18 APRIL 2025
Poster Competition Abstract Packet
AUTHOR: Ines Pulido Endrino
Integrative analysis of acquired drug resistance to KRAS(G12D) inhibitors.
KRAS is the most frequently mutated oncogene in cancer and, until recently, targeted therapies against mutant KRAS haven’t been successful. However, new breakthroughs in pharmacological design have led to the FDA-approval of two KRAS G12C inhibitors and one inhibitor specific to target the KRAS glycine to aspartic acid in codon 12 (G12D) mutation has shown strong responses in vitro and has entered phase I in clinical trials, MRTX1133. KRAS(G12D) is the most common KRAS mutation, being found in 37% of pancreatic ductal adenocarcinomas (PDACs), as well as in 12.5% of colorectal cancer and 4.9% of lung adenocarcinoma (LUAD) patients. MRTX1133, a selective non-covalent inhibitor of the KRAS(G12D) mutation, has demonstrated promising preclinical efficacy and has entered phase 1 clinical trials; however, the acquired drug resistance is expected to emerge. We have generated an acquired resistant model to KRASG12D inhibitors by drug escalation of MRTX1133. Our results show that although the IC50 for parental AsPC-1 cells is 0.2nM, drug resistant AsPC-1M-R cells can grow normally at 10 µM. Expression of KRAS, NRAS but more significantly HRAS are increased in the drug-resistant phenotype, likely to bypass inhibition of KRAS(G12D). Oncogenic KRAS(G12D) signaling isn’t essential in AsPC-1M-R cells as determined by a persistent RAS-GTP activity after MRTX1133 treatment due to the emergence of a secondary mutation in KRAS. In fact, tumor proliferation and survival in the resistant phenotype is sustained by increased receptor tyrosine kinase (RTK) phosphorylation. Evaluation of RTK activity demonstrated that resistant tumor cells activated several RTK such as the ErbB and Eph families and MET, predominantly. Dual inhibition with SHP2, SOS or ErbB in combination with MRTX113 didn’t exhibit a synergistic effect. Novel therapeutic approaches such as RAS(ON) inhibitors like RMC-6236 or specific KRAS(G12D) PROTAC degraders can significantly inhibit cell proliferation in vitro not only in MRTX1133 acquired resistance but also in adagrasib resistance models. To determine the underlying mechanisms of acquired drug resistance to MRTX1133, RNAseq analysis was performed. Complex transcriptomic reorganization shows the upregulation of CD24 -a potential cancer stem cell related gene- and the epithelial to mesenchymal signature highlight the difficulty to target drug resistant cells. These findings emphasize the necessity to design combinatorial treatments to prevent and delay the acquisition of drug resistance in KRAS-driven cancers.
AUTHOR: Jordan Vellky
GLP1R Expression is Prevalent in Prostate Cancer Patients and GLP1R Agonism (Ozempic) has Inhibitory Effects on Prostate Cancer Cells
Purpose As a disease of aging, metabolic co-morbidities are managed concurrently with prostate cancer. Recent FDA approvals have launched widespread use of GLP1 receptor agonist semaglutide for Type II Diabetes Mellitus and obesity. Mechanistically, semaglutide primarily activates GLP1 receptors in the pancreas. However, GLP1R is also expressed in advanced prostate cancer tissues where GLP1 agonism could affect signaling and disease progression. Understanding the effects of semaglutide treatment in prostate cancer is critical to gain a complete picture of the systemic effects this drug and potentially expose additional contexts where semaglutide can be therapeutically valuable in this disease space. Hypothesis Because GLP1R is expressed in advanced prostate cancer, exposure to GLP1R agonists could directly affect growth and signaling of cells in the prostate. Direct regulation of prostate cell signaling by incretin mimetics is clinically significant because many prostate cancer patients have metabolic co-morbidities (i.e. diabetes, obesity, metabolic syndrome) that are managed in parallel. Understanding the “off-target” effects of these drugs in the prostate could be an important clinical consideration. Patients and Methods RNA-sequencing data was accessed from dbGaP (consolidated, n=664) and Gene Expression Omnibus (GSE204057, n=55) to assess GLP1R expression in prostate cancer patients. Bioinformatics analyses were utilized to identify signaling pathways associated with GLP1R expression. Proliferation, metabolism, and signaling after GLP1R agonism with semaglutide were assessed in vitro in prostate cancer cell line models using incucyte, Seahorse, and a kinase proteome profiler array. Results We identified GLP1R expression in advanced prostate cancer samples including 145/310 (46.8%) of metastases, 59/115 (51.3%) of castration-resistant prostate cancer (CRPC), and 9/15 (60%) of neuroendocrine prostate cancer (NEPC), where it was associated with Notch and Hedgehog Signaling. Semaglutide treatment in pre-clinical prostate cancer models resulted in decreased cell proliferation, decreased glycolytic function, and decreased activation of kinase-mediated signaling. This overall suppression of signaling downstream of GLP1R is consistent with Gaicoupled GPCR signaling, which was confirmed using a cAMP assay. Interestingly, in a transdifferentiation model there was a significant negative correlation between GLP1R and AR expression. Further, proliferation of prostate cancer cells was significantly decreased with combination treatment of enzalutamide with semaglutide compared to either treatment alone. Conclusions GLP1R was expressed in advanced prostate cancer patients and treatment with semaglutide suppressed growth, metabolism, and signaling. This is clinically significant due to concurrent treatment for metabolic co-morbidities in men with prostate cancer. Our preliminary data shows that co-targeting AR and GLP1R decreased cell growth, suggesting GLP1R agonism may be therapeutically useful in combination with androgen receptor antagonists in advanced prostate cancer.
AUTHOR: Sai Santosh Babu Komakula
The role of myeloid cell-derived HMGB1 in the development of hepatocellular carcinoma
Background: Liver cancer is the 5th most common cancer and the 2nd most frequent cause of cancer-related death worldwide. Hepatocellular carcinoma (HCC) represents 80% of all primary liver cancers and is a major public health concern. The tumor microenvironment (TME) plays a central role in HCC onset, progression, and prognosis. The immune system usually identifies and eliminates tumor cells, yet tumors develop strategies to impair immune cell function by reducing antigenicity and exploiting feedback inhibition, thereby facilitating neoplastic progression. Among the mechanisms contributing to immune escape are impaired antigen presentation by dendritic cells (DCs), CD8+ cytotoxic T lymphocytes (CTLs) dysfunction, and immune checkpoints. High mobility group box 1 (HMGB1) is a non-histone chromatin-associated protein involved in the pathogenesis of chronic liver disease. HMGB1 is expressed in myeloid cells, including conventional dendritic cells (cDCs), which play a major role in the TME. However, whether myeloid cell-derived HMGB1 is involved in HCC is unknown. We hypothesize that intracellular HMGB1 drives cDC maturation towards LAMP3+ DCs, allowing effective cytotoxic CD8+ T cell responses to reduce HCC. Methods: We analyzed scRNA-seq datasets from human HCC for the expression of HMGB1 in all subsets of DCs in HCC tumor and non-tumor tissues and in hepatic draining lymph nodes. We generated mice with conditional ablation or overexpression of Hmgb1 in myeloid cells (Hmgb1_Mye and Hmgb1KI Mye). To induce HCC, 14-day-old male mice were injected i.p. with diethylnitrosamine (DEN). Immune cell populations were analyzed in tumor and non-tumor tissues using flow cytometry. cDCs were isolated from tumor and non-tumor tissues by FACS, and RNA-seq analysis was performed. Bone marrowderived dendritic cells (BMDCs) were used to analyze the DCs activation and maturation status. Results: Analysis of scRNA-seq datasets revealed LAMP3+ DCs increased in human HCC tumor tissue and hepatic dLNs. LAMP3+ DCs displayed increased activation and migration scores. Moreover, HMGB1 was highly expressed in cDCs (CLEC9A+ and CD1C+), which can differentiate to LAMP3+ DCs. Hmgb1KI Mye mice were protected from HCC, whereas control mice developed HCC after 8 months, and Hmgb1_Mye mice started developing HCC at 5 months. Macroscopic analysis and H&E staining of the livers from Hmgb1_Mye mice showed more tumors and higher tumor volume than control and Hmgb1KI Mye mice. Immunohistochemistry of HCC tumor sections revealed that Hmgb1_Mye mice have fewer LAMP3+ DCs in tumor tissues than control and Hmgb1KI Mye mice. Flow cytometry analysis showed that Hmgb1KI Mye mice had significantly increased cDCs in tumor tissues and dLNs than control and Hmgb1_Mye mice. RNA-seq data of cDCs unveiled that pathways involved in antigen presentation were downregulated in Hmgb1_Mye mice and upregulated in Hmgb1KI Mye mice compared to control mice. BMDCs of Hmgb1KI Mye mice showed enhanced DC activation and maturation markers and promoted CTLs proliferation and activation. Conclusions: The ablation of myeloid-derived HMGB1 accelerates HCC development in mice. Enhancing HMGB1 expression in myeloid cells increases the antigen presentation activity of cDCs. Therefore, increasing HMGB1 expression in cDCs could be a therapeutic approach to protect against HCC, paving the way for novel DC vaccine development.
AUTHOR: Purab Pal
Exploring
Pro-Apoptotic
Vulnerabilities in Endocrine Therapy-Resistant Breast Cancer Due to Sphingolipidomic Alterations
Background Endocrine therapy (ET) is a common and effective treatment for many patients with hormone receptor-positive breast cancer. Unfortunately, about 40% of these patients eventually develop resistance to ET, causing cancer to return or spread. Finding new ways to treat ET-resistant breast cancer is urgently needed. Our research focuses on a group of fat molecules called sphingolipids, which play important roles in how cells grow, survive, and die. One type of sphingolipid, called ceramide, is known to promote cell death in cancer cells. In our earlier studies, we found that ET-resistant breast cancer cells have unusually low levels of ceramide. Interestingly, when we added ceramide back to these cells, they were much more sensitive to dying than the non-resistant cancer cells. This raised an important question: why are ET-resistant cancer cells so vulnerable to ceramide, and can we use this to develop new treatments? Methods To answer this question, we used laboratory models of ET-resistant breast cancer cells. We treated these cells with ceramide and performed total RNA sequencing, which measures changes in gene expression. We also used advanced tools, including a special version of ceramide that acts like a probe, to identify which proteins in the cells directly interact with ceramide. By combining this probe with a method called quantitative proteomics, we were able to pinpoint which proteins mediate the unique ceramide action in ET-resistant cells. Results We discovered that adding ceramide to ET-resistant breast cancer cells triggers a specific kind of stress inside the cell, called endoplasmic reticulum stress (or, EnRS). This stress is normally a way for cells to manage misfolded proteins, but in these resistant cells, ceramide causes too much stress, leading to cell death. We found that this stress happens through a particular pathway involving proteins named PERK, p-eIF2_, and ATF4. When we blocked this pathway, the cells were less sensitive to ceramide, suggesting it plays a key role in the process. In addition, our proteomics analysis showed that ceramide interacts with several proteins located in the endoplasmic reticulum - the part of the cell where proteins are folded and processed. One of the top proteins we identified is called TRAM1. When we reduced the amount of TRAM1 in the ET-resistant cells, it mimicked the effects of ceramide treatment, causing the same kind of stress and cell death. Interestingly, we also found that patients with higher levels of these ceramide-interacting proteins tend to have more aggressive breast cancer and worse outcomes. Conclusions Our study shows that ceramide has a unique ability to kill ET-resistant breast cancer cells by overwhelming their stress management systems. This happens through interactions with specific proteins in the cells, leading to lethal levels of endoplasmic reticulum stress. Understanding how ceramide works in these resistant cells could help us design new treatments aimed at overcoming ET resistance and improving survival for patients facing this challenging form of breast cancer.
AUTHOR: Elie Abi Khalil
Immune landscape analysis of solid tumors via spatially guided cell isolation for enhanced immunotherapeutic development
BACKGROUND, PURPOSE, or OBJECTIVES: The tumor-immune microenvironment (TIME) in solid tumors is a complex ecosystem consisting of diverse interactions between cellular populations. Deeper investigation into the TIME reveals a prominent role of immune cells in regulating tumor progression- a novel phenomenon that has encouraged the evaluation of the crosstalk between cell types. The development of spatial omic technologies has thus enabled the exploration of these nuances at previously inaccessible resolutions, but the currently available platforms come at the cost of introducing bias, excluding molecular information, and incompatibility with single cell technologies. HYPOTHESIS/AIMS: Here, we leverage a photoactivatable (PA) dye Janelia-Fluor-646 in combination with a digital light processing (DLP) microscope to address these issues by developing a platform that enables the spatially guided isolation of cell populations from a region of interest (ROI) for downstream multiomic and single cell analyses. MATERIALS or METHODS: The tissue is initially harvested, fixed in 4% PFA, and sectioned into 400_m slices. This is followed by fluorescent antibody staining to define the ROI as well as incubation with the PA dye. Regions of interest are masked and photolabeled with the DLP microscope and the tissue is dissociated into a single-cell suspension for subsequent cell sorting. Collected cell populations are then lysed to extract nucleic acids and proteins for downstream analysis. RESULTS: As proof of concept, we show that ROIs identified via immunofluorescence could successfully be barcoded in mouse spleen and tumor sections using the dye and distinguished from the background by fluorescence activated cell sorting (FACS). We also demonstrate the ability to extract high quality DNA, RNA, and protein- as well as maintain high cell quality post-sorting. We have also developed a pipeline for the analysis of the sequenced RNA to identify differential patterns of gene expression present in immune cells from spatially distinct regions. CONCLUSIONS: Moving forward, we hope to establish this as a platform for immune landscape analysis within the TIME to uncover unique targets that could be explored within the context of tumor-immune interactions as well as immunotherapeutic development. Our findings will also help us generate the first single cell atlas of niche-specific immune cells.
AUTHOR: Paige, K. Malmrose
Evaluating the Role of MEIS1 in HOXB13-Dependent Prostate Cancer Progression and AR Signaling
BACKGROUND: Prostate Cancer (PrCa) is one of the leading causes of cancer-related death in men. Our lab has identified the HOX protein cofactor MEIS1 as a potential tumor suppressor in prostate biology. We have demonstrated that loss of MEIS1 protein expression strongly correlates with a higher likelihood of biochemical recurrence and metastatic progression (Bhanvadia et al., Clin Can Res 2018). Further, MEIS1 suppresses proliferation and cancer progression in a HOXB13-dependent manner (Van Opstall, ELife 2021). Androgen Receptor (AR), a key driver of prostate cancer, has been documented to interact with HOXB13 to promote prostate cancer progression. Despite the evidence that MEIS proteins are likely tumor-suppressive in PrCa, the interaction and functional impact between MEIS1, HOXB13, and AR has not been clearly defined. HYPOTHESIS: We hypothesize that increased MEIS1 expression reduces oncogenic AR-HOXB13 interactions and decreases oncogenic AR signaling.
