amoebaplasmamembrane. Ann.N. YAcad.Sci. 195,142–146(1972).

450.Liu,Y.etal.Aninfectivity-enhancingsiteontheSARS-CoV-2spikeprotein targetedbyantibodies. Cell 184,3452–3466(2021).

451.Mlcochova,P.etal.SARS-CoV-2B.1.617.2Deltavariantreplicationandimmune evasion. Nature 599,114–119(2021).

452.Peacock,T.P.etal.ThefurincleavagesiteintheSARS-CoV-2spikeproteinis requiredfortransmissioninferrets. Nat.Microbiol. 6,899–909(2021).

453.Planas,D.etal.ReducedsensitivityofSARS-CoV-2variantDeltatoantibody neutralization. Nature 596,276–280(2021).

454.Starr,T.N.etal.Prospectivemappingofviralmutationsthatescapeantibodies usedtotreatCOVID-19. Science 371,850–854(2021).

455.V’Kovski,P.,Kratzel,A.,Steiner,S.,Stalder,H.&Thiel,V.Coronavirusbiologyand replication:implicationsforSARS-CoV-2. Nat.Rev.Microbiol 19,155–170(2021).

456.Zhao,M.M.etal.CathepsinLplaysakeyroleinSARS-CoV-2infectionin humansandhumanizedmiceandisapromisingtargetfornewdrugdevelopment. SignalTransduct.TargetTher. 6,134(2021).

457.Zhou,B.etal.SARS-CoV-2spikeD614Gchangeenhancesreplicationand transmission. Nature 592,122–127(2021).

458.Filbin,M.R.etal.LongitudinalproteomicanalysisofsevereCOVID-19reveals survival-associatedsignatures,tissue-speciﬁccelldeath,andcell-cellinteractions. CellRep.Med. 2,100287(2021).

459.Jeong,K.,Kim,Y.,Jeon,J.&Kim,K.SubtypingofCOVID-19samplesbasedon cell-cellinteractioninsinglecelltranscriptomes. Sci.Rep. 13,19629(2023).

460.Lin,Y.etal.Scalablework ﬂowforcharacterizationofcell-cellcommunicationin COVID-19patients. PLoSComput.Biol. 18,e1010495(2022).

461.Rebelatto,C.L.K.etal.Safetyandlong-termimprovementofmesenchymal stromalcellinfusionincriticallyCOVID-19patients:arandomizedclinicaltrial. StemCellResTher. 13,122(2022).

462.Chaudhari,P.,Ghate,V.,Nampoothiri,M.&Lewis,S.Multifunctionalroleof exosomesinviraldiseases:Fromtransmissiontodiagnosisandtherapy. Cell Signal 94,110325(2022).

463.Dubey,A.etal.Exosomes:emergingimplementationofnanotechnologyfor detectingandmanagingnovelcoronavirus-SARS-CoV-2. AsianJ.Pharm.Sci. 17, 20–34(2022).

464.Babaei,G.,Zare,N.,Mihanfar,A.&Ansari,M.H.K.ExosomesandCOVID-19: challengesandopportunities. Comp.Clin.Path 31,347–354(2022).

465.Gunnels,T.F.,Stranford,D.M.,Mitrut,R.E.,Kamat,N.P.&Leonard,J.N.Elucidatingdesignprinciplesforengineeringcell-derivedvesiclestoinhibitSARSCoV-2infection. Small 18,e2200125(2022).

466.El-Shennawy,L.etal.CirculatingACE2-expressingextracellularvesiclesblock broadstrainsofSARS-CoV-2. Nat.Commun. 13,405(2022).

467.Ching,K.L.etal.ACE2-containingdefensosomesserveasdecoystoinhibit SARS-CoV-2infection. PLoSBiol. 20,e3001754(2022).

468.Akbar,N.,Azzimato,V.,Choudhury,R.P.&Aouadi,M.Extracellularvesiclesin metabolicdisease. Diabetologia 62,2179–2187(2019).

469.Agouni,A.etal.Endothelialdysfunctioncausedbycirculatingmicroparticles frompatientswithmetabolicsyndrome. Am.J.Pathol. 173,1210–1219(2008).

470.Eguchi,A.etal.Circulatingadipocyte-derivedextracellularvesiclesarenovel markersofmetabolicstress. J.Mol.Med. 94,1241–1253(2016).

471.Eitan,E.etal.Age-relatedchangesinplasmaextracellularvesiclecharacteristics andinternalizationbyleukocytes. Sci.Rep. 7,1342(2017).

472.Freeman,D.W.etal.Alteredextracellularvesicleconcentration,cargo,and functionindiabetes. Diabetes 67,2377–2388(2018).

473.Hulsmans,M.&Holvoet,P.MicroRNA-containingmicrovesiclesregulating inﬂammationinassociationwithatheroscleroticdisease. CardiovascRes. 100, 7–18(2013).

474.Khalyfa,A.etal.Exosomesandmetabolicfunctioninmiceexposedtoalternatingdark-lightcyclesmimickingnightshiftworkschedules. Front.Physiol. 8, 882(2017).

475.Kobayashi,Y.etal.Circulatingextracellularvesiclesareassociatedwithlipidand insulinmetabolism. Am.J.Physiol.Endocrinol.Metab. 315,E574–E582(2018).

476.Lakhter,A.J.&Sims,E.K.Minireview:emergingrolesforextracellularvesiclesin diabetesandrelatedmetabolicdisorders. Mol.Endocrinol. 29,1535–1548(2015).

477.Martínez,M.C.&Andriantsitohaina,R.Extracellularvesiclesinmetabolicsyndrome. Circ.Res. 120,1674–1686(2017).

478.O’Neill,S.,Bohl,M.,Gregersen,S.,Hermansen,K.&O’Driscoll,L.Blood-based biomarkersformetabolicsyndrome. TrendsEndocrinol.Metab. 27,363–374 (2016).

479.Pomatto,M.A.,Gai,C.,Deregibus,M.C.,Tetta,C.&Camussi,G.NoncodingRNAs carriedbyextracellularvesiclesinendocrinediseases. Int.J.Endocrinol. 2018, 4302096(2018).

480.Akbar,N.etal.Endothelium-derivedextracellularvesiclespromotesplenic monocytemobilizationinmyocardialinfarction. JCIInsight 2,e93344(2017).

481.Couch,Y.etal.Circulatingendothelialcell-derivedextracellularvesiclesmediate theacutephaseresponseandsicknessbehaviourassociatedwithCNSinﬂammation. Sci.Rep. 7,9574(2017).

482.Javeed,N.Sheddingperspectiveonextracellularvesiclebiologyindiabetesand associatedmetabolicsyndromes. Endocrinology 160,399–408(2019).

483.Lawson,C.,Vicencio,J.M.,Yellon,D.M.&Davidson,S.M.Microvesiclesand exosomes:newplayersinmetabolicandcardiovasculardisease. J.Endocrinol. 228,R57–R71(2016).

484.Eichner,N.Z.,Erdbrügger,U.&Malin,S.K.Extracellularvesicles:anoveltarget forexercise-mediatedreductionsintype2diabetesandcardiovasculardisease risk. J.DiabetesRes. 2018,7807245(2018).

485.Eichner,N.Z.etal.Lowcardiorespiratory ﬁtnessisassociatedwithhigher extracellularvesiclecountsinobeseadults. Physiol.Rep. 6,e13701(2018).

486.Balaphas,A.etal.Plateletsandplatelet‐derivedextracellularvesiclesinliver physiologyanddisease. Hepatol.Commun. 3,855–866(2019).

487.Randriamboavonjy,V.&Fleming,I.Plateletfunctionandsignalingindiabetes mellitus. Curr.Vasc.Pharm. 10,532–538(2012).

488.Bennett,C.L.etal.Theimmunedysregulation,polyendocrinopathy,enteropathy,X-linkedsyndrome(IPEX)iscausedbymutationsofFOXP3. Nat.Genet. 27,20–21(2001).

489.Khattri,R.,Cox,T.,Yasayko,S.-A.&Ramsdell,F.AnessentialroleforScurﬁnin CD4+ CD25+ Tregulatorycells. Nat.Immunol. 4,337–342(2003).