METHODS: Our laboratory has derived cell lines overexpressing lentiviral constructs of MEIS1 and/or CRISPR-generated knockouts of HOXB13 in both castration-resistant CWR-22Rv1 and androgen-sensitive LAPC4 cell lines. The nuclear interaction between MEIS1 and AR was evaluated with co-immunoprecipitations and proximity ligation assays. IncuCyte Live-Cell Analysis Systems were used to elucidate the in vitro growth phenotype of these prostate cancer cells under AR pathway modulation. qPCR and RNA-sequencing were conducted to identify MEIS1-regulated genes that confer tumor suppression and modulate AR-associated gene targets. RESULTS: Proliferation assays demonstrate that under AR-stimulated conditions, increased MEIS1 expression decreases the proliferation of prostate cancer cells. In addition, the presence of MEIS1 decreases AR target gene expression and AR signaling. CONCLUSIONS: These data support our hypothesis that MEIS1 has a HOXB13-dependent tumor suppressive role in prostate cancer cell lines. However, the functional role of MEIS1 proteins and how the MEIS1AR complex regulates transcription in the prostate is not clear. Filling such knowledge gaps has the potential to reveal novel mechanisms of MEIS1 proteins to implement new biomarkers to predict prostate cancer progression and new targets to therapeutically block cancer metastasis.
AUTHOR: Andrew D. Gould
Race-Based Mutational Profiles and Survival Outcomes in Glioblastoma
Background: Glioblastoma (GBM) is the most common primary malignant brain cancer in adults and is characterized by significant molecular heterogeneity. Emerging evidence suggests that racial and ethnic differences in GBM mutational profiles may contribute to disparities in prognosis and treatment responses. Understanding these variations is critical for advancing personalized therapies and addressing disparities in patient outcomes. Objective: This study aims to identify differences in mutational profiles and survival outcomes of GBM among African American (AA), Hispanic (H), and Non-Hispanic White (NHW) patients. Methods: We retrospectively analyzed next-generation sequencing (NGS) reports from GBM tumors of patients diagnosed at the University of Illinois Hospital (UIH). Survival data were available for 114 patients (27 AAs, 23 Hs, and 64 NHWs). Among these, NGS data were available for 20 AA, 14 H, and 49 NHW patients. In 90% (75/83) of cases, mutation data were obtained from the UIH in-house NGS panel, which includes 161 driver mutations and 51 fusions; the remaining 10% (8/83) belonged to patients who had diagnosis craniotomy at other institutions with different NGS panels. Key genetic alterations-point mutations, copy number variations (gains and losses), and structural variants-were categorized by racial group. In addition, five known prognostic factors (age at diagnosis, gender, MGMT promoter methylation status, extent of resection [biopsy vs. gross total resection], and disease burden) were also assessed. KaplanMeier survival curves and Cox proportional hazards regression analyses were performed using GraphPad Prism. Results: Overall survival (OS), but not progression-free survival (PFS), differed significantly across racial groups, with Hispanic patients exhibiting longer OS than AAs and NHWs. Multivariate analysis identified male gender and multifocal disease as negative prognostic factors in H and NHW patients, but not in AAs. While the most common copy number alterations were similar across all groups, the distribution of key point mutations differed in AAs compared to Hs and NHWs. We further contextualized these mutations within three known gliomagenesis signaling pathways: receptor tyrosine kinase (RTK), cyclindependent kinase/retinoblastoma (CDK/Rb), and p53/MDM. Mutations not falling into these pathways were grouped as “Other.” The frequency of alterations within the RTK, CDK/Rb, and p53/MDM pathways was comparable across racial groups. However, distinct differences emerged within the “Other” category: SLX4 mutations were enriched in AAs, TERT mutations in Hs, and BRCA and FANC mutations in NHWs. Conclusion: Our findings highlight both shared and distinct molecular features of GBM across AA, H, and NHW patients. While core gliomagenesis pathways were similarly altered among groups, unique mutation patterns within the “Other” category suggest potential race-associated genomic differences. These disparities, along with variable prognostic factor impacts on survival, underscore the importance of incorporating racial and ethnic diversity in GBM research. A more nuanced understanding of these differences may inform future efforts in precision oncology and help reduce outcome disparities for GBM patients.
AUTHOR: Frida Pantoja
Targeting nicotinamide pathway to overcome drug resistance in KRAS Non-small cell lung cancer._
Despite being the most frequently mutated oncogene in cancer, KRAS has long been considered undruggable, and effective targeted therapies have only become available recently. The FDA has recently approved Sotorasib and Adagrasib specifically for NSCLC patients harboring the KRASG12C mutation. These drugs have demonstrated promising initial responses; however, many patients eventually relapse due to the development of drug resistance. Understanding the mechanisms underlying this resistance is crucial to improve therapeutic outcomes. Secondary mutations in KRAS itself or the activation of alternative signaling pathways that bypass KRAS inhibition are well-known mechanisms of resistance. To investigate this, we developed Adagrasib-resistant NSCLC cell lines carrying the KRASG12C mutation. Transcriptomic profiling using RNA-seq revealed significant upregulation of the epithelialto-mesenchymal transition (EMT) program, a cellular process associated with increased invasiveness and drug resistance. Notably, we observed elevated expression of nicotinamide N-methyltransferase (NNMT), a metabolic enzyme involved in methylation processes and NAD+ metabolism. Although NNMT has been implicated in tumor progression and metabolic reprogramming in other cancer types, its specific role in lung cancer drug resistance remains poorly understood. Our preliminary results suggest a strong association between NNMT overexpression and a mesenchymal-like, drug-resistant phenotype. Transcriptomic analysis of these resistant, EMT-positive models indicated that NNMT overexpression disrupts the nicotinamide metabolic pathway, leading to NAD+ depletion. NAD+ is a critical cofactor for cellular energy and the disruption of this pathway may contribute to metabolic vulnerabilities in drug-resistant cancer cells. Importantly, we identified Daporinad, a NAMPT inhibitor that targets NAD+ metabolism, as a potential therapeutic agent to target adagrasib resistant cells. Our data suggest that targeting NNMT or the NAD+ salvage pathway could represent a novel strategy to overcome resistance in KRASG12C mutant lung cancer. In conclusion, our study suggests that targeting these pathways may provide new avenues for improving treatment efficacy in NSCLC patients with drug-resistant tumors.
AUTHOR: Sanjay, S Ganesh
Uveal Melanoma with a commonly found mutation in GNA11 or GNAQ secretes VEGF to escape from the eyes into circulation
ABSTRACT: BACKGROUND, PURPOSE, or OBJECTIVES: Uveal melanoma (UM) is a highly aggressive ocular cancer with a high rate of metastasis. The endothelial barrier in the eyes plays a crucial role in preventing the escape of UM cells; however, the exact mechanisms by which UM breaches this barrier remain unclear. It is well established that metastatic UM tumors exhibit increased vascular permeability due to increased vascular endothelial growth factor (VEGF) production. This study investigated how UM cells with common mutations use VEGF to increase vascular permeability, allowing for transendothelial migration. Additionally, we explore the potential of VEGF inhibition as a therapeutic strategy to mitigate UM metastasis.
HYPOTHESIS/AIMS: We believe that UM cells rely on VEGF to disrupt the endothelial barrier and facilitate migration into systemic circulation. Specifically, we propose that VEGF increases endothelial permeability by interacting with its receptor, VEGFR2, which is trafficked to the endothelial surface by the kinesin motor protein KIF13B. We also suggest that blocking VEGF signaling with anti-VEGF therapy (Aflibercept) or inhibiting VEGFR2 trafficking could reduce UM transendothelial migration and subsequent metastasis. MATERIALS or METHODS: To assess endothelial permeability, we conducted a gelatin-trapping assay, using human retinal endothelial cells (hREC) cultured with conditioned media (CM) from UM cell lines, UM/endothelial cocultures, or VEGF-treated controls. Leakage areas were visualized using Alexa555-labeled streptavidin and VE-Cadherin co-staining. To evaluate UM cell transmigration, we used an organ-on-a-chip model, in which UM cells embedded in collagen gels were introduced adjacent to a pre-formed endothelial barrier. The effects of VEGF neutralization using Aflibercept and VEGFR2 trafficking inhibition via KAI-a novel peptide-were analyzed using in vivo studies using a murine orthotopic model where we injected UM cells into the suprachoroidal space of SCID mice. Treatment groups received either Aflibercept or KAI, and circulating tumor cells (CTCs) were quantified via qPCR eight weeks post-implantation. RESULTS: Our results demonstrated that CM from UM cells significantly increased endothelial permeability, comparable to VEGFtreated controls. VEGF neutralization with Aflibercept markedly reduced endothelial leakage, confirming that UM-induced permeability is VEGF-dependent. In the organ-on-a-chip model, Aflibercept successfully inhibited transmigration of MP41 and 92.1 UM cells lines, but not Mel202, which has additional mutations. KAI treatment effectively blocked VEGFR2 trafficking and impeded MP41 transendothelial migration. In vivo, KAI treatment reduced the number of mice with detectable CTCs, suggesting a potential role in preventing UM metastasis. CONCLUSIONS: This study highlights the critical role of VEGF in facilitating UM cell escape from the eye through increasing endothelial permeability. Our findings suggest that UM cells utilize VEGF to breach the endothelial barrier and promote metastasis. The efficacy of VEGF blockage with Aflibercept and VEGFR2 trafficking inhibition with KAI suggests promising therapeutic targets. Interestingly, UM cells with additional mutations may employ VEGF-independent mechanisms for transendothelial migration, necessitating further investigation into alternative metastatic pathways. This research provides a deeper understanding of UM metastasis mechanisms and potential strategies to prevent UM metastasis.
AUTHOR: Serena, C Thomas
Endothelial Ackr1 Knockdown Inhibits Neutrophil-Mediated Pulmonary Metastasis
Background: Metastasis is the leading cause of breast cancer-related mortality, however, the molecular mechanisms behind metastasis remain largely unknown. During metastasis, tumor cells detach from the primary tumor, enter the circulation, and spread to distant organs. However, the success of metastatic colonization is not solely determined by the tumor cells ability to disseminate; it is also critically influenced by the establishment of pre-metastatic niches in target organs. These pre-metastatic niches are specialized microenvironments that support the seeding, growth, and survival of incoming tumor cells. Neutrophils play a crucial role in forming these niches by promoting inflammation and releasing factors that facilitate tumor cell adhesion and extracellular matrix remodeling. In non-tumor contexts, extravasation of neutrophils into surrounding tissue is facilitated by expression of Atypical Chemokine Receptor 1(ACKR1) on endothelial cells, but this has not been examined in the context of metastasis. Hypothesis: We hypothesize that eliminating endothelial ACKR1 expression may reduce neutrophil extravasation into the lungs, thereby disrupting the formation of the premetastatic niche and decreasing metastasis. Methods: To study the role of ACKR1 in neutrophil extravasation, we investigated the expression of endothelial ACKR1 at the distant metastatic site of the lung, a common site of breast cancer metastasis. We analyzed the distribution of disseminating tumor cells and neutrophils relative to ACKR1-positive versus ACKR1-negative vessels in the lungs of orthotopic mammary tumor-bearing mice. To analyze disseminating tumor cells and extravasated neutrophils, we performed immunofluorescent staining of lung vasculature from immunocompetent mice implanted with E0771.LMB mammary tumor cells 28 days post-tumor injection. Lung samples were stained with ACKR1, CD31, and either PanCK (to label tumor cells) or Ly6G (to label neutrophils). A custom script was developed to outline venules and calculate the minimum distance between each target cell and the nearest vessel wall within 150 microns of a venule. These results were binned to categorize target cells based on their proximity within 50, 100, or 150 microns of an ACKR1-positive or -negative venule. Additionally, we analyzed lung sections collected from ACKR1 endothelial cell knockout (ACKR1ECKO) and control mice treated with tumor-conditioned media (TCM) for nine days. Results: Our analysis revealed a significant increase in the average number of tumor cells within 50 microns of ACKR1-positive venules compared to ACKR1-negative venules. Similarly, neutrophil analysis showed a significant increase of neutrophils within 50 microns of ACKR1positive venules. These results indicated that neutrophils are preferentially localized adjacent to ACKR1-positive vessels. Additionally, our findings revealed decreased neutrophil presence in the lungs of ACKR1ECKO mice compared to the control mice following TCM treatment. Conclusions: These results suggest that ACKR1 is a major player in neutrophil-mediated metastasis, enhancing the priming of the pre-metastatic niche and promoting subsequent metastatic colonization. Understanding this mechanism may open new avenues for therapeutic strategies aimed at mitigating breast cancer metastasis. For future experiments, we will further explore potential secreted factors responsible for Ackr1 upregulation in the lung.
AUTHOR: Vipin S Rawat
Serine starvation activates mTORC2 in ER+ breast cancer cells
Serine starvation activates mTORC2 in ER+ breast cancer cells Vipin Rawat1, Prarthana Prasanth1, Purab pal1, Mete Emir Ozgurses1, Godwin Sharpey1, Dayo Akinsiku1, Jonathan L Coloff1 1 University of Illinois at Chicago Background The non-essential amino acid L-serine (serine) plays an important role in cancer cell growth due to its diverse biosynthetic functions. In many cancer cells, serine can be taken up from the circulation or can be synthesized through the serine synthesis pathway (SSP). We have previously discovered that luminal breast cancers, which represents 40-70% of all breast tumors, are serine auxotrophic. Serine auxotrophy thus makes them completely dependent on the uptake of exogenous serine for growth and proliferation. Objective This project aims to identify the signaling changes in luminal cells after serine starvation. Results Strikingly, serine starvation leads to the activation of AKT, as evidenced by increased levels of p-AKT473 in luminal breast cancer cells. However, the activation mechanism of AKT under serine starvation is unknown. The S473 site on AKT can be phosphorylated in a PI3K and mTORC2-dependent manner. Similarly, activation of GCN2 by uncharged tRNA under amino acid starvation can also lead to the activation of mTORC2 which further increase the AKT phosphorylation at S473. To understand the mechanism of action how serine starvation increases the phosphorylation of AKT at S473, we treated the cells with Everolimus (mTORC1 inhibitor), Torin1 (mTORC1&2 inhibitor) and GCN2i. As expected, treatment with Torin1 and GCN2i inhibits the phosphorylation of AKT at S473, which proves that the increased phosphorylation of AKT under serine starvation is mTORC2 and GCN2 dependent. The mTORC2-mediated phosphorylation and activation of AKT is generally considered an anti-apoptotic response. Accordingly, when we treated the cells with Alpelisib (mutated PIK3CA), GDC0068 (Pan AKT inhibitor) and Torin1 under serine starvation, we observed increased cell death by PI staining and WB of cleaved PARP and anti-apoptotic protein XIAP. Conclusions In conclusion, we have discovered mTORC2 activation as a survival mechanism used by cancer cells under serine starvation. Our in-vitro data provide us an opportunity to target ER+ PIK3CA mutated breast cancer therapeutically in combination with serine-free diet with already approved therapy such as Alpelisib.
AUTHOR: Natalia A. Obacz
JAG1 expressed by breast tumor cells promotes lymphovascular invasion and lymph node metastasis
Metastasis to tumor draining lymph nodes (tdLNs) is a prognostic factor for breast cancer (BC) progression and correlates with poor patient outcomes. Lymph node metastasis acts as an immunomodulator and3 a source for further metastatic spread, indicating that more study is needed to understand the mechanisms of breast cancer invasion into lymphatic vasculature and lymph nodes. We discovered that BC expression of the Notch ligand JAG1 promotes lymphovascular invasion and increases lymph node metastasis. To understand the clinical significance of JAG1, we evaluated synchronous primary breast tumor and lymph node metastases from over 300 patients via multiplex staining and determined that tumor cells metastasized to the tdLNs have higher JAG1 expression than cells from the primary tumors of the same patient. In mice, BC cell JAG1 expression increases the frequency of tumor lymphovascular invasion and lymph node colonization by orthotopically implanted tumor cells. JAG1 expression in BC cells promotes migration of tumor cells across a lymphatic endothelial (LEC) monolayer, and this was inhibited by a Notch4 neutralizing antibody. Cultured LEC stimulated by immobilized JAG1 or JAG1-expressing BC cells showed upregulation of VEGFC/ VEGFR3 signaling and inflammatory cytokines. Lymph node metastasis of JAG1 expressing tumor cells in mice was inhibited by a soluble VEGFR3 antagonist, soluble Flt4Fc. We conclude that JAG1 promotes lymph node metastasis of breast cancer cells, potentially by increasing transmigration of tumor cells across the LEC barrier and increasing LEC VEGFR3 secretion. These results suggest that lymphatic Notch and VEGFR3 signaling may represent therapeutic targets to limit breast cancer metastasis.
AUTHOR: Rishi, H Patel
The Role of Endothelial ACKR1 in Breast Cancer Metastasis
Background: Breast cancer metastasis is the leading cause of death in breast cancer patients, responsible for 75% of breast cancer-related deaths. A critical step in metastasis is tumor cell extravasation, where circulating tumor cells exit blood vessels to establish metastases in distant organs. Atypical Chemokine Receptor 1 (ACKR1), a non-signaling chemokine receptor expressed on endothelial cells (EC) and red blood cells, has emerged as a potential regulator of breast cancer cell extravasation based on its ability to promote leukocyte extravasation. Hypothesis: We hypothesize that endothelial ACKR1 is upregulated at distant metastatic sites, resulting in increased breast cancer cell extravasation and, ultimately, increased metastatic spread and mortality. At the primary tumor, cancer cells secrete inflammatory factors into circulation. These secreted factors can then travel to distant organs, like the lung which is a common site of breast cancer metastasis, and cause the endothelial cells to upregulate ACKR1 expression. Once tumor cells eventually arrive at these distant organs, they will then be able to extravasate out of blood vessels more readily through the ACKR1+ endothelial cells. Methods: To investigate this hypothesis, we orthotopically implanted metastatic breast cancer cells into wildtype and Ackr1 Null mice and observed lung metastatic burden. We used immunofluorescent staining to analyze endothelial Ackr1 expression in the lungs tumor-bearing mice at different stages of tumor progression. We examined the timing of tumor cell arrival in the lung and their spatial association with ACKR1-positive vessels. Using an endothelial cell-specific ACKR1 conditional knockout mouse model (ACKR1 ECKO), we evaluated the role of endothelial ACKR1 in tumor cell extravasation and metastatic colonization Results: Our findings demonstrate that Ackr1 Null mice exhibit a significant reduction in lung metastatic burden compared to wild-type mice. The presence of a primary tumor markedly upregulates endothelial Ackr1 expression at distant metastatic sites in the lung, specifically localizing to pulmonary venules-a key site for cellular extravasation. In tumor-bearing mice, disseminated tumor cells preferentially localize near Ackr1-positive venules, suggesting these vessels as a primary entry route into the lung parenchyma. Interestingly, although tumor cell extravasation remains unchanged in ACKR1 ECKO mice, overall lung metastasis is reduced compared to control mice, indicating an alternative mechanism underlying decreased lung metastasis in these models. Conclusion/Future Directions: We have determined that pulmonary Ackr1 expression is induced by breast tumors and plays an important role in breast cancer metastasis progression. Investigating the factors responsible for endothelial ACKR1 upregulation during breast cancer progression promises new avenues for targeted treatments for patients at highrisk for metastatic disease.
AUTHOR: Kihak Lee
MRTFA Expression Shapes an Immune-Suppressing Tumor microenvironment via Upregulation of VSIR
Breast cancer ranks as the second most common cancer among women, comprising 31% of female cancer diagnoses. Despite similar incidence rates of breast cancer, African American women show a 4 to 5 times higher risk of deaths from breast cancer than White American women, highlighting a significant disparity in survival outcomes. Spread of breast cancer cells to other organs is the key cause of death of breast cancer patients. Thus, it is important to identify and understand the biological factors driving this disparity, particularly those influencing cancer metastasis. Transcription factors play a crucial role in metastasis, and the myocardin-related transcription factors A and B (MRTFA/B) serve as co-activators of serum response factor (SRF), and the MRTF-SRF complex facilitates breast cancer cell migration, invasion, and metastasis by regulating genes involved in actin cytoskeleton remodeling. MRTFA is also active in the tumor microenvironment (TME), where it aids tumor growth. Although MRTFA/B’s tumor-supporting roles are well-documented in experimental models, their specific impact on human cancer progression and metastasis remains to be fully elucidated. Therefore, interrogating MRTFA/B’s function in human breast cancer is important for deeper insights into metastatic mechanisms and improving patient outcomes. To examine the clinical, demographic, tumor-intrinsic, and tumor microenvironmental patterns of MRTFA/B expression and activation, we employed multiplex imaging techniques on racially diverse tissue microarrays (TMAs) and conducted bioinformatics analyses using data from The Cancer Genome Atlas (TCGA), the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), and available single-cell RNA sequencing datasets. Notably, MRTFA expression levels were higher in African American patients than in White American patients. Furthermore, our single-cell analysis revealed that MRTFA is enriched in dendritic cells (DCs) among other diverse cell subtypes within the breast cancer TME and higher MRTFA expression in bulk tumor samples correlated with increased DC infiltration in the TCGA dataset. Moreover, our multiplex-imaging of TMAs showed strong MRTFA expression in HLA-DRA+CD45+ cells, indicative of antigenpresenting cells. Notably, public single-cell RNA sequencing data showed that breast cancer DCs strongly express the V-set immunoregulatory receptor (VSIR), an immune checkpoint protein, suggesting an immunosuppressive tumor microenvironment. We also found that VSIR expression is regulated by the MRTFA-SRF complex. In addition, we found an increased T-cell infiltration, and T-cell dysfunction in African American patients with elevated VSIR gene expression in TCGA dataset. These findings suggest that MRTFA expression in DCs within breast cancer contributes to survival disparities between African American and White American women by promoting an immunosuppressive TME through the upregulation of VSIR expression. We will test this hypothesis by functional work in human and mouse dendritic cells.
AUTHOR: Mete E. Ozgurses
Modeling Physiological Lipid Availability of Tumors to Improve In Vitro Studies of Cancer Cell Lipid Metabolism
BACKGROUND, PURPOSE, or OBJECTIVES: Lipids are highly crucial in cellular physiology and pathology. They are the main structural components of cellular membranes and play critical roles in metabolism, signaling, and other core processes. Many studies of lipid metabolism, both in cancer and other fields, rely on the results of in vitro tissue culture experiments. Generally, in vitro culture systems use media containing non-physiological levels of nutrients, including high levels of critical nutrients like glucose and glutamine. Recently, several groups have generated culture media with physiological levels of polar metabolites, which can correct some tissue culture-related artifacts. However, these new-generation media still use traditional serum levels, representing severe lipid starvation conditions.
HYPOTHESIS/AIMS: We hypothesize that the Lipid-starved conditions in traditional tissue culture levels impact cancer cell behavior in vitro and that developing a more physiological lipid medium will maintain more accurate cell metabolic characteristics. MATERIALS or METHODS: We have generated Human Serum Lipid Media (HSLM) according to human serum lipoprotein levels and compared it to both human samples and RPMI 10% FBS with lipidomic analysis. To assess available lipoproteins in the tumor microenvironment, we isolated both plasma and Tumor Interstitial Fluid (TIF) from mouse breast tumor models and analyzed lipoprotein expression using proteomics and western blot. With the data obtained from animal experiments, we have supplemented our RPMI 10% FBS with high-density lipoprotein to match the TIF levels of Lipids. Cells are grown in HSLM and H-RPMI 10% FBS and obtained growth curves, including PI/Hoesct staining. We have also performed GC-MS C13 glucose tracing experiments on how cells change their metabolism in H-RPMI 10% FBS. RESULTS: Our lipidomic analysis showed that the HSLM is similar to human serum samples relative to RPMI 10% FBS. However, we found that HSLM induced cell death in multiple breast cancer cell lines. We found that TIF has lower lipoproteins present than serum levels. Cells adapted to H-RPMI 10% FBS were able to grow and significantly reduced SREBP score and de novo lipogenesis. Furthermore, Cells also had similar levels of Polyunsaturated fatty acid/Saturated fatty acid ratio when cultured in H-RPMI 10%FBS as an in vivo orthotopic breast cancer tumor model compared to cells cultured in regular media. CONCLUSIONS: With this project, we have attempted to adjust the in vitro cell culture conditions by supplementing cell culture media with lipoproteins. Overall, we believe adjusting the cell line metabolism by supplementing lipids in culture media will better represent tumor metabolism, potentially making better patient outcomes predictions. HYPOTHESIS/AIMS: We hypothesize that the Lipid-starved conditions in traditional tissue culture levels impact cancer cell behavior in vitro and that developing a more physiological lipid medium will maintain more accurate cell metabolic characteristics. MATERIALS or METHODS: We have generated Human Serum Lipid Media (HSLM) according to human serum lipoprotein levels and compared it to both human samples and RPMI 10% FBS with lipidomic analysis. To assess available lipoproteins in the tumor microenvironment, we isolated both plasma and Tumor Interstitial Fluid (TIF) from mouse breast tumor models and analyzed lipoprotein expression using proteomics and western blot.
AUTHOR: Qianxun Wang
ACKR1 as a regulator of pulmonary inflammatory pathophysiology in metastasis and respiratory distress
ABSTRACT BACKGROUND Cancer metastasis is a complex biological process in which an understanding of cellular mechanisms is central to an understanding of disease progression. Our lab recently discovered that Atypical Chemokine Receptor 1 (ACKR1) plays a critical role in facilitating breast cancer cell metastasis to the lung, particularly by promoting immune cell migration across the lung endothelial barrier in the metastatic niche. Based on these early observations, my work will clarify the mechanism(s) of ACKR1 activity in inflammatory contexts, particularly in the context of lung pathophysiology and metastasis. To investigate cell-cell interactions governed by ACKR1 in detail, I am developing an in vitro co-culture system using ACKR1 knockout (KO) human umbilical vein endothelial cells (HUVEC) and tumor-conditioned media to model metastatic interactions between immune, tumor, and endothelial cells. To extend these findings to other clinically relevant forms of pulmonary inflammation, I will examine ACKR1-regulated roles in mouse models of pulmonary distress with acute and chronic lung inflammation. METHODS To examine the metastasis- and inflammatory-relevant cell behaviors regulated by endothelial ACKR1, I will establish CRISPR-mediated ACKR1 KO and control HUVEC and lung microvascular endothelial cells (LMECs). I will culture these cells with freshly isolated neutrophils, monocytes, or tumor cells and determine the percentage of cells that bind the endothelial monolayer or undergo transendothelial migration. I will determine which steps of transendothelial migration are blocked in the absence of ACKR1. To examine endothelial ACKR1 function under pulmonary distress, I will induce sepsis-like acute or chronic inflammation using a single intraperitoneal injection of lipopolysaccharide (LPS) or continuous osmotic pump LPS administration, respectively, in a newly generated endothelialspecific ACKR1 knockout mouse model (ACKR1ECKO). I will examine immune cell dynamics using both immunohistochemistry and multiparameter flow cytometry to measure neutrophil and monocyte infiltration into the lung parenchyma. Vascular leakage will be quantified by pulmonary edema formation and Evans Blue extravasation assays. To complement the in vitro experiments, I will examine the ratios of recruited, attached, and extravasated cells using intravascular antibodies to distinguish intravascular from extravascular cell positions. I will examine the formation of high endothelial venule (HEV)-like vessels and critical regulators of the immune response by quantifying the percentage of vessels that have acquired HEV markers, including sequential upregulation of immune cell adhesion molecules and the transformation in shape from flat to cuboidal-like structures. RESULTS Our initial findings indicate that ACKR1 is an important regulator of immune cell extravasation and vascular remodeling and critical for neutrophil extravasation into the lung metastatic niche. In preliminary in vitro testing, we have determined that either tumor-conditioned media or LPS upregulate ACKR1 expression in HUVEC. Co-culture of HUVEC with neutrophils suggests that tumor-conditioned media increases the amount of neutrophil adhesion to the endothelial monolayer but not the percentage of transmigration. We are currently testing CRISPR constructs to excise ACKR1 and define its role in this process. CONCLUSIONS This research will elucidate the function of ACKR1 in immune cell trafficking and vascular remodeling during lung inflammation, presenting potential therapeutic targets in metastasis, acute respiratory distress syndrome (ARDS), and other pulmonary inflammatory processes.