490.Scherm,M.G.etal.miRNA142-3ptargetsTet2andimpairsTregdifferentiation andstabilityinmodelsoftype1diabetes. Nat.Commun. 10,5697(2019).

491.Serr,I.etal.Type1diabetesvaccinecandidatespromotehumanFoxp3+ Treg inductioninhumanizedmice. Nat.Commun. 7,10991(2016).

492.Serr,I.etal.AmiRNA181a/NFAT5axislinksimpairedTcelltoleranceinduction withautoimmunetype1diabetes. Sci.Transl.Med. 10,eaag1782(2018).

493.Scherm,M.G.etal.Betacellandimmunecellinteractionsinautoimmunetype 1diabetes:howtheymeetandtalktoeachother. Mol.Metab. 64,101565 (2022).

494.Salomon,B.etal.B7/CD28costimulationisessentialforthehomeostasisofthe CD4+ CD25+ immunoregulatoryTcellsthatcontrolautoimmunediabetes. Immunity 12,431–440(2000).

495.Feuerer,M.,Shen,Y.,Littman,D.R.,Benoist,C.&Mathis,D.Howpunctual ablationofregulatoryTcellsunleashesanautoimmunelesionwithinthe pancreaticislets. Immunity 31,654–664(2009).

496.Long,S.A.etal.DefectsinIL-2RsignalingcontributetodiminishedmaintenanceofFOXP3expressioninCD4+ CD25+ regulatoryT-cellsoftype1 diabeticsubjects. Diabetes 59,407–415(2010).

497.Grinberg-Bleyer,Y.etal.IL-2reversesestablishedtype1diabetesinNODmice byalocaleffectonpancreaticregulatoryTcells. J.Exp.Med. 207,1871–1878 (2010).

498.Koeck,E.S.etal.Adipocyteexosomesinducetransforminggrowthfactorbeta pathwaydysregulationinhepatocytes:anovelparadigmforobesity-related liverdisease. J.Surg.Res. 192,268–275(2014).

499.Thomou,T.etal.Adipose-derivedcirculatingmiRNAsregulategeneexpression inothertissues. Nature 542,450–455(2017).

500.Eguchi,A.etal.Microparticlesreleasebyadipocytesactas “ﬁnd-me” signalsto promotemacrophagemigration. PLoSONE 10,e0123110(2015).

501.Wadey,R.M.etal.Inﬂammatoryadipocyte-derivedextracellularvesiclespromoteleukocyteattachmenttovascularendothelialcells. Atherosclerosis 283, 19–27(2019).

502.Amano,S.U.etal.Localproliferationofmacrophagescontributestoobesityassociatedadiposetissueinﬂammation. CellMetab. 19,162–171(2014).

503.Russo,L.&Lumeng,C.N.Propertiesandfunctionsofadiposetissuemacrophagesinobesity. Immunology 155,407–417(2018).

504.Ying,W.etal.Adiposetissuemacrophage-derivedexosomalmiRNAscan modulateinvivoandinvitroinsulinsensitivity. Cell 171,372–384.e312(2017).

505.Delić,D.etal.UrinaryexosomalmiRNAsignatureintypeIIdiabeticnephropathy patients. PLoSONE 11,e0150154(2016).

506.LaMarca,V.&Fierabracci,A.InsightsintothediagnosticpotentialofextracellularvesiclesandtheirmiRNAsignaturefromliquidbiopsyasearlybiomarkersofdiabeticmicro/macrovascularcomplications. Int.J.Mol.Sci. 18,1974 (2017).

507.Chen,Y.etal.miR-155regulatesdifferentiationofbrownandbeigeadipocytes viaabistablecircuit. Nat.Commun. 4,1769(2013).

508.Ge,W.etal.Single-celltranscriptomeproﬁlingrevealsdermalandepithelialcell fatedecisionsduringembryonichairfollicledevelopment. Theranostics 10, 7581–7598(2020).

509.Zhong,S.etal.Asingle-cellRNA-seqsurveyofthedevelopmentallandscapeof thehumanprefrontalcortex. Nature 555,524–528(2018).

510.Voss,A.J.etal.Identiﬁcationofligand-receptorpairsthatdrivehumanastrocyte development. Nat.Neurosci. 26,1339–1351(2023).

511.Li,L.etal.Single-cellRNA-Seqanalysismapsdevelopmentofhumangermline cellsandgonadalnicheinteractions. CellStemCell 20,858–873e854(2017).

512.Li,L.etal.Dissectingtheepigenomicdynamicsofhumanfetalgermcell developmentatsingle-cellresolution. CellRes. 31,463–477(2021).

513.Liu,J.Q.etal.Wnt/β-cateninsignalling:function,biologicalmechanisms,and therapeuticopportunities. SignalTransduct.TargetTher. 7,3(2022).

514.Wang,R.etal.Dissectinghumangonadalcelllineagespeciﬁcationandsex determinationusingasingle-cellRNA-seqapproach. GenomicsProteom.Bioinforma. 20,223–245(2022).

515.Wang,P.etal.Dissectingtheglobaldynamicmolecularproﬁlesofhumanfetal kidneydevelopmentbysingle-cellRNAsequencing. CellRep. 24,3554–3567(2018).

516.Shi,Z.,Liu,J.,Wang,F.&Li,Y.IntegratedanalysisofSolutecarrierfamily-2 membersrevealsSLC2A4asanindependentfavorableprognosticbiomarkerfor breastcancer. Channels 15,555–568(2021).

517.Chai,Y.J.etal.UpregulationofSLC2(GLUT)familygenesisrelatedtopoor survivaloutcomesinpapillarythyroidcarcinoma:analysisofdatafromThe CancerGenomeAtlas. Surgery 161,188–194(2017).

518.Flavahan,W.A.etal.Braintumorinitiatingcellsadapttorestrictednutrition throughpreferentialglucoseuptake. Nat.Neurosci. 16,1373–1382(2013).

519.Ancey,P.B.,Contat,C.&Meylan,E.Glucosetransportersincancer fromtumor cellstothetumormicroenvironment. FEBSJ. 285,2926–2943(2018).

520.Ricard-Blum,S.Thecollagenfamily. ColdSpringHarb.Perspect.Biol. 3,a004978 (2011).

521.Wang,Y.etal.Thedouble-edgedrolesofROSincancerpreventionandtherapy. Theranostics 11,4839–4857(2021).

522.Zhang,J.etal.ROSandROS-mediatedcellularsignaling. Oxid.Med.CellLongev. 2016,4350965(2016).

523.Adjei,A.A.Signaltransductionpathwaytargetsforanticancerdrugdiscovery. Curr.Pharm.Des. 6,361–378(2000).

524.Barrantes,F.J.Structureandfunctionmeetatthenicotinicacetylcholine receptor-lipidinterface. Pharm.Res. 190,106729(2023).

525.Marsh,D.,Watts,A.&Barrantes,F.J.Phospholipidchainimmobilizationand steroidrotationalimmobilizationinacetylcholinereceptor-richmembranes from Torpedomarmorata BiochimBiophys.Acta 645,97–101(1981).

526.Marsh,D.&Barrantes,F.J.Immobilizedlipidinacetylcholinereceptor-rich membranesfromTorpedomarmorata. Proc.Natl.Acad.Sci.USA 75,4329–4333 (1978).

527.Rousselet,A.,Devaux,P.F.&Wirtz,K.W.Freefattyacidsandesterscanbe immobilizedbyreceptorrichmembranesfrom Torpedomarmorata butnot phospholipidacylchains. BiochemBiophys.Res.Commun. 90,871–877(1979).

528.Ellena,J.F.,Blazing,M.A.&McNamee,M.G.Lipid-proteininteractionsin reconstitutedmembranescontainingacetylcholinereceptor. Biochemistry 22, 5523–5535(1983).

529.Gavva,N.R.etal.RepeatedadministrationofvanilloidreceptorTRPV1 antagonistsattenuateshyperthermiaelicitedbyTRPV1blockade. J.Pharm.Exp. Ther. 323,128–137(2007).

530.Caterina,M.J.etal.Impairednociceptionandpainsensationinmicelackingthe capsaicinreceptor. Science 288,306–313(2000).

531.Yang,F.etal.Theconformationalwaveincapsaicinactivationoftransient receptorpotentialvanilloid1ionchannel. Nat.Commun. 9,2879(2018).

532.Purvis,J.E.&Lahav,G.Encodinganddecodingcellularinformationthrough signalingdynamics. Cell 152,945–956(2013).

533.Scheel,C.etal.Paracrineandautocrinesignalsinduceandmaintain mesenchymalandstemcellstatesinthebreast. Cell 145,926–940(2011).

534.Hynes,R.O.Theextracellularmatrix:notjustpretty ﬁbrils. Science 326, 1216–1219(2009).

535.Gerdes,H.H.,Rustom,A.&Wang,X.Tunnelingnanotubes,anemerging intercellularcommunicationrouteindevelopment. Mech.Dev. 130,381–387 (2013).

536.Ariazi,J.etal.Tunnelingnanotubesandgapjunctions-theirroleinlong-range intercellularcommunicationduringdevelopment,health,anddiseaseconditions. Front.Mol.Neurosci. 10,333(2017).

537.Rustom,A.,Saffrich,R.,Markovic,I.,Walther,P.&Gerdes,H.H.Nanotubular highwaysforintercellularorganelletransport. Science 303,1007–1010(2004).

538.Vignais,M.L.,Caicedo,A.,Brondello,J.M.&Jorgensen,C.Cellconnectionsby tunnelingnanotubes:effectsofmitochondrialtrafﬁckingontargetcellmetabolism,homeostasis,andresponsetotherapy. StemCellsInt. 2017,6917941 (2017).

539.Patheja,P.etal.Theuseofopticaltrapandmicrobeamtoinvestigatethe mechanicalandtransportcharacteristicsoftunnelingnanotubesintumor spheroids. J.Biophotonics 8,694–704(2015).

540.Pasquier,J.etal.Preferentialtransferofmitochondriafromendothelialto cancercellsthroughtunnelingnanotubesmodulateschemoresistance. J.Transl. Med. 11,94(2013).

541.Pontes,B.etal.Structureandelasticpropertiesoftunnelingnanotubes. Eur. Biophys.J. 37,121–129(2008).

542.Yang,H.etal.Biochip-basedstudyofunidirectionalmitochondrialtransferfrom stemcellstomyocytesviatunnelingnanotubes. Biofabrication 8,015012(2016).

543.Zhang,J.etal.Directobservationoftunnelingnanotubeswithinhuman mesenchymalstemcellspheroids. J.Phys.Chem.B 122,9920–9926(2018).

544.Sandison,D.R.,Piston,D.W.,Williams,R.M.&Webb,W.W.Quantitative comparisonofbackgroundrejection,signal-to-noiseratio,andresolutionin confocalandfull-ﬁeldlaserscanningmicroscopes. ApplOpt. 34,3576–3588 (1995).

545.Denk,W.,Strickler,J.H.&Webb,W.W.Two-photonlaserscanning ﬂuorescence microscopy. Science 248,73–76(1990).

546.Dyba,M.,Hell,S.W.&Jakobs,S.Conceptsfornanoscaleresolutionin ﬂuorescencemicroscopy. Curr.Opin.Neurobiol. 14,599–609(2004).

547.Donnert,G.etal.Macromolecular-scaleresolutioninbiological ﬂuorescence microscopy. Proc.Natl.Acad.Sci.USA 103,11440–11445(2006).

548.Dyba,M.&Hell,S.W.Focalspotsofsize λ/23openupfar-ﬁeld ﬂorescence microscopyat33nmaxialresolution. Phys.Rev.Lett. 88,163901(2002).

549.Pellett,P.A.etal.Two-colorSTEDmicroscopyinlivingcells. Biomed.Opt.Express 2,2364–2371(2011).

550.Mueller,V.etal.STEDnanoscopyrevealsmoleculardetailsofcholesterol-and cytoskeleton-modulatedlipidinteractionsinlivingcells. Biophys.J. 101, 1651–1660(2011).

551.Mueller,V.etal.FCSinSTEDmicroscopy:studyingthenanoscaleoflipid membranedynamics. MethodsEnzymol. 519,1–38(2013).

552.Rust,M.J.,Bates,M.&Zhuang,X.Sub-diffraction-limitimagingbystochastic opticalreconstructionmicroscopy(STORM). Nat.Methods 3,793–795(2006).

553.Huang,B.,Wang,W.,Bates,M.&Zhuang,X.Three-dimensionalsuper-resolution imagingbystochasticopticalreconstructionmicroscopy. Science 319,810–813 (2008).

554.Dani,A.,Huang,B.,Bergan,J.,Dulac,C.&Zhuang,X.Superresolutionimagingof chemicalsynapsesinthebrain. Neuron 68,843–856(2010).

555.Nagy,P.,Vereb,G.,Post,J.N.,Friedländer,E.&Szölloősi,J.Novelsinglecell ﬂuorescenceapproachesintheinvestigationofsignalingatthecellularlevel.In BiophysicalAspectsofTransmembraneSignaling, (edDamjanovichS.)33–70 (SpringerBerlinHeidelberg,2005).

556.Szöllosi,J.,Damjanovich,S.&Mátyus,L.Applicationof ﬂuorescenceresonance energytransferintheclinicallaboratory:routineandresearch. Cytometry 34, 159–179(1998).

557.Jares-Erijman,E.A.&Jovin,T.M.FRETimaging. Nat.Biotechnol. 21,1387–1395 (2003).

558.Hildebrandt,N.,Wegner,K.&Algar,W.Luminescentterbiumcomplexes: superiorFörsterresonanceenergytransferdonorsfor ﬂexibleandsensitive multiplexedbiosensing. Coord.Chem.Rev. 273,125–138(2014).

559.Lakowica,J. PrinciplesofFluorescenceSpectroscopy,3rded.;SpringerScience& BusinessMedia:NewYork,NY,USA,205–235(2006).

560.Chan,F.T.,Kaminski,C.F.&KaminskiSchierle,G.S.HomoFRET ﬂuorescence anisotropyimagingasatooltostudymolecularself‐assemblyinlivecells. Chemphyschem 12,500–509(2011).

561.Lidke,D.etal.Imagingmolecularinteractionsincellsbydynamicandstatic ﬂuorescenceanisotropy(rFLIMandemFRET). BiochemSoc.Trans. 31,1020–1027 (2003).

562.Shrestha,D.,Jenei,A.,Nagy,P.,Vereb,G.&Szöllősi,J.UnderstandingFRETasa researchtoolforcellularstudies. Int.J.Mol.Sci. 16,6718–6756(2015).

563.Epe,B.,Woolley,P.,Steinhäuser,K.G.&Littlechild,J.Distancemeasurementby energytransfer:the3′ endof16‐SRNAandproteinsS4andS17oftheribosome of Escherichiacoli Eur.J.Biochem. 129,211–219(1982).

564.Jovin,T.M.&Arndt-Jovin,D.J.FRETmicroscopy:digitalimagingof ﬂuorescence resonanceenergytransfer.Applicationincellbiology.In CellStructureand FunctionbyMicrospectroﬂuorometry,(edKOHENE.)99–117(AcademicPress, 1989).

565.Stryer,L.&Haugland,R.P.Energytransfer:aspectroscopicruler. Proc.Natl.Acad. Sci.USA 58,719–726(1967).

566.Chan,S.S.,Arndt-Jovin,D.J.&Jovin,T.M.Proximityoflectinreceptorsonthe cellsurfacemeasuredby ﬂuorescenceenergytransferina ﬂowsystem. J.Histochem.Cytochem. 27,56–64(1979).

567.Szöllósi,J.etal.Fluorescenceenergytransfermeasurementsoncellsurfaces:a criticalcomparisonofsteady‐state ﬂuorimetricand ﬂowcytometricmethods. Cytometry 5,210–216(1984).

568.Gordon,G.W.,Berry,G.,Liang,X.H.,Levine,B.&Herman,B.Quantitative ﬂuorescenceresonanceenergytransfermeasurementsusing ﬂuorescence microscopy. Biophys.J. 74,2702–2713(1998).