AUTHOR: Hammad Farooq
A New Approach for Discovering Functional Links Connecting Non-Coding Regulatory Variants to Gene Targets
Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with complex traits or diseases. However, the majority of these variants exhibit modest individual effects, are linked through to linkage disequilibrium (LD) with nearby polymorphisms, and are predominantly located within non-coding cis-regulatory elements (CREs). These features present significant challenges in pinpointing the causal variants and elucidating their tissue-specific regulatory effects on gene expression and phenotype. To address these challenges, we developed a novel algorithm that integrates polymer folding principles and 3D chromatin interaction data to prioritize putative causal variants and their target genes. By analysing millions of eQTL-Gene pairs from the GTEx dataset across human somatic tissues, we identified a subset (~10-20%) of putative functional eQTL-Gene pairs, further validated by phenotypic associations and CRISPR deletion experiments. Our results demonstrate that functional eQTL-Gene pairs predominantly reside within the same topologically associating domain (TAD) and exhibit strong associations with cell-type specific CREs enriched for tissue-specific transcription factor binding sites. Unlike conventional approaches that prioritize variants based on linear genomic distance or chromatin features such as histone modifications or accessibility, our algorithm emphasizes the critical role of physical 3D chromatin interactions. Specifically, the identified functional pairs are confined to the small subset of chromatin interactions sufficient for chromatin locus folding, highlighting the importance of spatial genome organization in gene regulation. By reducing false-positive associations commonly encountered in eQTL studies, our approach uncovers novel variantgene regulatory relationships. These findings suggest a mechanism wherein a limited number of regulatory variants drive tissue-specific gene expression through their physical interactions with target genes within shared TADs. Our work provides new insights into the molecular underpinnings of GWAS-identified phenotypes and advances our understanding of the 3D genome’s role in gene regulation.
AUTHOR: Timur Rusanov
Macrophage polarization in the oral cancer immune microenvironment
Macrophage polarization in the oral cancer immune microenvironment PRESENTING AUTHOR: Timur Rusanov1 CO-AUTHORS: Olga Karginova1, Farah Yousef1, Jalees Rehman1,2, and Ameen A. Salahudeen1,2 1 - Department of Biochemistry and Molecular Genetics, University of Illinois Chicago 2 - University of Illinois Cancer Center BACKGROUND: Oral squamous cell carcinomas are responsible for over 12,000 deaths in the US yearly and for patients with oral precancer (premalignant) lesions there have been no targeted therapeutic strategies that reduce the overall risk of progression to carcinoma. Abnormal Wnt signaling has been associated with progression of early premalignancy to invasive carcinoma, while inhibition of Wnt in animal models was shown to halt precancer progression. However, the underlying mechanisms of how Wnt signaling drives premalignant progression remain unclear and represents an untapped area for therapeutic intervention. HYPOTHESIS: I believe that oral tumor precancer cells modulate macrophages via Wnt upregulation to promote anti-inflammatory polarization. This, in turn, creates a permissive immune microenvironment that drives premalignant progression to carcinoma. METHODS: We used the 4-Nitroquinoline 1-oxide carcinogen model of mouse oral carcinoma generation to capture a spectrum of disease stages. Expert pathologist annotation confirmed acquisition of early tissue dysplasia through to invasive carcinoma at distinct time-points, representing oral cancer formation and progression (tumorigenesis). RESULTS: With this data we defined an early dysplasia to severe dysplasia transition window, usable as intervention time-point. We initiated a pilot Wnt inhibition study by intervention during the premalignant transition window with smallmolecule inhibitor C59 and recombinant expression of negative regulator of Wnt activation; DKK1. Preliminary findings assessed by H&E lesion grading indicate DKK1 intervention may have reduced progression to carcinoma, while C59 intervention at the chosen dosage had an inconclusive effect. Finally, for downstream processing of our carcinogenesis and intervention models, we prepared a single-cell and spatial transcriptomics workflow of oral tumor tissue. This workflow pairs single-cell sequencing of murine tumor samples to annotate cell-types in our spatial transcriptomics sequencing datasets. This allows for characterizing the gene signatures of normal, premalignant, and tumor cells across stromal, mesenchymal and immune cells relevant in the oral tumor microenvironment. CONCLUSIONS: The 4-NQO mouse model was successfully used to capture distinct oral cancer disease stages and our next steps will be to probe for macrophage polarization across tumor progression. We will determine the timeline of tumor cell Wnt upregulation and probe for the activity of pro-tumorigenic, anti-inflammatory macrophage immune cell populations (M2-like) across disease states. This will indicate if premalignant cells are dependent on Wnt signaling for the polarization of macrophages towards pro-tumorigenic states. Immunostaining and in situ approaches are being used to validate our mechanistic intervention strategy. Downstream analysis aims to uncover the role of the Wnt pathway in creating an immune-permissive environment during oral tumorigenesis. Wnt signaling among epithelial, fibroblast and immune cells will be assessed by our established single-cell and spatial transcriptomics workflows to generate a timeline and source of abnormal Wnt pathway activation. Models inhibiting Wnt signaling will indicate if this pathway is relevant for macrophage polarization and ultimately as a target to prevent premalignant progression.
AUTHOR: Debdutta Mandal
Impact of Notch4 blockade on macrophage recruitment to the breast tumor microenvironment
BACKGROUND, PURPOSE, or OBJECTIVES: The Notch signaling pathway is a highly conserved pathway that regulates cell fate and cell differentiation in multicellular organisms. The Notch pathway plays a vital role in tumor angiogenesis. Notch1 is broadly expressed throughout the body, and preclinical studies demonstrate that targeting Notch1 limits tumor vessel perfusion. However, Notch1 blockade has been linked to gastric toxicity and tissue hypoxia, impeding the pharmaceutical development of Notch1 antagonists. In contrast, Notch4 is primarily expressed in the vascular endothelium and is known to be upregulated in human patients with triplenegative breast cancer (TNBC). High levels of Notch4 expression have been associated with poor overall survival in breast cancer. In mice, implantation of mammary tumors into Notch4 null animals reduces mammary tumor angiogenesis, perfusion, and volume. Notch4 inhibition could enable targeting of the Notch signaling pathway in tumor stroma without disrupting normal organ function, making it a valuable potential therapeutic target in the treatment of breast cancer. Although Notch4 is mainly expressed on the endothelium, some publications have observed Notch4 expression on immune cells, particularly macrophages. We found that treatment with a novel anti-Notch4 neutralizing antibody, E7011, caused a significant increase in the number of tumor-associated macrophages (TAMs). To test the functional importance of TAMs in the mechanism of E7011, we depleted TAMs with an intra-tumoral injection of liposomal clodronate before administering E7011. Depletion of macrophages via clodronate abrogated the tumor volume reduction of E7011 treatment, suggesting that TAMs are an important player in E7011 efficacy. HYPOTHESIS/AIMS: We hypothesize that tumor-associated macrophage Notch4 is responsible for breast cancer metastasis and tumor progression. MATERIALS or METHODS: I will section cryopreserved primary tumors from two syngeneic murine mammary carcinoma models, Py8119 and E0771.LMB, implanted orthotopically into wildtype C57BL6/J mice. I will perform immunofluorescent staining to quantify macrophages (CD68+) and Notch4 in tumor-bearing mice treated with E7011 or control lgG. In preliminary experiments, I have optimized Notch4 and CD68 double staining. I will use this technique to examine mammary tumors excised at different time points to determine how macrophage recruitment changes with tumor progression. RESULTS: We found that at endpoint, day 22 post tumor implantation, tumors treated with E7011 have fewer macrophages than control IgG treated tumors. Single cell RNA sequencing performed in the lab showed that tumors treated with E7011 have more macrophages at midpoint, day 10 post tumor implantation. Immunofluorescent staining with tumors implanted in mice with Notch4 genetically ablated from the endothelium showed that blockade of Notch4 increases the macrophage populations in endpoint tumors. CONCLUSIONS: We see a correlation between biological tumor size and macrophage populations in the breast cancer tumor microenvironment. Targeting tumor associated macrophage Notch4 may allow us to develop therapeutics to reduce tumor volume.
AUTHOR: Monica A. Haughan
Adrenergic Signaling in Adipocytes Drives Ovarian Cancer Cell Invasion
BACKGROUND: Ovarian cancer (OC) is the 6th leading cause of cancer death in women. Part of why OC is particularly deadly is that it is frequently not diagnosed until late stages when it has spread in body. The omentum, a fatty tissue that covers the intestine is an extremely common site of spread therefore expanding our understanding of the drivers of omental spread is of particular interest. High grade serous OC (HGSOC) is the most common and lethal OC subtype. Our investigation of molecules produced when HGSOC cells are in proximity to omentum uncovered increased epinephrine, a stress hormone also called adrenaline. Epinephrine is known to drive lipid release from omentum feeding HGSOC. Excitingly, propranolol, an FDA approved medication which blocks epinephrine signaling, has been shown to potentially have positive impacts in preclinical and retrospective clinical HGSOC studies. HYPOTHESIS: Increased epinephrine signaling in omental adipocytes drives lipid release promoting HGSOC cell invasion. METHODS: To understand the molecular signaling between HGSOC cells and omentum we employ a cutting-edge technology known as imaging mass spectrometry (IMS) which identifies molecules in a sample with spatial resolution. We applied IMS to co-cultures of a mouse HGSOC cell model (MOE PTENshRNA) with mouse omental explants in addition to human HGSOC cell models (OVCAR4 and OVCAR8), human omental explants, and a unique human adipocyte model which forms 3D structures known as spheroids. The co-cultures are compared to monoculture and non-tumorigenic co-culture controls. Additionally, conditioned media including any secreted factors were collected from mouse omental explants and human adipocyte spheroids treated with epinephrine (10mM), propranolol (1mM), or control for 24 hours. MOE PTENshRNA, OVCAR4, and OVCAR8 were treated with these conditioned media and their invasiveness was evaluated via Boyden chamber assay. The conditioned media was also subjected to liquid chromatography mass spectrometry (LC-MS). RESULTS: Epinephrine was increased in co-culture of HGSOC models with omental explants and human adipocyte spheroids. In MOE PTENshRNA, OVCAR4, and OVCAR8 treatment with adipocyte conditioned media increased invasion. Further, this invasiveness was decreased in HGSOC cells treated with conditioned media of propranolol treated adipocyte cells. Only epinephrine treated omentum conditioned media increased MOE PTENshRNA invasion beyond the baseline. LCMS of epinephrine treated conditioned media revealed several increased lipids. CONCLUSIONS: Overall, we aim to deepen our understanding of the molecular signaling driving omental spread of HGSOC using our IMS co-culture system. We demonstrate that epinephrine is produced in co-culture and modification of epinephrine signaling in adipocytes alters their secreted factors which can drive HGSOC invasion. We have also begun to characterize the lipids released by adipocytes with epinephrine and propranolol treatment which may be important in disease progression.
AUTHOR: Caitlyn, C Castillo
Nanoparticle-engineered macrophages to target brain metastatic breast cancer cells
Background Breast cancer metastasis to the brain is a devastating stage of breast cancer that presents with cognitive decline and seizures, and currently there are no treatments available. Brain metastases are resistant to immunotherapy, chemotherapy, and targeted therapies. This is partly because the blood-brain barrier and the blood-tumor barriers prevent effective delivery of these treatment modalities at clinically safe doses and partly because we do not know enough about the molecular mechanisms that govern breast cancer brain metastasis. Recent work in mouse models of breast cancer brain metastases revealed that the interaction between Yes Associated Protein (YAP) and Transcriptional Enhanced Association Domain (TEAD) family of transcription factors is an essential step in outgrowth of disseminated cancer cells. Hypothesis We hypothesize that nanoparticle-engineered macrophages may act as an effective delivery to brain metastatic regions at a clinically safe dose. Methods Here, we demonstrate a series of luminescence based viability assays and time-lapse imaging assays that we optimized to investigate the impact of YAP-TEAD inhibitors and chemotherapeutic agents on the viability of brain metastatic breast cancer cells in vitro. Results The luminescence based viability assay shows less than 50% viability after 48 hours with Paclitaxel, a chemotherapeutic agent. Timelapse imaging shows cancer cell death when Paclitaxel is delivered via nanoparticle-engineered macrophages. Our results will guide the prioritization of these treatment modalities in preclinical in vivo models. Conclusion We will specifically investigate the efficiency of one or more of these agents being loaded onto nanoparticle-engineered macrophages to better overcome blood-brain and blood-tumor barriers for effective delivery and targeted destruction of brain metastases.
AUTHOR: Courtney R Coleman
Local Control of Genome Editing and Tumorigenesis In Vivo
Background: Precise spatial control of site-specific DNA recombination in vivo remains a significant challenge due to unintended background activity and the limited tunability of current tools. One of the major research areas affected by the lack of local genome editing tools is the modeling of localized tumor initiation. Primary tumors are typically known to originate at a specific site and gradually progress to spread and metastasize. However, existing animal models utilizing conditional oncogene expression or carcinogen treatment often result in multiple tumors developing simultaneously at different locations within an organ. Alternative strategies, such as focal implantation of tumor cells or tissue, can produce local tumors but bypass the critical early stages of tumor development. As a result, researchers are left with either rapidly growing multiple tumors or locally restricted advanced tumors. Recent advancements in optogenetic technologies have led to the emergence of photoactivatable Cre recombinases as a promising solution for regulation of such localized genome editing. An ideal optogenetic Cre system for spatiotemporal control in vivo must demonstrate strong light-induced recombination activity while avoiding background recombination in the absence of light. However, existing photoactivatable Cre enzymes typically suffer from suboptimal performance and/or non-specific background activity. Additionally, successful spatiotemporal control in vivo relies on an illumination device capable of delivering precise control over light intensity, duration, area, and timing, while ensuring no physical damage to the targeted tissue and maintaining stable operation. Overcoming these challenges is crucial for developing more accurate models of tumor initiation and progression, advancing cancer research and therapeutic strategies. Methods and Results: In this study, we introduce a technology for localized conditional genome editing, using the engineered blue light-regulated recombinase, E-LightR-Cre. This system meets the key requirements for spatially controlled gene manipulation in vivo, with no detectable activity in the dark and robust activation upon exposure to blue light. To facilitate local activation of E-LightR-Cre in mouse lungs, we developed an implantable wireless µ-ILED device that allows precise, focal illumination of the lungs without causing damage to the target organ. By adjusting the intensity and duration of the light, we can control the size of the activated area. Light-controlled local expression of oncogenic KRas-G12D in a subset of cells induces early tumorigenic changes in vitro and leads to the formation of localized oncogenic lesions in an inducible lung adenocarcinoma mouse model. Conclusions: This study presents E-LightR-Cre as a transformative tool for genome engineering, demonstrating precise activation with no detectable background activity and exceptional compatibility with in vivo applications. The integration of flexible, wireless µ-ILED systems enables spatiotemporal optogenetic manipulation in the lungs and potentially other internal organs that cannot be accessed by external illumination or peripheral implantation. The successful application of this methodology in modeling localized lung cancer offers new opportunities for investigating early tumor evolution and the associated microenvironmental interactions. Beyond early tumor initiation, this platform has broad applications for enhancing our understanding of tumor progression, metastasis, and other spatially restricted biological processes.
AUTHOR: Avdhi J Tripathi
Targeted Drug Delivery for Prostate Cancer Treatment Using RAP-Decorated DHNALoaded Liposomes
Background: Prostate cancer is one of the most common cancers among men in the United States and the second leading cause of cancer-related deaths, following lung cancer. The standard treatment, androgen deprivation therapy, often leads to the development of castration-resistant prostate cancer (CRPC) and a loss of bone mineral density. Traditional therapies like chemotherapy and radiotherapy can cause significant side effects due to their lack of specificity and are often ineffective against metastatic prostate cancer. This scenario underscores the need for targeted drug delivery systems to enhance drug bioavailability and therapeutic efficacy. Hypothesis/Aims: This study proposes that a drug delivery system using liposomes encapsulating 1,4-Dihydroxy-2-Naphthoic Acid (DHNA)-a prebiotic metabolite with anti-cancer and anti-inflammatory properties-can effectively inhibit prostate cancer cell viability. Additionally, we hypothesize that the RAGE antagonist peptide (RAP) can be conjugated to DHNA-loaded liposomes to specifically bind to the RAGE receptor on prostate cancer cells, improving drug localization and reducing cell viability. Study Design: We optimized the formulation of DHNA-loaded liposomes for high encapsulation efficiency and controlled release. The liposome surface was modified with RAP to target RAGE, which is overexpressed in prostate cancer. We evaluated particle size, encapsulation efficiency, and release rates using Dynamic Light Scattering and UV spectrometry. Flow cytometry quantified RAGE receptor expression in VCaP prostate cancer cells versus the non-cancerous WPMY-1 cell line. We also assessed RAP conjugation efficiency and performed cell viability assays to evaluate toxicity against metastatic prostate cancer cells. Results: The DHNA-loaded liposomes demonstrated a 94.7% encapsulation efficiency, with a particle size of 81.3 nm and a Polydispersity Index (PI) of 0.32. They released 60% of their content within 24 hours at pH 6.5, resembling the prostate cancer tumor microenvironment. Following RAP peptide attachment, particle size increased to 236 nm, and PI improved to 0.18, indicating uniform distribution. RAP conjugation efficiency was 80%. In the VCaP prostate cancer cell line, RAGE receptor expression was confirmed, and dose-dependent binding of RAP to the receptor was noted. Cell viability assays revealed significant toxicity to VCaP cells (IC50 ~1 _M) with minimal effects on controls, while Western blotting showed decreased expression of RAGE and Androgen Receptor. Conclusion: Our preclinical findings indicate that the RAP-decorated, DHNA-loaded liposomal formulation effectively targets and inhibits the growth of prostate cancer cells. Although these results are promising, further preclinical studies are needed to enhance our understanding of its potential to improve therapeutic outcomes in prostate cancer treatment.
AUTHOR: Joseph R. Dominguez
Monocyte-derived omentum-resident macrophages provide anti-tumor immunity in early-stage ovarian metastasis
Background: Ovarian cancer is one of the leading causes of cancer-related deaths among women, with an expected 20,000 women to receive a diagnosis in 2025. Recent studies indicate that majority of cases of high-grade serous ovarian cancer (HGSOC) originate from epithelial cells of the fallopian tube. Once the tumor cells disseminate, they migrate to the omentum to use fat-storing adipocytes to promote tumor growth. Immune cells such as macrophages promote inflammation as a protective response, however prolonged inflammation can lead to cancer. For this reason, researchers are interested in targeting tumor-associated macrophages (TAM), which are phenotypically different than resident tissue macrophages (RTM). RTM can be defined based on their origin, either derived from embryonic precursors or developed from monocytes during adulthood. As cancer progresses, the tumor microenvironment modifies monocytes into TAMs. Our goal is to understand the function of embryonic-derived macrophages and monocyte-derived macrophages in the omentum during early-stage ovarian cancer. Hypothesis: We hypothesize that CX3CR1+ monocytederived omental macrophages provide anti-tumor support. Methods: We used the transgenic mouse model CX3CR1gfp/+ to label a novel subset of omentum-resident macrophages. We confirmed the origin of each macrophage population by using the tamoxifen-inducible mouse model CX3CR1CreER/gfp: R26TdTomato/+, where labeled cells are derived from embryonic progenitors. To examine how omentum macrophages respond to tumor cells, we injected adult female mice with 1x106 ID8 cells, a cancer cell line derived from mouse ovarian epithelial cells, and harvested the omentum two weeks after injection. We injected ID8 tumor cells into Wt1cre/+: Csf1fl/fl mice, which have 80% reduction in omental and peritoneal macrophages, and CX3CR1CreER/+: Csf1rfl/fl mice, which deplete only monocyte-derived CX3CR1+ macrophages. The tumor of the omentums were quantified by measuring their bioluminescence with an LagoX instrument. Results were gathered using either confocal microscopy or flow cytometry. Results: We found that Tim4+ LYVE1+ omentum resident macrophages were embryonicderived macrophages while CX3CR1+ omentum resident macrophages were monocyte-derived macrophages. Confocal microscopy imaging showed monocyte-derived macrophages surround CK19+ tumor cells two weeks post-tumor challenge. Depletion of peritoneal and omental macrophages led to significant increases in tumor cells in both the omentum and peritoneal cavity. Depletion of CX3CR1+ macrophages led to a significant, two-fold increase in tumor burden. Conclusions: We conclude that both peritoneal and omental macrophages can provide early anti-tumor immunity in an ID8-challenged ovarian cancer model. Monocyte-derived CX3CR1+ macrophages surround the tumor during early-metastasis and reduction of these macrophages led to increased tumors in the omentum, but not peritoneal fluid, implying sitespecific roles for each macrophage population during early metastasis. While we are examining the specific function of omental CX3CR1+ macrophages, we conclude that these cells possess anti-tumor properties during early ID8 challenge.
AUTHOR: Mohamed Haloul
ISG15/ISGylation Modulate Breast Cancer Cell Mechanics and Metastasis
ISG15/ISGylation Modulate Breast Cancer Cell Mechanics and Metastasis PRESENTING
AUTHOR: Mohamed A. Haloul CO-AUTHORS: Priya Shah1, Vinay Pai1, Caitlyn Castillo1, Aditi Upadhye2, Mark A Sanborn2, James Lee3, Jalees Rehman2, Ekrem Emrah Er1 1 University of Illinois at Chicago, Department of Physiology & Biophysics, 2 University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics, 3 University of Illinois at Chicago, Department of Biomedical Engineering. ABSTRACT: BACKGROUND, PURPOSE, or OBJECTIVES: Breast cancer metastasis is driven by profound biophysical changes in malignant cells, with cortical deformability playing a crucial role in tumor progression. By enhancing their ability to squeeze through dense stromal barriers, evade immune cells and intravasate into circulation. We recently showed that reducing cancer cell deformability enhances cytotoxic lymphocyte activation, a process we termed mechanosurveillance, wherein immune cells exploit mechanical vulnerabilities to suppress metastasis. Despite its therapeutic potential, the molecular regulators of mechanosurveillance remain largely unknown. One promising candidate is Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein frequently overexpressed in breast cancer and linked to poor prognosis, higher tumor grade, and increased invasiveness. Notably, ISG15 interacts with actin cytoskeleton regulators, suggesting a role in modulating cancer cell mechanics.
HYPOTHESIS/AIMS: ISG15/ISGylation softens cancer cells to escape mechanosurveillance MATERIALS or METHODS: To investigate this, we utilized in vitro and in vivo loss-of-function models. RESULTS: Our findings revealed that loss of ISG15 or disruption of its post-translational modification, ISGylation, led to higher F-actin/G-actin ratio, larger cell size and increased cellular stiffness. More importantly, ISG15/ISGylation-deficient cells exhibited enhanced mechanosurveillance, rendering cancer cells more susceptible to immune-mediated cytotoxicity. Further, our scRNA-seq analysis indicated a shift toward an M1 macrophage phenotype in ISG15-deficient mice, suggesting a more pro-inflammatory and anti-tumor immune landscape. Consistent with these findings, PyMT-ISG15_/_ mice exhibited delayed tumor onset and significantly lower tumor volume compared to PyMT wild-type mice, reinforcing the role of ISG15 in promoting tumor progression. CONCLUSIONS: By bridging tumor mechanics and immune surveillance, our study position ISG15 and ISGylation as a critical regulator of metastasis. Targeting ISG15 or disrupting ISGylation may not only stiffen cancer cells but also reshape the immune microenvironment, offering a novel therapeutic strategy to exploit mechanosurveillance for combating metastatic disease.
AUTHOR: Manoela Lima Oliveira
Feasibility of a Remote Time-Restricted Eating and Mindfulness Intervention Focused on Risk Factors Associated with Early-Onset Colorectal Cancer Development in Young Adults: A Randomized Controlled Pilot Trial
Purpose: Increased adiposity and chronic psychosocial stress (CPS) are under investigation as drivers of the recent uptick in early onset colorectal cancer (EOCRC) in the United States and other Westernized countries. Methods: We conducted a randomized controlled pilot trial evaluating time restricted eating (TRE) (daily ad libitum eating between 12-8pm and fast the remaining hours) and Mindfulness (complete the “Mindfulness for Beginners” course available in the Calm.com® app) among young adults with obesity and CPS to evaluate its feasibility and acceptability. This was an 8-week, parallel_arm study. Young adults (18-39 years old) with obesity (BMI ≥ 30 and ≤ 49.9 kg/m2) and a Perceived Stress Score (PSS) ≥14 indicating moderate to severe perceived stress were randomized to the following groups: TRE; Mindfulness; TRE & Mindfulness; or Control. The focus of study was on feasibility and acceptability, but we also explored changes in body weight, body composition; circulating metabolic, inflammation, and oxidative stress biomarkers; and intestinal inflammation. Results: Forty-three participants completed the baseline data collection and were randomized. Overall acceptability of the trial was high. Participants were enrolled within a short period (10 months) and attrition rates were low (7%). Participants completed between 90-94% of weekly sessions across all study arms. Participants in the TRE group lost a mean of -0.85 (SD 5.58) lbs; Mindfulness group lost a mean of -4.62 (SD 10.19) lbs; TRE & Mindfulness group lost a mean of -4.21 (SD 7.49) lbs; and Control group lost a mean of -0.2 (SD 3.53) lbs. PSS scores decreased significantly within all arms except for Control; no between group differences were observed. There was a statistically significant decrease in high sensitivity C-reactive protein (hs-CRP) from baseline within the TRE arm only (-1.58; 95% CI: -3.08, -0.09). No other within or between group changes were found for total fat mass, visceral fat mass; circulating biomarkers; or intestinal inflammation. Conclusion: This study was feasible and well accepted by the participants. Results were promising for showing decreases in body weight among the Mindfulness and TRE & Mindfulness arms, PSS for all intervention arms and hs-CRP within the TRE arm.
AUTHOR: Xiaoying Cai
Neoantigen peptide-loaded anti-CD40-spycatcher antibody as a novel cancer vaccine platform
Precision cancer therapy opened up an attractive strategy by therapeutic vaccines targeting neoantigens. Neoantigens are immunogenic and HLA-bound epitopes capable of activating helper and cytotoxic T lymphocytes and not subject to immunological tolerance (1-3). However, personalized peptide-based cancer vaccines still have the limitations to formulate and deliver (4). For example, most vaccine technology was initially based on the use of adjuvants (e.g., alum), which only provides moderate therapeutic effects (5). To address these issues, we utilize the engineered anti-CD40 agonistic antibody expressing Spy-Catcher003 (_CD40-SC) as a novel antigen presenting cells (APCs)-targeted peptide vaccine delivery platform for effective cancer immunotherapy. Since targeting and activating the CD40 receptor on APCs via antiCD40 agonistic antibody has been shown to elicit superior T cells priming and boost antigenspecific CD8+ T (6). Besides, the use of SpyCatcher003, which is a 113-residue protein with reactive Lys31, could trigger spontaneous and irreversible amidation reaction with SpyTag003. SpyTag003/SpyCatcher003 is a protein coupling approach, which can form an intermolecular isopeptide bond between Lys31 and Asp117. Moreover, the protein pair can react with a rate of 5.5 x 105 M-1 s-1, which is helpful for potential therapeutic application (7). Therefore, we hypothesize the efficient and specific delivery, accomplished via SpyCatcher003-expressed anti-mouse CD40 agonistic antibody and SpyTag003-conjugated neoantigen peptide, will ensure the present of neoantigens to T cells and elicit robust and durable specific CD8+ T cell response. Our goal is to directly deliver neoantigen peptide-loaded _CD40-SC to APCs in draining lymph nodes (dLN) as a therapeutic vaccine.
AUTHOR: Riley, C Hunt
Leveraging the Emergency Department to Address Health Inequities in Breast Cancer Screening
BACKGROUND, PURPOSE, or OBJECTIVES: Breast cancer (BC) is the second leading cause of cancer death among women in Illinois, with black women facing the highest mortality rate. At UI Health, only 54% of women aged 40-74 visiting the Emergency Department (ED) report a history of mammograms, and the majority aged 25-39 lack documented BC risk assessments. In April 2024, an ED BC screening program began offering women aged 25-39 and women aged 40-74 with no mammogram history (ever or >2 years) BC risk assessments and linkage to recommended BC screenings.
HYPOTHESIS/AIMS: Describe preliminary outcomes of a pilot breast cancer screening program in the ED. MATERIALS or METHODS: BC screening outcomes and patient history were collected in a REDCap database by BC patient navigators using EPIC chart review and patient-reported information. A descriptive analysis was conducted to evaluate demographics, risk assessment scores, and volume of patients outreached and linked to services through the ED BC program. RESULTS: From April 1 to Dec 4, 2024, 270 women were contacted (15% in-person, 59% by phone, 26% via text/email). Most were Non-Hispanic (78%) and African-American (67%). Of these, 131 completed BC risk assessments. 21.3% were identified as intermediate to high risk with 68% of those women being under age 40. 32 women aged 40+ were scheduled for mammograms, with 16 (50%) receiving their first mammogram ever.
CONCLUSIONS: Linking ED patients to BC risk assessments and screening is feasible. Many women had no prior risk assessments or screenings and were found to be at higher risk for BC, highlighting the need for more targeted BC preventive services.