SignalTransductionandTargetedTherapy(2024)9:196

569.Zal,T.&Gascoigne,N.R.Photobleaching-correctedFRETefﬁciencyimagingof livecells. Biophys.J. 86,3923–3939(2004).

570. Żal,T., Żal,M.A.&Gascoigne,N.R.InhibitionofTcellreceptor-coreceptor interactionsbyantagonistligandsvisualizedbyliveFRETimagingofthe T-hybridomaimmunologicalsynapse. Immunity 16,521–534(2002).

571.Zeug,A.,Woehler,A.,Neher,E.&Ponimaskin,E.G.Quantitativeintensity-based FRETapproaches acomparativesnapshot. Biophys.J. 103,1821–1827(2012).

572.Mittag,A.etal.Sequentialphotobleachingof ﬂuorochromesforpolychromatic slide‐basedcytometry. Cytom.A 69,139–141(2006).

573.Szabà,G.,Pine,P.S.,Weaver,J.L.,Kasari,M.&Aszalos,A.Epitopemappingby photobleaching ﬂuorescenceresonanceenergytransfermeasurementsusinga laserscanningmicroscopesystem. Biophys.J. 61,661–670(1992).

574.Szalóki,N.etal.HighthroughputFRETanalysisofprotein–proteininteractions byslide‐basedimaginglaserscanningcytometry. Cytom.A 83,818–829(2013).

575.Roy,R.,Hohng,S.&Ha,T.Apracticalguidetosingle-moleculeFRET. Nat. Methods 5,507–516(2008).

576.Mills,J.D.etal.Illuminatingproteininteractionsintissueusingconfocaland two-photonexcitation ﬂuorescentresonanceenergytransfermicroscopy. J. Biomed.Opt. 8,347–356(2003).

577.McGinty,J.etal.Invivo ﬂuorescencelifetimetomographyofaFRETprobe expressedinmouse. Biomed.Opt.express 2,1907–1917(2011).

578.Depry,C.,Mehta,S.,Li,R.&Zhang,J.Visualizationofcompartmentalizedkinase activitydynamicsusingadaptableBimKARs. Chem.Biol. 22,1470–1479(2015).

579.Sample,V.,Mehta,S.&Zhang,J.Geneticallyencodedmolecularprobesto visualizeandperturbsignalingdynamicsinlivingbiologicalsystems. J.CellSci. 127,1151–1160(2014).

580.Banerjee,S.,Versaw,W.K.&Garcia,L.R.Imagingcellularinorganicphosphatein Caenorhabditiselegans usingageneticallyencodedFRET-basedbiosensor. PLoS ONE 10,e0141128(2015).

581.Bins,A.D.etal.Intravitalimagingof ﬂuorescentmarkersandFRETprobesby DNAtattooing. BMCBiotechnol. 7,1–7(2007).

582.Jin,K.etal.Intravitaltwo-photonimagingofCa2+ signalinginsecretoryorgans ofYellowCameleontransgenicmice. Sci.Rep. 8,15880(2018).

583.Tao,W.etal.ApracticalmethodformonitoringFRET-basedbiosensorsinliving animalsusingtwo-photonmicroscopy. Am.J.Physiol.CellPhysiol. 309, C724–C735(2015).

584.Thunemann,M.etal.CorrelativeintravitalimagingofcGMPsignalsandvasodilationinmice. Front.Physiol. 5,394(2014).

585.Radbruch,H.etal.IntravitalFRET:probingcellularandtissuefunctioninvivo. Int.J.Mol.Sci. 16,11713–11727(2015).

586.Groves,J.T.&Dustin,M.L.Supportedplanarbilayersinstudiesonimmunecell adhesionandcommunication. J.Immunol.Methods 278,19–32(2003).

587.Sackmann,E.Supportedmembranes:scientiﬁcandpracticalapplications. Science 271,43–48(1996).

588.Dustin,M.L.etal.Anoveladaptorproteinorchestratesreceptorpatterningand cytoskeletalpolarityinT-cellcontacts. Cell 94,667–677(1998).

589.Groves,J.T.&Boxer,S.G.Micropatternformationinsupportedlipidmembranes. Acc.Chem.Res. 35,149–157(2002).

590.Dustin,M.L.,Bromley,S.K.,Davis,M.M.&Zhu,C.Identiﬁcationofselfthrough two-dimensionalchemistryandsynapses. Annu.Rev.CellDev.Biol. 17,133–157 (2001).

591.Kim,J.etal.mGRASPenablesmappingmammaliansynapticconnectivitywith lightmicroscopy. Nat.Methods 9,96–102(2012).

592.Carpenter,M.A.etal.Proteinproximityobservedusing ﬂuorogenactivating proteinanddyeactivatedbyproximalanchoring(FAP-DAPA)system. ACS Chem.Biol. 15,2433–2443(2020).

593.Szent-Gyorgyi,C.etal.Malachitegreenmediateshomodimerizationofantibody VLdomainstoforma ﬂuorescentternarycomplexwithsingularsymmetric interfaces. J.Mol.Biol. 425,4595–4613(2013).

594.Telmer,C.A.etal.Rapid,specific,no-wash,far-red fluorogenactivationin subcellularcompartmentsbytargeted fluorogenactivatingproteins. ACSChem. Biol. 10,1239–1246(2015).

595.Pratt,C.P. StudiesofBKChannelTrafﬁckingandSynapticVesicleRecyclingwith Fluorogen-ActivatingPeptidesApplicationsofFAPsforNeurobiologicalImaging (CarnegieMellonUniversity,2017).

596.He,J.etal.Anear-infraredgeneticallytargetableandactivatablephotosensitizer. Nat.Methods 13,263(2016).

597.Kuljis,D.A.etal.Fluorescence-basedquantitativesynapseanalysisforcelltypespeciﬁcconnectomics. eNeuro 6,ENEURO.0193 –19(2019).

598.Bulgari,D.etal.Activity-evokedandspontaneousopeningofsynapticfusion pores. Proc.Natl.Acad.Sci.USA 116,17039–17044(2019).

599.Stack,E.C.,Wang,C.,Roman,K.A.&Hoyt,C.C.Multiplexedimmunohistochemistry,imaging,andquantitation:areview,withanassessmentofTyramidesignalampliﬁcation,multispectralimagingandmultiplexanalysis. Methods 70,46–58(2014).

600.Werner,M.,VonWasielewski,R.&Komminoth,P.Antigenretrieval,signal ampliﬁcationandintensiﬁcationinimmunohistochemistry. HistochemCellBiol. 105,253–260(1996).

601.Pasqual,G.etal.MonitoringTcell-dendriticcellinteractionsinvivobyintercellularenzymaticlabelling. Nature 553,496–500(2018).

602.Ge,Y.etal.Enzyme-mediatedintercellularproximitylabelingfordetectingcellcellinteractions. J.Am.Chem.Soc. 141,1833–1837(2019).

603.Liu,Z.etal.Detectingtumorantigen-speciﬁcTcellsviainteraction-dependent fucosyl-biotinylation. Cell 183,1117–1133e1119(2020).

604.Schumacher,T.N.&Schreiber,R.D.Neoantigensincancerimmunotherapy. Science 348,69–74(2015).

605.Kim,D.I.&Roux,K.J.Fillingthevoid:proximity-basedlabelingofproteinsin livingcells. TrendsCellBiol. 26,804–817(2016).

606.Lobingier,B.T.etal.Anapproachtospatiotemporallyresolveproteininteractionnetworksinlivingcells. Cell 169,350–360e312(2017).

607.Roux,K.J.,Kim,D.I.,Raida,M.&Burke,B.Apromiscuousbiotinligasefusion proteinidentiﬁesproximalandinteractingproteinsinmammaliancells. J.Cell Biol. 196,801–810(2012).

608.Guo,Z.etal.E-cadherininteractomecomplexityandrobustnessresolvedby quantitativeproteomics. Sci.Signal 7,rs7(2014).

609.Li,Y.etal.TheN-cadherininteractomeinprimarycardiomyocytesasdeﬁned usingquantitativeproximityproteomics. J.CellSci. 132,jcs221606(2019).