AUTHOR: Nivida Dinesh Shete
The ER partial agonist TTC-352 inhibits a novel epithelial-mesenchymal transition pathway in tamoxifen-resistant breast cancer cells
Background: Endocrine therapy (ET) is the standard of care for estrogen receptor-positive (ER+) breast cancer. The primary treatment challenges are endocrine therapy resistance and metastasis. Epithelial mesenchymal transition (EMT) is integral to migration and invasion. Our lab has previously shown a correlation between protein kinase C alpha (PKCa) overexpression and tamoxifen resistance (TR) (Tonetti, 2003) and further reported a novel EMT signaling in TR breast cancer cells. This pathway involves FOXC2-mediated repression of the p120catenin promoter transcription, resulting in loss of E-cadherin at the plasma membrane and enhanced cell migration and invasion (Pham, 2017). Estrogen-induced apoptosis and tumor regression in long-term estrogen-deprived (LTED) breast cancer is explained by the induction of unfolded protein response (UPR), activation of the death receptor pathway and mitochondrial pathway, and down-regulation of pro-survival, cell growth pathways (Shete, 2023). Although 17betaestradiol (E2) is efficacious following exhaustive ET, it is associated with adverse effects. A novel Selective Human Estrogen Receptor Partial Agonist (ShERPA), TTC-352, induces UPR and shrinks TR tumors (Molloy, 2014; Abderrahman, 2021). The Phase I clinical trial results of TTC-352 demonstrated efficacy without reaching the maximum tolerated dose (Dudek, 2020). Objective: To investigate the impact of E2 and TTC-352 treatment on the novel EMT pathway in TR cells. Results: TR, PKCa-overexpressing cell lines used in this study include MCF-7/PKCa and LTED MCF-7:5C cells. They are more migratory and invasive than tam-sensitive MCF-7 cells. 3D spheroid invasion assay revealed that MCF-7/PKCa and MCF-7:5C spheroids are more invasive than MCF-7 spheroids. E2 and TTC-352 treatment lowered FOXC2 at the transcript level in MCF-7/PKCa cells compared to the control group. Chromatin immunoprecipitation with FOXC2 antibody showed that E2 and TTC-352 treatment lowered FOXC2 binding on the p120catenin promoter, resulting in increased p120catenin promoter transcription in MCF-7/PKCa and MCF-7:5C cells. This effect was correlated with decreased cell migration and invasion. Strong intensity of E-cadherin immunofluorescence staining at the plasma membrane was observed post-E2 and TTC-352 treatment in TR cells. Conclusion: Our findings suggest that patients with endocrine-resistant ER+ breast cancer may benefit from TTC-352 therapy by inhibiting the activation of the PKCa-FOXC2-p120catenin signaling and cell migration identified in TR cell lines. Strategizing less toxic treatment options may reduce metastasis and prolong patient survival with a better quality of life.
AUTHOR: Srishti Kolla
The Impact of Social and Environmental Factors on the Presentation of Uveal Melanoma
Purpose: Uveal melanoma (UM) is the most common intraocular malignancy in adults and is known to have a poor prognosis.1 Although the genetic profiling of UM is well characterized, there is emerging evidence regarding other factors that can influence the presentation and prognostication of UM. Other factors, such as social determinants of health (SDOH), geography, and environmental factors, play an important role in contextualizing not only a patient’s presentation at diagnosis, but also the prognosis and potential management of their care. Thus, such factors are important to identify and investigate to further understand how to tailor treatment and management for UM patients. Hypothesis: Worse social and environmental factors correlate to a more severe presentation of uveal melanoma. Methods: A retrospective chart review of patients at the University of Illinois Hospital diagnosed with uveal melanoma between 2010 and 2023 was performed. Patient demographic information, such as age, race, sex, and address, was collected for each patient. Tumor presentation characteristics (largest basal diameter, tumor thickness, extraocular extension) was also recorded. Using zip code, environmental and socioeconomic characteristics was recorded for every patient. Additionally, patients were geocoded using ArcGIS Pro software in order to compare the presentation of uveal melanoma between patients within and outside of a 60-minute drive time radius of University of Illinois Hospital. Results: Geography was not found to affect the presenting tumor volume or treatment method of uveal melanoma between patients within and outside of a 60-minute drive time radius of UI Hospital (p > 0.999). Of the environmental and social determinants of health analyzed, none were found to be significantly correlated with tumor size, extraocular extension, or treatment plan. However, several trends were present. For instance, being from a zip code with a higher number of naturalized citizens had a trend towards being associated with a smaller tumor thickness (p = 0.068). Areas with higher prostate cancer diagnosis rates and higher blood pressures had a trend towards being associated with a larger tumor thickness (p = 0.09 and p = 0.09, respectively). Lastly, there was a trend for areas with a higher number of households below the Asset Limited, Income Constrained, Employed (ALICE) threshold to be associated with larger tumor thicknesses (p = 0.093). Conclusion: Given the trends identified, further studies with larger sample sizes in the Chicagoland area are needed to identify potential relationships between SDOH from one’s place of residence and the presentation of uveal melanoma. Further research is also required to understand if geography plays a role in delays in the diagnosis of uveal melanoma.
AUTHOR: Sujoy Upadhyay
Representation in Clinical Trials Evaluating
Lifestyle Interventions Targeting Energy Balance in Cancer Survivors: A Scoping Review
BACKGROUND, PURPOSE, or OBJECTIVES: The rate of clinical trial participation in any cancer research study is 22%, with 7% participating in cancer treatment trials (drugs, devices, or behavioral interventions). However, data on clinical trial enrollment in lifestyle interventions among cancer survivors remain limited. Understanding demographic gaps in enrollment is crucial for improving the generalizability of lifestyle interventions aimed at enhancing survival, quality of life, and reducing cancer recurrence risk. This scoping review applies PRISMA guidelines to assess representation of cancer survivors by sex, race, ethnicity, age, and cancer type in clinical trials evaluating lifestyle interventions. HYPOTHESIS/AIMS: We hypothesize that underrepresentation exists for racial/ethnic minorities, men with prostate cancer, and young adult cancer survivors in lifestyle intervention trials. Overrepresentation is expected among breast cancer survivors compared to other cancer types. Targeted recruitment efforts are needed to increase inclusivity in lifestyle interventions for cancer survivors. MATERIALS or METHODS: Eligible studies met the following criteria: Participants included adults aged 18 years or older with a cancer diagnosis. Interventions were aimed at modifying lifestyle behaviors such as diet, physical activity, or weight control. Exclusion criteria included observational studies, qualitative studies, protocol papers, and studies involving children, pharmacologic, or surgical interventions. A systematic search strategy was applied using Medical Subject Headings (MESH) and terms related to cancer survivorship and lifestyle interventions. Data extraction and review processes involved importing articles into Rayyan for title and abstract screening. Two independent reviewers conducted full-text reviews for inclusion, and extracted data were entered into REDCap. Means and proportions were calculated for respective quantitative and categorical variables. RESULTS: A total of 117 articles were included in this review. In terms of intervention type, 11% of studies focused on diet, 70% targeted physical activity, and 19% incorporated both diet and physical activity interventions. The demographic characteristics of study participants revealed that the mean sample age was 56.4 years (SD = 8.4), with a range of 33 to 75 years. Most participants were female (80%), and only 52% of studies reported information on race and ethnicity. Among those that did, the majority of participants were White (71%), followed by Black/African American (11%) and Hispanic/Latino (6%). Regarding cancer types represented, breast cancer was the most commonly studied (72%), followed by colorectal cancer (22%), prostate cancer (15%), endometrial cancer (12%), ovarian cancer (10%), and lung cancer (10%). CONCLUSIONS: Breast cancer survivors comprised 51% of all studies. Despite rising cancer rates in women under 50, few lifestyle intervention trials included young adult survivors. Prostate cancer remains the most common cancer among men, yet trial enrollment is low. Hispanic/Latino populations, who have the highest prevalence of modifiable cancer risk factors, were also underrepresented. Targeted recruitment is needed to improve representation in lifestyle intervention trials, particularly among young women cancer survivors, men with prostate cancer, and Hispanic/Latino populations. Hence, more inclusive research strategies are essential to ensure lifestyle interventions are generalizable and effective across diverse cancer survivor populations.
AUTHOR: Tina Khajeh
Evaluation of multilevel risk factors for lung cancer risk using machine learning models
Background: Lung cancer is the leading cause of cancer deaths in the U.S. Smoking and age have been identified as risk factors for lung cancer, however, these individual factors do not fully explain the differences in lung cancer rates among different race/ethnicity and gender groups. Neighborhood conditions influence one’s health outcome, and yet, limited studies have systematically assessed the relative contributions of multilevel factors for lung cancer disparity. Aims: This study aims to explore multilevel risk factors for lung cancer by integrating individual and neighborhood-level variables using machine learning approaches. Methods: We used electronic medical records (EMR) of individuals over 40 years old who received lung-related diagnostic testing at the University of Illinois Hospital (UIH) between 2013 and 2017 (N=7,184). The lung cancer incidence rate was 10.3% for White, 11.02% for Black, and 6.1% for Hispanic patients. Individual variables (age, smoking status, gender, race/ethnicity) and neighborhood variables (homicide rate, % poverty, racial composition) were included in the analysis. The machine learning (ML) model, Elastic Net, was used. The area under the ROC curve (AUC) was calculated on the test set, and the Shapley Additive Explanations (SHAP) value was computed on the best model to assess the contribution of each variable to prediction. The average AUC and SHAP values were reported over 100 different data splits. Results: The ML models with neighborhood and individual variables achieved considerable performance, with an average AUC of 0.664. At the specificity of 0.80, the average sensitivity was 0.337. The ranking of variables based on SHAP showed that smoking and age were the most contributing variables, followed by neighborhood %Black, %Hispanic, %poverty, and homicide rate. Performance bias was observed across different race/ethnicity groups. For Blacks, the models had the highest average AUC (0.668) compared to Hispanics (0.614, p=8.08e-08) and Whites (0.595, p=4.24e-10). The model performed almost equally for females (AUC = 0.664) and males (0.66, p=0.132) with no significant differences. In addition, at the default threshold of 0.5, sensitivity was lower for Hispanics (0.239) compared to Whites (0.619, p<2.45e-34) and Blacks (0.734, p<2.49e-34). In contrast, specificity was higher for Hispanics (0.878) compared to Whites (0.543, p<2.45e-34) and Blacks (0.517, p<2.49e-34). Conclusion: Our analysis confirmed that the ML models can successfully integrate individual and neighborhood variables for lung cancer prediction in a cohort from the highly segregated city of Chicago. Several neighborhood variables were identified to be significant risk factors for lung cancer. Additional analysis is warranted for understanding the interplay between neighborhood and individual characteristics explaining the risk of lung cancer.
AUTHOR: Hannah Choi
Patient Expectancy in Cancer Symptom Management: A Concept Analysis
BACKGROUND/PURPOSE/OBJECTIVES: Cancer survivors hold diverse health- and treatmentrelated expectancies that influence their health outcomes. Assessing patient expectancy is challenging due to inconsistent terminology and definitions in the literature. This study aims to clarify the concept of patient expectancy in cancer symptom management by identifying existing definitions, measurement methods, and key attributes and by proposing a theoretical definition to guide development of valid, reliable assessment tools and effective interventions. HYPOTHESIS/AIMS: We hypothesize that a clear, theoretical definition of patient expectancy in cancer symptom management will help standardize measurement approaches and improve the reliability of expectancy assessments. By identifying key attributes and existing definitions, this study aims to enhance understanding of how expectancy influences health outcomes and inform the development of effective interventions for cancer survivors. MATERIALS/METHODS: Rodgers’ evolutionary model was used to examine how the concept of patient expectancy in cancer symptom management has evolved over time. A comprehensive literature search was conducted across MEDLINE, CINAHL, PsycInfo, and Google. RESULTS: Among 1,845 sources screened, 31 were included in this analysis. Symptom management strategies ranged from standard care to complementary and integrative approaches such as acupuncture, exercise, and cognitive-behavioral intervention. Three main attributes were identified to define patient expectancy: awareness, future-directedness, and value-based expression. Key antecedents included patient demographics, disease characteristics, prior information, and unmet needs. Consequences consisted of patients’ health status and satisfaction regarding symptom management. The proposed definition of patient expectancy in cancer symptom management is “cancer survivors’ cognitive awareness, future-directed beliefs, and value-based expression of desired symptom management outcomes that influence their health status and treatment satisfaction.” CONCLUSIONS: The findings provide a clear and context-specific definition of patient expectancy in cancer symptom management. Understanding this concept can facilitate development of patient expectancy measures as well as interventions to optimize patient expectancies, ultimately improving cancer-related health outcomes.
AUTHOR: Hannah Choi
Predictors of Acupuncture Expectancy in Breast Cancer Survivors
BACKGROUND/PURPOSE/OBJECTIVES: Acupuncture has shown promise in alleviating symptoms among breast cancer survivors, including pain, fatigue, and anxiety. Patient expectancy-the belief that a treatment will be effective-is an important driver of treatment effectiveness and adherence. Understanding the factors shaping expectancy and perceptions regarding acupuncture is essential for addressing its under-utilization and improving treatment outcomes and access. This study aimed to identify factors influencing acupuncture expectancy among breast cancer survivors. HYPOTHESIS/AIMS: We hypothesize that demographic, psychosocial, and clinical factors influence acupuncture expectancy among breast cancer survivors. MATERIALS/METHODS: This secondary analysis examined baseline data from 74 women with breast cancer who participated in two acupuncture trials involving a 5-week, 10-session intervention. Our primary outcome was acupuncture expectancy, measured using a sum score from a 4-item subscale of the validated Attitudes and Beliefs about Complementary and Alternative Medicine Questionnaire, with higher scores indicating greater expectancy. Other measures include the 11-item Social Determinants of Health (SDOH) Questionnaire, assessing eight domains of SDOH needs (e.g., financial resource, transportation, and social connections), and the PROMIS-29 v1.0, evaluating seven physical and emotional health domains using 4-item sum scores for each domain. Bivariate and multivariate linear regression analyses were conducted to identify significant factors influencing acupuncture expectancy. RESULTS: Participants were on average 54 years old; 49% identified as Black, and 45% as White. Most (82%) had some college education or higher, and 65% were single. The majority (88%) were diagnosed with stage 0, I, or II breast cancer. Multivariate regression analysis identified factors positively associated with acupuncture expectancy: 1) identifying as White (compared to non-White; _=0.56, p=0.009), 2) higher education levels (some college or above: _=1.07, p<0.001; graduate school: _=0.96, p=0.002; reference: high school or below), 3) greater financial difficulty paying for basic living needs (_=0.32, p=0.004), and 4) experiencing arm pain (_=0.47, p=0.023). CONCLUSIONS: Despite financial challenges, many participants reported high acupuncture expectancy, reflecting its perceived value in symptom management. Future research should address socioeconomic challenges to enhance accessibility and guide interventions, helping integrate acupuncture into oncology care to enhance symptom relief and quality of life for breast cancer survivors.