610.VanItallie,C.M.etal.BiotinligasetaggingidentiﬁesproteinsproximaltoEcadherin,includinglipomapreferredpartner,aregulatorofepithelialcell–cell andcell–substrateadhesion. J.CellSci. 127,885–895(2014).

611.Fredriksson,K.etal.Proteomicanalysisofproteinssurroundingoccludinand claudin-4revealstheirproximitytosignalingandtrafﬁckingnetworks. PLoSONE 10,e0117074(2015).

612.Na,Y.etal.Fbxo45bindsSPRYmotifsintheextracellulardomainofN-cadherin andregulatesneuronmigrationduringbraindevelopment. Mol.CellBiol. 40, e00539–19(2020).

613.Shafraz,O.,Xie,B.,Yamada,S.&Sivasankar,S.Mappingtransmembranebinding partnersforE-cadherinectodomains. Proc.Natl.Acad.Sci.USA 117, 31157–31165(2020).

614.Branon,T.C.etal.Efﬁcientproximitylabelinginlivingcellsandorganismswith TurboID. Nat.Biotechnol. 36,880–887(2018).

615.Iskratsch,T.,Wolfenson,H.&Sheetz,M.P.Appreciatingforceandshape-therise ofmechanotransductionincellbiology. Nat.Rev.Mol.CellBiol. 15,825–833 (2014).

616.Sanford,K.K.,Likely,G.D.&Earle,W.R.Thedevelopmentofvariationsin transplantabilityandmorphologywithinacloneofmouse ﬁbroblaststransformedtosarcoma-producingcellsinvitro. J.Natl.CancerInst. 15,215–237 (1954).

617.Temin,H.M.&Rubin,H.CharacteristicsofanassayforRoussarcomavirusand Roussarcomacellsintissueculture. Virology 6,669–688(1958).

618.Sheetz,M.P.&Singer,S.Biologicalmembranesasbilayercouples.Amolecular mechanismofdrug-erythrocyteinteractions. Proc.Natl.Acad.Sci.USA 71, 4457–4461(1974).

619.Gauthier,N.C.,Fardin,M.A.,Roca-Cusachs,P.&Sheetz,M.P.Temporary increaseinplasmamembranetensioncoordinatestheactivationofexocytosis andcontractionduringcellspreading. Proc.Natl.Acad.Sci.USA 108, 14467–14472(2011).

620.Juliano,R.L.&Haskill,S.Signaltransductionfromtheextracellularmatrix. J.Cell Biol. 120,577–585(1993).

621.Martin,P.Woundhealing-aimingforperfectskinregeneration. Science 276, 75–81(1997).

622.Bernstein,L.R.&Liotta,L.A.Molecularmediatorsofinteractionswithextracellularmatrixcomponentsinmetastasisandangiogenesis. Curr.Opin.Oncol. 6, 106(1994).

623.Lauffenburger,D.A.&Horwitz,A.F.Cellmigration:aphysicallyintegrated molecularprocess. Cell 84,359–369(1996).

624.Dembo,M.&Wang,Y.-L.Stressesatthecell-to-substrateinterfaceduring locomotionof ﬁbroblasts. Biophys.J. 76,2307–2316(1999).

625.Huse,M.Mechanicalforcesintheimmunesystem. Nat.Rev.Immunol. 17, 679–690(2017).

626.Polacheck,W.J.&Chen,C.S.Measuringcell-generatedforces:aguidetothe availabletools. Nat.Methods 13,415–423(2016).

627.Roca-Cusachs,P.,Conte,V.&Trepat,X.Quantifyingforcesincellbiology. Nat. CellBiol. 19,742–751(2017).

628.Style,R.W.etal.Tractionforcemicroscopyinphysicsandbiology. SoftMatter 10,4047–4055(2014).

629.Colin-York,H.etal.Spatiotemporallysuper-resolvedvolumetrictractionforce microscopy. NanoLett. 19,4427–4434(2019).

630.Colin-York,H.etal.Super-resolvedtractionforcemicroscopy(STFM). NanoLett. 16,2633–2638(2016).

631.Plotnikov,S.V.,Sabass,B.,Schwarz,U.S.&Waterman,C.M.High-resolution tractionforcemicroscopy. MethodsCellBiol. 123,367–394(2014).

632.Stubb,A.etal.Fluctuation-basedsuper-resolutiontractionforcemicroscopy. NanoLett. 20,2230–2245(2020).

633.Heinz,W.F.&Hoh,J.H.Spatiallyresolvedforcespectroscopyofbiologicalsurfaces usingtheatomicforcemicroscope. TrendsBiotechnol. 17,143–150(1999).

634.Binnig,G.,Quate,C.F.&Gerber,C.Atomicforcemicroscope. Phys.Rev.Lett. 56, 930–933(1986).

635.Giessibl,F.J.Advancesinatomicforcemicroscopy. Rev.Mod.Phys. 75,949 (2003).

636.Radmacher,M.,Fritz,M.,Kacher,C.M.,Cleveland,J.P.&Hansma,P.K.Measuring theviscoelasticpropertiesofhumanplateletswiththeatomicforcemicroscope. Biophys.J. 70,556–567(1996).

637.Hofmann,U.G.,Rotsch,C.,Parak,W.J.&Radmacher,M.Investigatingthe cytoskeletonofchickencardiocyteswiththeatomicforcemicroscope. J.Struct. Biol. 119,84–91(1997).

638.Rotsch,C.,Braet,F.,Wisse,E.&Radmacher,M.AFMimagingandelasticity measurementsonlivingratlivermacrophages. CellBiol.Int. 21,685–696(1997).

639.Perez,R.,Garcia,R.&Schwarz,U.High-resolutionnoncontactatomicforce microscopy. Nanotechnology 20,260201(2009).

640.Casuso,I.etal.Characterizationofthemotionofmembraneproteinsusing high-speedatomicforcemicroscopy. Nat.Nanotechnol. 7,525–529(2012).

641.Kodera,N.,Yamamoto,D.,Ishikawa,R.&Ando,T.Videoimagingofwalking myosinVbyhigh-speedatomicforcemicroscopy. Nature 468,72–76(2010).

642.Miyagi,A.,Chipot,C.,Rangl,M.&Scheuring,S.High-speedatomicforce microscopyshowsthatannexinVstabilizesmembranesonthesecondtimescale. Nat.Nanotechnol. 11,783–790(2016).

643.Uchihashi,T.,Iino,R.,Ando,T.&Noji,H.High-speedatomicforcemicroscopy revealsrotarycatalysisofrotorlessF1-ATPase. Science 333,755–758(2011).

644.Heath,G.R.&Scheuring,S.High-speedAFMheightspectroscopyrevealsμsdynamicsofunlabeledbiomolecules. Nat.Commun. 9,4983(2018).

645.Ando,T.High-speedatomicforcemicroscopycomingofage. Nanotechnology 23,062001(2012).

646.Heath,G.R.&Scheuring,S.Advancesinhigh-speedatomicforcemicroscopy (HS-AFM)revealdynamicsoftransmembranechannelsandtransporters. Curr. Opin.Struct.Biol. 57,93–102(2019).

647.Sakiyama,Y.,Mazur,A.,Kapinos,L.E.&Lim,R.Y.Spatiotemporaldynamicsof thenuclearporecomplextransportbarrierresolvedbyhigh-speedatomicforce microscopy. Nat.Nanotechnol. 11,719–723(2016).

648.Sun,Z.,Hashemi,M.,Warren,G.,Bianco,P.R.&Lyubchenko,Y.L.Dynamicsof theinteractionofRecGproteinwithstalledreplicationforks. Biochemistry 57, 1967–1976(2018).

649.Vert,G.&Chory,J.Crosstalkincellularsignaling:backgroundnoiseorthereal thing? Dev.Cell 21,985–991(2011).

650.Lee,H.-W.etal.Real-timesingle-moleculeco-immunoprecipitationanalyses revealcancer-speci ﬁcRassignallingdynamics. Nat.Commun. 4,1505(2013).

651.Grigoriev,A.Onthenumberofprotein–proteininteractionsintheyeastproteome. NucleicAcidsRes. 31,4157–4161(2003).