AUTHOR: Mingyue Lu
Sexual Orientation Differences in Current Tobacco Use
BACKGROUND, PURPOSE, or OBJECTIVES: In December 2019, federal Tobacco 21 (T21) raised the federal minimum legal sales age to purchase tobacco products from age 18 to age 21. Higher use of tobacco products among sexual minority adults compared to heterosexual adults prior to T21 implementation is well documented. HYPOTHESIS/AIMS: The aim of this study was to estimate current tobacco use, including current smoker or e-cigarette user status, following implementation of T21 policy stratified by age group and sexual orientation. MATERIALS or METHODS: This pooled cross-sectional study used Behavioral Risk Factor Surveillance System data for 2021 - 2023. Data analysis was performed from January 2025 to March 2025. The primary outcome was current tobacco use which included e-cigarette user or current smoking. Respondents were separately asked if they used e-cigarettes or if they smoked some day, every day, not right now, or not ever). Responses were binarized with responses some days and everyday categorized as “Yes” and not at all and not ever categorized as “No.” We used logistic regression models to estimate the marginal effect on the interaction between sexual orientation and age group to estimate unadjusted prevalence of current tobacco use, current e-cigarette use, and current smoker status. All estimates include survey weights reflecting BRFSS complex cluster-stratified sampling design. RESULTS: The analytic sample included n= 596,197 respondents, 8.5% (survey weighted) of whom identified as sexual minority, representing 100 million adults living in 25 states. Among respondents ages 18-20, sexual minority young adults were 28% more likely (95% CI 13-45%) to reported current tobacco use compared to heterosexual peers, (22.1% versus 17.3%); 29% more likely (95% CI 14-47%) to report current e-cigarette use compared to heterosexual peers (20.6% versus 15.9%); and 70% more likely (95% CI 28-126%) to be current smokers (5.8% versus 3.4%). Current tobacco use peaked in older age groups among sexual minority adults with 32.6% among respondents between ages 40-44, compared to heterosexual adults with 23.4% among respondents between ages 2124. Current e-cigarette use peaked in both group between ages 21-24, with 24.4% of sexual minority young adults and 18.8% of heterosexual young adults. Current smoker status peaked in both groups between ages 40-44, with 24.8% of sexual minority young adults and 17.5% of heterosexual young adults. CONCLUSIONS: After implementation of T21, sexual minority populations continued to report higher levels of current tobacco use compared to heterosexual peers of all age groups. Disparities in current tobacco use were present in early adulthood driven by e-cigarette use and were widest in mid-life driven by smoking.
AUTHOR: Tahniat Nadeem
Decision-Making and Patient Experiences in Stopping or Pausing HER2+ Treatment in Late-Stage
Breast Cancer
Background: HER2-Positive Breast Cancer, occurring in 15-20% of diagnoses, is indicated when the human epithelial growth factor receptor 2 (HER2) is overproduced, promoting the proliferation of malignant cells. At the time of diagnosis and progression, testing for HER2 status is critical in providing the right treatments. To combat HER2-Positive Metastatic Breast Cancer (HER2+ MBC), commonly referred to as Stage IV cancer, there are various treatments, including HER2-targeting treatments and chemotherapy drugs. The progression, hormonal influences, treatment efficacy, and other factors differ from patient to patient. Aim: The study intends to characterize patient experiences with HER2-targeting drugs, particularly in those with sustained disease stability and decision making in maintaining treatment. Methods: A questionnaire with closed- and open-ended questions was developed to elicit experiences of patients diagnosed with HER2+ MBC, with answers submitted anonymously. Particular attention was directed at whether patients considered stopping or pausing HER2-targeting treatment after achieving no evidence of disease (NED) and the associated decision-making process. The questionnaire was administered online via outreach through social media and nonprofit digital newsletters to MBC patients in the U.S. who have received HER-2 targeting drugs. Results: From the 124 respondents, 42 (34%) were originally diagnosed with early-stage MBC and 82 (66%) with MBC that had spread beyond the breast to distant locations at initial diagnosis. 85 (68%) had reached NED after a median treatment time of 6 months; Among these, 45% stated they had either discussed or thought about discussing with their oncologist the possibility of stopping HER2-targeting treatment. A patient with more education was more likely to consider a discussion about ending treatment. Only 7 patients with NED stopped treatment. Most patients who achieved NED status reported that insufficient safety evidence led them to continue treatment, despite being uncertain of its necessity. Those who considered stopping often indicated side effects, followed by the emotional toll of continuous treatment. Quality of life (QoL) challenges and financial impacts were reported, too. Overall, the decision-making was varied both in reasoning and who the decision maker was (the oncologist, the patient, or both). Conclusion: The experiences in treatment are distinct for each patient, and it is impactful to understand patients’ perspectives on ongoing therapy. Nearly half of surveyed patients who achieved NED considered stopping HER2-targeting treatment, and this was often brought up in discussions with higher educated patients. The findings indicate a potential treatment gap that is not based on treatment efficacy nor QoL. Patients seek to comprehend and be involved in decision making conversations with their clinician, so they can be well-informed of the risks in continuing, pausing, or stopping treatment. Conversations about potential risks to staying on treatment or going off treatment based on what is currently known are deserved by all patients. By addressing patients’ experiences with therapy, future researchers can recognize concerns and design effective treatments that keep the patient’s total wellbeing in mind.
AUTHOR: Kate, E Klein
Implementation Science Research to Understand the Readiness of a Mobile Clinic Intervention to Screen for High-Risk HPV Infections and Associated Morbidity in Mali and Nigeria
ABSTRACT: Access to and community acceptance of point-of-care testing for high-risk human papillomavirus (hrHPV) are two important factors in cervical cancer (CC) prevention in low and middle income countries (LMICs). As a result, HPV transmission persists, leading to cervical cancer, which, in turn, is undiagnosed or diagnosed at a late stage for which few treatments are available, leading to many preventable deaths. These prevention challenges contribute to the large burden of morbidity and mortality associated with CC in LMICs. BACKGROUND, PURPOSE, or OBJECTIVES: In 2024, Northwestern University was funded to develop a mobile clinic innovation to conduct point of care rapid DNA testing to identify women with high risk oncogenic strains of HPV in rural parts of Mali and Nigeria. The author (Klein) conducted this study prior to implementation to help identify the context specific barriers and facilitators among leaders and the intervention target communities. HYPOTHESIS/AIMS: The specific aims of the project are: 1. To understand the potential barriers and facilitating factors from the Individuals domain ‘high level leaders’ - political/administrative, religious, and community leaders - around the HPV screening mobile clinic intervention to inform the implementation of the HPV-Associated Cancers Prevention and Control Program 2. To understand the potential barriers and facilitating factors from the Individuals domain ‘innovation recipients’- women of high risk age - around the HPV screening mobile clinic intervention to inform the implementation of the HPV-Associated Cancers Prevention and Control Program MATERIALS or METHODS: Using implementation science methodology surveys and focus groups were conducted amongst political/administrative, religious, business and community leaders, as well as women in the target testing age range in the four intervention target communities in Mali and Nigeria. Data collected was analyzed using Excel for quantitative data using descriptive statistics. For qualitative data, MAXQDA and a collaborative coding and thematic analysis between collaborators at Northwestern University, United States and University of Science, Technique and Technologies of Bamako, Mali and the University of Ibadan, Nigeria. Themes will then be used to characterize community readiness for such an intervention and to develop recommendations for intervention design. RESULTS: From January 2024 to January 2025, this study was planned, alongside the manufacturing and shipping of the vehicle for the mobile clinic, and the initial planning for the implementation of the intervention. A total of 18 focus groups were conducted between January and March 2025, ten in Mali and eight in Nigeria. The Malian focus groups separated marketplace leaders from community leaders, which added two extra focus groups. CONCLUSIONS: The introduction of innovative, contextually adapted, and culturally competent strategies can bridge the disparities in access to prevent CC and other HPV-associated cancers. Intervention science methods can help researchers to ascertain the level of readiness for the intervention within target communities, as well as the context specific determinants that must be taken into account prior to and during the intervention. Implementation science research methods can help to ascertain the level of readiness for the intervention within target communities, as well as the context specific determinants that must be considered.
AUTHOR: Kaitlin Chakos
Impact of Diet Modifications on Body Weight, Body Composition, Treatment Outcomes, and Quality of Life During Primary Treatment for Breast Cancer: A Systematic Review
BACKGROUND, PURPOSE, or OBJECTIVES: Breast cancer is a significant public health challenge, with 290 000 new cases annually and significant healthcare costs. Treatment advancements have led to improvements in survival, but common adverse effects include weight gain, fatigue, nausea, and taste changes, decreasing quality of life. HYPOTHESIS/ AIMS: This review aims to assess the impact of diet and lifestyle interventions during primary treatment for breast cancer and their effects on body weight, body composition, treatmentrelated adverse outcomes, and patient-reported quality of life. MATERIALS or METHODS: A search of PubMed, CINAHL, and EMBASE conducted through May 10, 2023, identified 31 publications describing 27 interventions including diet or diet plus exercise. The Cochrane Risk of Bias tool assessed the quality of publications. RESULTS: The findings suggest that whole foods, aerobic and strength-training exercises, and intermittent fasting during treatment may improve body weight and composition, treatment-related outcomes, and quality of life. Limitations include variation in study duration, small sample sizes, and limited sociodemographic data. CONCLUSIONS: Improvements seen with increased diet quality and reduced caloric intake, with or without exercise, challenge current standard-of-care recommendations during treatment for breast cancer. While there is a need for additional research, healthcare teams can confidently promote healthy diets and exercise during primary treatment for breast cancer to manage weight and improve treatment-related side effects and quality of life.
AUTHOR: Sydney DeJonge
Associations of Exercise on Colorectal Cancer Incidence and Mortality in Chicago, IL
BACKGROUND, PURPOSE, or OBJECTIVES: Colorectal cancer (CRC) is the third most common cancer diagnosis in both males and females and is the second leading cause of cancer-related death in the United States. Recent evidence has demonstrated that physically inactive individuals have a 50% higher risk of developing CRC compared to those who are physically active. Additionally, CRC survivors who remain active following their diagnosis have a 30% lower risk of CRC-related death and nearly 40% lower risk of all-cause death. There are numerous physiological benefits that stem from physical activity that can be beneficial toward lowering CRC incidence and mortality rates; however, individuals who live in underresourced, high-poverty communities in Chicago, IL are presented with barriers such as lack of greenspace, violent crime, and poor public facilities that impact their engagement in physical activity. HYPOTHESIS/AIMS: To examine the association between low exercise engagement (i.e., physical inactivity) and CRC incidence and mortality in the 77 community areas of Chicago, IL. We hypothesized that CRC incidence and mortality would be positively associated with low exercise engagement. METHODS: We utilized population data from 77 community areas of Chicago between 2017 and 2022 to examine bivariate correlations between CRC incidence, mortality, and engagement in exercise. A linear regression model was used to analyze the predictive effects of low exercise on CRC incidence and mortality. Significance level was set at p<.05. RESULTS: Low exercise was significantly correlated with both CRC incidence (r=.370, p<.001) and mortality (r=.355, p<.01) in Chicago. The regression analysis explained that low exercise was a stronger positive predictor for CRC incidence (_= .519, p<.001) compared to CRC mortality (_= .256, p<.01). CONCLUSIONS: Being physically inactivity has a greater impact on CRC incidence for individuals living in Chicago communities, in addition to significantly contributing to CRC mortality for current survivors.
AUTHOR: Abhery Das
Neighborhood deprivation and suicide among adolescent and young adult cancer patients
Background/Purpose/Objectives: In 2024, approximately 84,100 adolescent and young adults (AYAs) between 15-39 years old received a cancer diagnosis. Hypothesis/Aims: Given their unique psychosocial, economic, and clinical stressors, we examined whether AYA cancer patients living in deprived neighborhoods have a higher risk of suicide when compared to those in the least deprived neighborhoods. Materials/MethodsOur sample comprised 486,374 AYA cancer patients from the Surveillance, Epidemiology, and End Results (SEER) dataset between 2006-2020. We use Cox proportional hazard models to test the association between quintiles of neighborhood deprivation, from Q1 (most deprived) to Q5 (least deprived), and survival months until suicide mortality from the time of cancer diagnosis. Results: We find that AYA cancer patients living in more deprived neighborhoods (Q1, Q2, Q4) have a higher risk of suicide when compared to those in the least deprived neighborhoods (Q5) (Q1: HR - 1.82 [1.142.90]; Q2: HR - 1.95 [1.35-2.81]; Q4: HR - 1.48 [1.05 - 2.07]). Conclusions: Mental health services and monitoring from treatment through survivorship may support suicide prevention efforts for young cancer patients. Early identification of high-risk AYA cancer patients living in deprived areas may help target suicide prevention interventions.
AUTHOR: Li-Ting, H. Longcoy
Strategies for Engaging in Advance Care Planning Discussions Among Patients with Cancer and Their Family Caregivers: A Qualitative Study
BACKGROUND, PURPOSE, or OBJECTIVES: Patients with cancer often avoid advance care planning (ACP) discussions with their family caregivers, resulting in end-of-life care that does not align with their preferences. ACP is a process that helps patients make informed decisions about end-of-life care by enabling them to clarify and communicate their values and goals to family members and healthcare providers. ACP benefits patients and families by (1) enhancing care satisfaction, patient-family communication, and quality of death and (2) alleviating family caregiver depression, anxiety, and caregiving burden. However, most patients with cancer do not initiate ACP discussions or complete an advance directive - a document outlining their wishes regarding medical treatment. In the US, only 18% of patients with advanced cancer have completed an advance directive, and only 24% have discussed ACP with a healthcare provider following their cancer diagnosis. Factors contributing to the low completion rate of advance directives include (1) discomfort confronting end-of-life, (2) limited understanding of ACP, (3) lack of readiness for end-of-life discussions, and (4) high emotional burden of these discussions. The absence of ACP discussions may delay access to palliative and hospice care, resulting in costly and aggressive treatments that conflict with patient goals and increase caregiver distress. Such challenges underscore the necessity of building resilience to help patients and family caregivers overcome barriers to ACP. To guide the development of a resilience-building intervention, this study explored the strategies and resources used by patients with cancer and their family caregivers during ACP discussions. HYPOTHESIS/AIMS: The aim of this qualitative study was to explore the strategies and resources employed by patients with cancer and their family caregivers during ACP discussions. MATERIALS or METHODS: A qualitative study was conducted using 45-to 60-minute semistructured interviews. Patients with cancer and/or their family caregivers who have completed advance directives were recruited through clinical visits at the University of Illinois Cancer Center and via social media. Two nursing researchers independently conducted conventional content analyses to systematically identify the strategies and resources participants used during ACP discussions. RESULTS: A total of 18 participants completed the interviews. The strategies reported by the participants were organized into 5 categories reflecting key stages of the ACP process: 1) preparation for ACP discussions; 2) identification of decision-making surrogates; 3) initiation of ACP discussions; 4) engagement in ACP discussions; and 5) resolution of disagreements. The most frequently used strategies were those aimed at preparing for ACP discussions, such as clarifying personal values regarding end-of-life care. Strategies focused on resolving disagreements were the next most frequently used. When conflicts arose, caregivers often prioritized patient autonomy as a key strategy, underscoring how resilience helps overcome communication barriers in ACP. CONCLUSIONS: There is a need to implement effective interventions to facilitate meaningful ACP discussions between patients with cancer and their caregivers. Based on qualitative analysis of patient and caregiver interviews regarding ACP discussions, an effective resilience-building intervention should 1) provide education to foster preparation and self-awareness around end-oflife care and 2) incorporate communication strategies to resolve ACP-related disagreements and support meaningful engagement.