652.Kerrien,S.etal.IntAct opensourceresourceformolecularinteractiondata. NucleicAcidsRes. 35,D561–D565(2007).

653.Monti,M.,Orrù,S.,Pagnozzi,D.&Pucci,P.Interactionproteomics. Biosci.Rep. 25, 45–56(2005).

654.Phee,B.K.etal.Identiﬁcationofphytochrome‐interactingproteincandidatesin Arabidopsisthaliana byco‐immunoprecipitationcoupledwithMALDI‐TOFMS. Proteomics 6,3671–3680(2006).

655.Ren,L.,Emery,D.,Kaboord,B.,Chang,E.&Qoronﬂeh,M.W.Improvedimmunomatrixmethodstodetectprotein:proteininteractions. J.BiochemBiophys. Methods 57,143–157(2003).

656.Wang,W.,Miao,F.,Wu,D.,Yang,J.&Wang,Z.Applicationofproteomics technologyinstudyofcellsignalingtransduction. Biotechnol.Bull. 11,46(2013).

657.Alberts,B.Thecellasacollectionofproteinmachines:preparingthenext generationofmolecularbiologists. Cell 92,291–294(1998).

658.Paul,F.E.,Hosp,F.&Selbach,M.Analyzingprotein–proteininteractionsby quantitativemassspectrometry. Methods 54,387–395(2011).

659.Jia,J.etal.Eukaryoticexpression,Co-IPandMSidentifyBMPR-1Bproteinproteininteractionnetwork. Biol.Res. 53,24(2020).

660.Cao,J.-Y.,Xu,Y.-P.&Cai,X.-Z.TMT-basedquantitativeproteomicsanalyses revealnoveldefensemechanismsofBrassicanapusagainstthedevastating necrotrophicpathogen Sclerotiniasclerotiorum J.Proteom. 143,265–277(2016).

661.Peng,Y.etal.AngiogenininteractswithribonucleaseinhibitorregulatingPI3K/ AKT/mTORsignalingpathwayinbladdercancercells. CellSignal 26,2782–2792 (2014).

662.Li,L.etal.Ribonucleaseinhibitorup-regulationinhibitsthegrowthandinduces apoptosisinmurinemelanomacellsthroughrepressionofangiogeninandILK/ PI3K/AKTsignalingpathway. Biochimie 103,89–100(2014).

663.Shu,J.etal.Downregulationofangiogenininhibitsthegrowthandinduces apoptosisinhumanbladdercancercellsthroughregulatingAKT/mTORsignalingpathway. J.Mol.Histol. 46,157–171(2015).

664.Ito,T.etal.Acomprehensivetwo-hybridanalysistoexploretheyeastprotein interactome. Proc.Natl.Acad.Sci.USA 98,4569–4574(2001).

665.VonMering,C.etal.Comparativeassessmentoflarge-scaledatasetsof protein–proteininteractions. Nature 417,399–403(2002).

666.Patel,S.J.etal.Identiﬁcationofessentialgenesforcancerimmunotherapy. Nature 548,537–542(2017).

667.Dunn,G.P.,Koebel,C.M.&Schreiber,R.D.Interferons,immunityandcancer immunoediting. Nat.Rev.Immunol. 6,836–848(2006).

668.Vredevoogd,D.W.etal.Augmentingimmunotherapyimpactbylowering tumorTNFcytotoxicitythreshold. Cell 178,585–599.e515(2019).

669.Morsut,L.etal.Engineeringcustomizedcellsensingandresponsebehaviors usingsyntheticnotchreceptors. Cell 164,780–791(2016).

670.Roybal,K.T.etal.EngineeringTcellswithcustomizedtherapeuticresponse programsusingsyntheticNotchreceptors. Cell 167,419–432e416(2016).

671.Toda,S.,Blauch,L.R.,Tang,S.K.Y.,Morsut,L.&Lim,W.A.Programmingselforganizingmulticellularstructureswithsyntheticcell-cellsignaling. Science 361, 156–162(2018).

672.Gordon,W.R.etal.Mechanicalallostery:evidenceforaforcerequirementinthe proteolyticactivationofNotch. Dev.Cell 33,729–736(2015).

673.Artavanis-Tsakonas,S.,Rand,M.D.&Lake,R.J.Notchsignaling:cellfatecontrol andsignalintegrationindevelopment. Science 284,770–776(1999).

674.Barrett,D.M.,Teachey,D.T.&Grupp,S.A.Toxicitymanagementforpatients receivingnovelT-cellengagingtherapies. Curr.Opin.Pediatr. 26 ,43 – 49 (2014).

675.Gajewski,T.F.,Schreiber,H.&Fu,Y.-X.Innateandadaptiveimmunecellsinthe tumormicroenvironment. Nat.Immunol. 14,1014–1022(2013).

676.Xu,C.,Ma,D.,Ding,Q.,Zhou,Y.&Zheng,H.L.PlantPhoneDB:Amanually curatedpan-plantdatabaseofligand-receptorpairsinferscell-cellcommunication. PlantBiotechnol.J. 20,2123–2134(2022).

677.Thurley,K.etal.Reliableencodingofstimulusintensitieswithinrandom sequencesofintracellularCa2+ spikes. Sci.Signal 7,ra59(2014).

678.Liang,D.,Minikes,A.M.&Jiang,X.Ferroptosisattheintersectionoflipid metabolismandcellularsignaling. Mol.Cell 82,2215–2227(2022).

679.Guo,P.etal.Reassemblyofpeptidenanoﬁbrilsonlivecellsurfacespromotes cell-cellinteractions. NanoLett. 23,6386–6392(2023).

680.Bondos,S.E.,Dunker,A.K.&Uversky,V.N.Ontherolesofintrinsicallydisorderedproteinsandregionsincellcommunicationandsignaling. CellCommun.Signal 19,88(2021).

681.vanNiel,G.etal.Challengesanddirectionsinstudyingcell-cellcommunication byextracellularvesicles. Nat.Rev.Mol.CellBiol. 23,369–382(2022).

682.Klein,A.M.&Macosko,E.InDropsandDrop-seqtechnologiesforsingle-cell sequencing. LabChip 17,2540–2541(2017).

683.Ziegenhain,C.etal.Comparativeanalysisofsingle-cellRNAsequencing methods. Mol.Cell 65,631–643e634(2017).

684.Stoeckius,M.etal.Simultaneousepitopeandtranscriptomemeasurementin singlecells. Nat.Methods 14,865–868(2017).

685.M,P.N.etal.Estimatingtheallele-speciﬁcexpressionofSNVsfrom10x genomicssingle-cellRNA-sequencingdata. Genes 11,240(2020).

686.Jin,S.etal.Inferenceandanalysisofcell-cellcommunicationusingCellChat. Nat.Commun. 12,1088(2021).

687.Buccitelli,C.&Selbach,M.mRNAs,proteinsandtheemergingprinciplesofgene expressioncontrol. Nat.Rev.Genet. 21,630–644(2020).

688.Vento-Tormo,R.etal.Single-cellreconstructionoftheearlymaternal-fetal interfaceinhumans. Nature 563,347–353(2018).

689.Noel,F.etal.DissectionofintercellularcommunicationusingthetranscriptomebasedframeworkICELLNET. Nat.Commun. 12,1089(2021).

690.Cabello-Aguilar,S.etal.SingleCellSignalR:inferenceofintercellularnetworks fromsingle-celltranscriptomics. NucleicAcidsRes. 48,e55(2020).

691.Zhang,Y.etal.CellCall:integratingpairedligand-receptorandtranscription factoractivitiesforcell-cellcommunication. NucleicAcidsRes. 49,8520–8534 (2021).

692.Hou,R.,Denisenko,E.,Ong,H.T.,Ramilowski,J.A.&Forrest,A.R.R.Predicting cell-to-cellcommunicationnetworksusingNATMI. Nat.Commun. 11,5011 (2020).

693.Efremova,M.,Vento-Tormo,M.,Teichmann,S.A.&Vento-Tormo,R.CellPhoneDB:inferringcell-cellcommunicationfromcombinedexpressionofmultisubunitligand-receptorcomplexes. Nat.Protoc. 15,1484–1506(2020).

694.Wang,Y.etal.iTALK:anRpackagetocharacterizeandillustrateintercellular communication.Preprintat https://www.biorxiv.org/content/10.1101/507871v1 (2019).

695.Bernhagen,J.etal.MIFisanoncognateligandofCXCchemokinereceptorsin inﬂammatoryandatherogeniccellrecruitment. Nat.Med. 13,587–596(2007).

696.Khozyainova,A.A.etal.Complexanalysisofsingle-cellRNAsequencingdata. Biochemistry 88,231–252(2023).

697.Tyler,S.R.etal.PyMINEr ﬁndsgeneandautocrine-paracrinenetworksfrom humanIsletscRNA-seq. CellRep. 26,1951–1964e1958(2019).

698.Cillo,A.R.etal.Immunelandscapeofviral-andcarcinogen-drivenheadand neckcancer. Immunity 52,183–199e189(2020).

699.Browaeys,R.,Saelens,W.&Saeys,Y.NicheNet:modelingintercellularcommunicationbylinkingligandstotargetgenes. Nat.Methods 17,159–162(2020).

700.Turei,D.etal.Integratedintra-andintercellularsignalingknowledgeformulticellularomicsanalysis. Mol.Syst.Biol. 17,e9923(2021).

701.Choi,H.etal.Transcriptomeanalysisofindividualstromalcellpopulations identiﬁesstroma-tumorcrosstalkinmouselungcancermodel. CellRep. 10, 1187–1201(2015).

702.Cheng,J.,Zhang,J.,Wu,Z.&Sun,X.Inferringmicroenvironmentalregulationof geneexpressionfromsingle-cellRNAsequencingdatausingscMLnetwithan applicationtoCOVID-19. Brief.Bioinform 22,988–1005(2021).

703.Wang,S.,Karikomi,M.,MacLean,A.L.&Nie,Q.Celllineageandcommunication networkinferenceviaoptimizationforsingle-celltranscriptomics. NucleicAcids Res. 47,e66–e66(2019).

704.Wilk,A.J.,Shalek,A.K.,Holmes,S.&Blish,C.A.Comparativeanalysisofcell-cell communicationatsingle-cellresolution. Nat.Biotechnol. 42,470–483(2024).

705.Hu,Y.,Peng,T.,Gao,L.&Tan,K.CytoTalk:denovoconstructionofsignal transductionnetworksusingsingle-celltranscriptomicdata. Sci.Adv. 7, eabf1356(2021).

706.Baccin,C.etal.Combinedsingle-cellandspatialtranscriptomicsrevealthe molecular,cellularandspatialbonemarrownicheorganization. Nat.CellBiol. 22,38–48(2020).

707.Li,J.etal.Non-cell-autonomouscancerprogressionfromchromosomal instability. Nature 620,1080–1088(2023).

708.Quail,D.F.&Joyce,J.A.Themicroenvironmentallandscapeofbraintumors. CancerCell 31,326–341(2017).

709.Turei,D.,Korcsmaros,T.&Saez-Rodriguez,J.OmniPath:guidelinesandgateway forliterature-curatedsignalingpathwayresources. Nat.Methods 13,966–967 (2016).

710.Langfelder,P.&Horvath,S.WGCNA:anRpackageforweightedcorrelation networkanalysis. BMCBioinforma. 9,559(2008).

711.Camp,J.G.etal.Multilineagecommunicationregulateshumanliverbud developmentfrompluripotency. Nature 546,533–538(2017).

712.Cohen,M.etal.Lungsingle-cellsignalinginteractionmaprevealsbasophilrole inmacrophageimprinting. Cell 175,1031–1044e1018(2018).

713.vanDijk,D.etal.Recoveringgeneinteractionsfromsingle-celldatausingdata diffusion. Cell 174,716–729e727(2018).

714.Yasukawa,H.,Sasaki,A.&Yoshimura,A.Negativeregulationofcytokinesignalingpathways. Annu.Rev.Immunol. 18,143–164(2000).

715.Tsuyuzaki,K.,Ishii,M.&Nikaido,I.Sctensordetectsmany-to-manycell-cell interactionsfromsinglecellRNA-sequencingdata. BMCBioinforma. 24,420 (2023).

716.Zheng,R.etal.Epsinnanotherapyregulatescholesteroltransporttofortify atheromaregression. Circ.Res. 132,e22–e42(2023).

717.Hatzimanikatis,V.,Richelle,A.,Joshi,C.&Lewis,N.E.Assessingkeydecisionsfor transcriptomicdataintegrationinbiochemicalnetworks. PLoSComput.Biol. 15, e1007185(2019).

718.Komurov,K.Modelingcommunity-widemolecularnetworksofmulticellular systems. Bioinformatics 28,694–700(2012).

719.Richelle,A.etal.Model-basedassessmentofmammaliancellmetabolicfunctionalitiesusingomicsdata. CellRep.Methods 1,100040(2021).

720.Zheng,G.X.etal.Massivelyparalleldigitaltranscriptionalproﬁlingofsingle cells. Nat.Commun. 8,14049(2017).

721.Genshaft,A.S.etal.Multiplexed,targetedproﬁlingofsingle-cellproteomesand transcriptomesinasinglereaction. GenomeBiol. 17,188(2016).

722.Stahlberg,A.,Thomsen,C.,Ruff,D.&Aman,P.QuantitativePCRanalysisofDNA, RNAs,andproteinsinthesamesinglecell. Clin.Chem. 58,1682–1691(2012).

723.Ponten,F.etal.Aglobalviewofproteinexpressioninhumancells,tissues,and organs. Mol.Syst.Biol. 5,337(2009).

724.Jin,J.,Yu,S.,Lu,P.&Cao,P.Decipheringplantcell-cellcommunicationsusing single-cellomicsdata. Comput.Struct.Biotechnol.J. 21,3690–3695(2023).

725.Lee,J.H.etal.HighlymultiplexedsubcellularRNAsequencinginsitu. Science 343,1360–1363(2014).

726.Lee,J.H.etal.Fluorescentinsitusequencing(FISSEQ)ofRNAforgene expressionproﬁlinginintactcellsandtissues. Nat.Protoc. 10,442–458(2015).

727.CuiZhou,D.etal.Spatiallyrestricteddriversandtransitionalcellpopulations cooperatewiththemicroenvironmentinuntreatedandchemo-resistantpancreaticcancer. Nat.Genet. 54,1390–1405(2022).

728.Kuppe,C.etal.Spatialmulti-omicmapofhumanmyocardialinfarction. Nature 608,766–777(2022).

729.Zhang,Q.etal.Leveragingspatialtranscriptomicsdatatorecovercelllocations insingle-cellRNA-seqwithCeLEry. Nat.Commun. 14,4050(2023).

730.Wang,G.,Mofﬁtt,J.R.&Zhuang,X.Multiplexedimagingofhigh-densitylibraries ofRNAswithMERFISHandexpansionmicroscopy. Sci.Rep. 8,4847(2018).

731.Fang,R.etal.Conservationanddivergenceofcorticalcellorganizationin humanandmouserevealedbyMERFISH. Science 377,56–62(2022).

732.Eng,C.L.etal.Transcriptome-scalesuper-resolvedimagingintissuesbyRNA seqFISH. Nature 568,235–239(2019).

733.Rao,A.,Barkley,D.,Franca,G.S.&Yanai,I.Exploringtissuearchitectureusing spatialtranscriptomics. Nature 596,211–220(2021).

734.Eisenstein,M.Howtomakespatialmapsofgeneactivity downtothecellular level. Nature 606,1036–1038(2022).

735.Chen,A.etal.Spatiotemporaltranscriptomicatlasofmouseorganogenesis usingDNAnanoball-patternedarrays. Cell 185,1777–1792e1721(2022).

736.Wei,X.etal.Single-cellStereo-seqrevealsinducedprogenitorcellsinvolvedin axolotlbrainregeneration. Science 377,eabp9444(2022).

737.Cang,Z.&Nie,Q.Inferringspatialandsignalingrelationshipsbetweencells fromsinglecelltranscriptomicdata. Nat.Commun. 11,2084(2020).

738.Tang,Z.,Zhang,T.,Yang,B.,Su,J.&Song,Q.spaCI:decipheringspatialcellular communicationsthroughadaptivegraphmodel. Brief.Bioinform 24,bbac563 (2023).

739.Pham,D.etal.stLearn:integratingspatiallocation,tissuemorphologyandgene expressionto ﬁndcelltypes,cell-cellinteractionsandspatialtrajectorieswithin undissociatedtissues.Preprintat https://www.biorxiv.org/content/10.1101/ 2020.05.31.125658v1 (2020).

740.Dries,R.etal.Giotto:atoolboxforintegrativeanalysisandvisualizationof spatialexpressiondata. GenomeBiol. 22,78(2021).

741.Tanevski,J.,Flores,R.O.R.,Gabor,A.,Schapiro,D.&Saez-Rodriguez,J. Explainablemultiviewframeworkfordissectingspatialrelationshipsfromhighly multiplexeddata. GenomeBiol. 23,97(2022).

742.Arnol,D.,Schapiro,D.,Bodenmiller,B.,Saez-Rodriguez,J.&Stegle,O.Modeling cell-cellinteractionsfromspatialmoleculardatawithspatialvariancecomponentanalysis. CellRep. 29,202–211e206(2019).

743.Shao,X.etal.Knowledge-graph-basedcell-cellcommunicationinferencefor spatiallyresolvedtranscriptomicdatawithSpaTalk. Nat.Commun. 13,4429 (2022).

744.Armingol,E.etal.Context-awaredeconvolutionofcell-cellcommunicationwith Tensor-cell2cell. Nat.Commun. 13,3665(2022).

745.Li,H.etal.Decodingfunctionalcell-cellcommunicationeventsbymulti-view graphlearningonspatialtranscriptomics. Brief.Bioinform 24,bbad359(2023).

746.Cang,Z.etal.Screeningcell-cellcommunicationinspatialtranscriptomicsvia collectiveoptimaltransport. Nat.Methods 20,218–228(2023).

747.Zhao,W.,Johnston,K.G.,Ren,H.,Xu,X.&Nie,Q.Inferringneuron-neuron communicationsfromsingle-celltranscriptomicsthroughNeuronChat. Nat. Commun. 14,1128(2023).

748.Rieckmann,J.C.etal.Socialnetworkarchitectureofhumanimmunecells unveiledbyquantitativeproteomics. Nat.Immunol. 18,583–593(2017).

749.Schapiro,D.etal.histoCAT:analysisofcellphenotypesandinteractionsin multipleximagecytometrydata. Nat.Methods 14,873–876(2017).

750.Keren,L.etal.Astructuredtumor-immunemicroenvironmentintriplenegative breastcancerrevealedbymultiplexedionbeamimaging. Cell 174,1373–1387 e1319(2018).

751.Li,D.,Ding,J.&Bar-Joseph,Z.Identifyingsignalinggenesinspatialsingle-cell expressiondata. Bioinformatics 37,968–975(2021).

752.Yuan,Y.&Bar-Joseph,Z.GCNG:graphconvolutionalnetworksforinferringgene interactionfromspatialtranscriptomicsdata. GenomeBiol. 21,300(2020).

753.Longo,S.K.,Guo,M.G.,Ji,A.L.&Khavari,P.A.Integratingsingle-cellandspatial transcriptomicstoelucidateintercellulartissuedynamics. Nat.Rev.Genet. 22, 627–644(2021).

754.Bienkowski,M.S.etal.Integrationofgeneexpressionandbrain-wideconnectivityrevealsthemultiscaleorganizationofmousehippocampalnetworks. Nat.Neurosci. 21,1628–1643(2018).

755.Fornito,A.,Arnatkeviciute,A.&Fulcher,B.D.Bridgingthegapbetweenconnectomeandtranscriptome. TrendsCogn.Sci. 23,34–50(2019).

756.Brown,S.P.&Hestrin,S.Intracorticalcircuitsofpyramidalneuronsreﬂecttheir long-rangeaxonaltargets. Nature 457,1133–1136(2009).

757.Kornberg,T.B.&Roy,S.Communicatingbytouch-neuronsarenotalone. Trends CellBiol. 24,370–376(2014).

758.Leong,A.T.etal.Long-rangeprojectionscoordinatedistributedbrain-wide neuralactivitywithaspeci ﬁcspatiotemporalproﬁle. Proc.Natl.Acad.Sci.USA 113,E8306–E8315(2016).

759.Xiao,Y.&Yu,D.Tumormicroenvironmentasatherapeutictargetincancer. Pharm.Ther. 221,107753(2021).

760.He,S.etal.High-pleximagingofRNAandproteinsatsubcellularresolutionin ﬁxedtissuebyspatialmolecularimaging. Nat.Biotechnol. 40,1794–1806(2022).

761.Derry,J.M.J.etal.Trackableintratumormicrodosingandspatialproﬁling provideearlyinsightsintoactivityofinvestigationalagentsintheintacttumor microenvironment. Clin.CancerRes. 29,3813–3825(2023).

762.Liu,Y.etal.High-spatial-resolutionmulti-omicssequencingviadeterministic barcodingintissue. Cell 183,1665(2020).

763.Bressan,D.,Battistoni,G.&Hannon,G.J.Thedawnofspatialomics. Science 381, eabq4964(2023).

764.Tracey,L.J.,An,Y.&Justice,M.J.CyTOF:anemergingtechnologyforsingle-cell proteomicsinthemouse. Curr.Protoc. 1,e118(2021).

765.Giesen,C.etal.Highlymultiplexedimagingoftumortissueswithsubcellular resolutionbymasscytometry. Nat.Methods 11,417–422(2014).

766.Angelo,M.etal.Multiplexedionbeamimagingofhumanbreasttumors. Nat. Med. 20,436–442(2014).

767.Blow,N.Tissuepreparation:tissueissues. Nature 448,959–963(2007).

768.Slavov,N.Unpickingtheproteomeinsinglecells. Science 367,512–513(2020).

769.Black,S.etal.CODEXmultiplexedtissueimagingwithDNA-conjugatedantibodies. Nat.Protoc. 16,3802–3835(2021).

770.Hansen,J.etal.Areferencetissueatlasforthehumankidney. Sci.Adv. 8, eabn4965(2022).

771.Mongia,A.etal.AnnoSpatannotatescelltypesandquantiﬁescellular arrangementsfromspatialproteomics. Nat.Commun. 15,3744(2024).

772.Schurch,C.M.etal.Coordinatedcellularneighborhoodsorchestrateantitumoralimmunityatthecolorectalcancerinvasivefront. Cell 183 ,838 (2020).

773.Huo,L.etal.Single-cellmulti-omicssequencing:applicationtrends,COVID-19, dataanalysisissuesandprospects. Brief.Bioinform 22,bbab229(2021).

774.Troulé,K.etal.CellPhoneDBv5:inferringcell-cellcommunicationfromsinglecellmultiomicsdata.Preprintat https://arxiv.org/abs/2311.04567 (2023).

775.Vandereyken,K.,Sifrim,A.,Thienpont,B.&Voet,T.Methodsandapplicationsfor single-cellandspatialmulti-omics. Nat.Rev.Genet. 24,494–515(2023).

776.Pang,L.etal.Microﬂuidics-basedsingle-cellresearchforintercellularinteraction. Front.CellDev.Biol. 9,680307(2021).

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s41392-024-01888-z

Signal Transduction and Targeted Therapy

Cell

cellcommunication:newinsightsandclinical implications

INTRODUCTION

RESEARCHHISTORYANDMILESTONEEVENTSOFCCC

MOLECULARMECHANISMOFCCC

MechanismsofCCCinphysiologicalhomeostasis

THECLINICALAPPLICATIONANDRESEARCHPROGRESSOF CCC

Cancers

Centralnervoussystemdiseases

Otherapplications

ChemicallytaggingCCCs

DOWNSTREAMANALYSISANDEXPERIMENTALVALIDATION

COMPUTATIONALMETHODSFORINFERRINGCCC

CHALLENGESANDPERSPECTIVES

ACKNOWLEDGEMENTS

AUTHORCONTRIBUTIONS

ADDITIONALINFORMATION

REFERENCES