AUTHOR: Kate Cares
Feasibility and acceptability of Time Restricted Eating with and without a prebiotic supplement in Pediatric Cancer Survivors
Background The majority of pediatric cancer survivors (PCS) will develop long-term health conditions by the age of 50, with 96% developing at least one critical, chronic health condition. Quality of life and long-term health among survivors of pediatric cancer can be greatly enhanced with the reduction of obesity and an improved dietary lifestyle. Time restricted eating (TRE) may improve body composition and cardiometabolic disease risk through spontaneous caloric restriction and an improved cardiometabolic profile. Prebiotics have also been known to have an additive effect on caloric restriction by upregulating satiating hormones and decreasing inflammation by harnessing the microbiome. The effect of TRE, with or without a prebiotic supplement, could improve the life of survivors of pediatric cancers due to the early onset of cardiometabolic diseases. Methods Adult survivors of a pediatric cancer were randomized 8 hour TRE alone or 8 hour TRE with a prebiotic supplement for 12 weeks. Feasibility was measured by compliance to the eating window and ingestion of the prebiotic supplement while acceptability was measured by a validated survey at the beginning and end of the intervention. Secondary measures included body weight, body composition, and cardiometabolic markers. Results Thirteen participants were enrolled and randomized into TRE alone (n=6) and TRE with a prebiotic supplement (n=7). Feasibility was high with all randomized participants completing the intervention and data collection. Compliance was high, the average eating window of 458.87 minutes for the TRE group and 444.71 minutes for the TRE with prebiotic group. Participants attended 87-92% of weekly intervention meetings and the prebiotic groups ingested 85% of the recommended supplement. Overall acceptability of the trial was high with participants in TRE scoring >3 average across all 5 out of 7 domains on the diet satisfaction survey and the TRE with prebiotic scored >3 across all 7 domains. Additionally, improvement in cardiometabolic risk factors were observed in both arms from the beginning to the end of the intervention but no between group differences were observed. Conclusions TRE with and without a prebiotic supplement is a feasible and acceptable dietary intervention in adult survivors of a pediatric cancer. Additionally, this salient and accessible intervention may be efficacious in improving cardiometabolic risk in PCS.
AUTHOR: Rashwitha J. Saldanha
Electrochemical Biosensors in the Detection of Pancreatic Cancer Biomarkers: AI Driven Model & Patient Cohort Study.
BACKGROUND: Detection of cancer-specific biomarkers has significantly enhanced early detection, leading to improved patient health and mortality. Developing biosensors to detect cancer-specific biomarkers has advanced through the years, making it a sensitive, inexpensive, and quicker approach to diagnosing cancer and monitoring disease prognosis. Pancreatic cancer, a disease with a survival rate of only 13% rate in the U.S, in which about 30-50% of deaths are due to late-stage detection, highlights the critical need for a quick and sensitive diagnostic method, especially for early diagnosis. Gold-screen-based electrochemical biosensors are analytical devices that satisfy the needs mentioned above due to their ability to detect lower concentrations of specific markers, indicating that they can be used for the early detection of pancreatic cancer. The sensitivity is further enhanced by nanoengineering the biosensor with a suitable conductive material like graphene oxide, which increases the surface area for a sample to bind. The addition of detection of multiple pancreatic cancer-specific protein biomarkers like CA125, CEA, CA242, and CA19-9 also enhances the diagnosis of patient samples. HYPOTHESIS: Using biosensors with electrochemical properties can be a quicker solution and a more sensitive approach in predicting the diagnosis of pancreatic cancer in patients by using multiple biomarkers like CA242, CEA, CA19-9, and CA125, which improve the specificity for the detection of pancreatic cancer in the samples. METHODS: Nanoengineering of Sensor: The gold working electrode surface of the biosensor is coated with graphene oxide nano colloidal solution and kept in UV overnight for crosslinking. Electrochemical Biosensor: The nanoengineered sensors are coated with a DSP crosslinker, followed by the addition of biomarker-specific antibodies. A blocking agent, TBS, is added to prevent non-specific binding. The protein and patient samples are added, and the sensors run in a potentiostat. Data from the analysis is added to the AI model. ELISA is performed: A capture antibody is coated onto a plate and binds to the target antigen from the sample. Western Blot: Proteins in the serum sample are separated using gel electrophoresis and transferred to a membrane where specific antibodies to detect the biomarker are added and visualized using chemiluminescence.
RESULTS: Patient samples were individually run on the graphene-coated sensors for all four pancreatic biomarkers (IRB no:360124-28). Electrochemical spectroscopy calculated each patient sample’s impedance(1700 to 2000 Ω), capacitance (7.0-06 - 8.0-06 C) , and resistance (1.0+06 - 9.0+0.6 Rp). Each parameter was compared to a risk table for each biomarker, labeling the results as high risk, low risk, and standard concentrations. Patients were divided into two categories: control non-cancer and cancer. Comparatively, most control samples showed lower concentrations of the cancer biomarkers than the cancer samples, and ELISA and Western Blot justified this data. Further, the data were analyzed using an early-stage AI model, specifically Support Vector Machine (SVM), showing accuracy of 50%. CONCLUSIONS: Pancreatic cancer diagnosis is challenging as detection is difficult during the earlier stages in patients. To solve this problem, targeting multiple cancer biomarkers by electrochemical sensors is a valuable tool in early diagnosis due to its portability, rapid detection and sensitivity.
AUTHOR: Sharmishthaa Gudetee Padmanaban
Collagen Matrix-Specific Peptide Decorated Nanocarriers for Tumor Microenvironment Targeted Drug-Delivery
BACKGROUND: Cancer remains a significant global health challenge due to its heterogeneity and resistance to therapy. Targeting the tumor microenvironment (TME), particularly the extracellular matrix (ECM), offers a promising therapeutic approach. Among ECM components, denatured collagen IV, degraded by matrix metalloproteinase-2 (MMP-2), is critical in tumor invasion and progression. Nanotechnology-based drug delivery systems, such as liposomal platforms, allow for therapeutic agents’ encapsulation and selective delivery. This study focuses on developing a Paclitaxel (PTX)-loaded liposomal formulation modified with a Denatured Collagen Binding Protein (DCBP) to enhance specificity toward altered ECM structures, potentially improving breast cancer treatment.
HYPOTHESIS: We hypothesize that PTX-loaded liposomes decorated with DCBP specifically target denatured collagen IV degraded by MMP2. By improving ECM-specific binding, these liposomes will enhance drug delivery efficiency, reducing tumor cell viability and inhibiting invasion and angiogenesis. METHODS: Empty and PTX-loaded liposomes were prepared via solvent evaporation, with CBP conjugation achieved using click chemistry. Liposome size, stability, zeta potential, and polydispersity index (PDI) were characterized using dynamic light scattering (DLS). PTX encapsulation efficiency and drug release kinetics were determined. Cytotoxicity, colony formation, scratch, and tube formation assays were conducted to evaluate therapeutic efficacy. Co-culture spheroids composed of endothelial cells, fibroblasts, and MCF-7 cells were used for 3D targeting studies. Binding affinity was assessed using spheroids coated with 10 µg/mL denatured and intact collagen IV. Live/dead assays, brightfield imaging, and immunofluorescence evaluated spheroid responses. Hepatotoxicity was assessed using zebrafish embryo assays. RESULTS: DLS revealed PTX-loaded liposomes were 66.5 ± 14.1 nm with a zeta potential of -5.52 ± 0.378 mV, and CBP PTX-loaded liposomes were 68.3 ± 34.7 nm with a zeta potential of -3.94 ± 2.28 mV respectively, and encapsulation efficiency ranged from 42.25% to 63.5%. PTX-liposomes (20 nM and 40 nM) significantly reduced MCF-7 cell viability after 6 hours, inducing G2/M cell cycle arrest. They also suppressed cell migration over 72 hours and inhibited colony formation. ICC images of co-cultured spheroids treated with PTX-loaded liposomes for 24 hours showed a noticeable reduction in endothelial cells. Nuclei (blue), endothelial cells, MCF-7 cells (green), and fibroblasts (red) were stained for visualization. The treatment led to significant disintegration of the spheroids, primarily due to endothelial cell depletion, indicating an inhibition of angiogenesis. This effect was more pronounced with CBP-bound PTX liposomes, suggesting their enhanced ability to disrupt vascularization within the tumor microenvironment. A 50 µg/mL CBP PTX-liposomes concentration prevented endothelial tube formation, indicating anti-angiogenic effects. CBP-liposomes demonstrated enhanced binding affinity to denatured collagen IV over intact collagen IV, confirming specificity. In 3D spheroids, PTX-liposomes and CBP PTX-liposomes disrupted spheroid morphology within 24 hours.
AUTHOR: Cecily Byrne
Food Insecurity and Clinical Biomarkers of Inflammation among Cancer Survivors in the All of Us Research Program
BACKGROUND, PURPOSE, OR OBJECTIVES: Food insecurity is associated with a 40% increase in the prevalence of chronic conditions, including cancer. Stress-evoked inflammation is a hypothesized mechanism driving these associations. HYPOTHESIS/AIMS: This study tested the association between food insecurity and inflammation in cancer survivors. We hypothesized that food insecurity was associated with inflammation in lung, breast, prostate, and colorectal cancer survivors. MATERIALS OR METHODS: Our sample included individuals with a history of lung, breast, prostate, and colorectal cancer from the All of Us Research Program. Food insecurity was measured using the validated Hunger Vital Sign questions, and inflammatory biomarkers were obtained from electronic health records. Our primary analysis tested the association between food insecurity and c-reactive protein (CRP; n=413) using multivariable regression models, controlling for sociodemographics and current cancer treatment. Secondarily, we tested the association between food insecurity and albumin (n=2,948) and neutrophil to lymphocyte ratio (NLR; n=2,497). RESULTS: The primary cohort was 69.8 ± 9.5 years in age, 61.0% female, 89.3% non-Hispanic White, and 9.9% had food insecurity. A higher proportion of racial/ethnic minorities (40.8%) and individuals with lower annual household income (33.3%) and education (29.4%) had food insecurity. Mean CRP was higher among those with food insecurity (14.5 ± 18.5) than food secure individuals (10.4 ± 17.8), but it was not significantly associated with CRP in our fully adjusted models (_=1.18; 95% CI: 0.77, 1.81). Food insecurity was not associated with lower albumin (<3.5 g/dL) nor elevated NLR (≥3) in logistic regression models. CONCLUSIONS: Lung, breast, prostate, and colorectal cancer survivors had moderate levels of inflammation measured by CRP; however, food insecurity was not associated with CRP in fully adjusted models. Future studies should explore this association in other cohorts of cancer survivors.
AUTHOR: Narjes Sweis
Difference in Presentation and Survival Outcomes Based on Country of Origin in Hispanic Patients with Colon Cancer
Introduction: Colorectal cancer (CRC) is the second leading cause of cancer-related death in the U.S., with rising incidence in early-onset cases. Hispanic populations are disproportionately affected, often diagnosed at younger ages compared to other groups. However, limited research stratifies Hispanic patients by country of origin. Aims: This study aims to analyze clinical presentations, treatment patterns, and outcomes among Hispanic subpopulations, compared to non-Hispanic Whites, to better understand disparities and improve care for these patients. Methods: This retrospective cohort study used NCDB data (2008-2021) to analyze Hispanic patients with colon cancer, stratified by country of origin (Mexico, Puerto Rico, Cuba, and other Latin American countries) and compared to non-Hispanic White patients. Chi-square tests (95% CI) assessed demographics, tumor characteristics, and treatment delays. Cox proportional hazards regression analyzed survival, adjusting for stage, Charlson-Deyo score, tumor size, age of onset, sex, insurance status, and origin. Statistical significance was set at p < 0.05. Results: total of 822,628 patients with colorectal cancer were analyzed, including 19,120 Hispanic patients across various subpopulations. Mexican patients represented the largest group (39.6%). Early-onset CRC (<45 years) was more prevalent among Mexicans (15.2%) and South/Central Americans (12.0%), compared to non-Hispanic Whites (5.4%). Treatment delays were noted in 7.4% of Hispanic patients, with Mexicans (9.2%) experiencing the highest rate. Cox regression revealed that advanced stage, higher Charlson-Deyo scores, and being uninsured were associated with worse survival. However, Mexican (HR: 0.748) and South/ Central American (HR: 0.728) patients had better survival than non-Hispanic Whites (p<0.001). Conclusion: This study identified significant disparities in the clinical characteristics, treatment patterns, and outcomes of colorectal cancer among Hispanic patients, stratified by country of origin, compared to non-Hispanic Whites. While some Hispanic subpopulations, such as Mexicans and South/Central Americans, demonstrated better survival outcomes, they were also more likely to experience treatment delays and early-onset disease. These findings highlight the need for targeted public health strategies to improve timely access to CRC screening and treatment for Hispanic patients, ensuring equitable care and better survival outcomes across all subpopulations.
AUTHOR: Svetlan Semina
Single-cell and spatial approaches to identify targets in endocrine therapy-resistant breast cancer
BACKGROUND: Even though most women with estrogen receptor positive (ER+) breast tumors can benefit from endocrine therapy (ET), up to 40% of these patients will experience relapse. Treatment of patients with ER+ tumors is complicated by variability in a number of ER+ cells among tumors. Multiple clinical studies have shown that tumors with a low fraction of ER+ cells (ERlow tumors, 1-10% ER+ cells) are less responsive to ET, recur earlier, and contribute to worse patient survival. A significant factor in ERlow tumors progression is their heterogeneity when various cell populations show different sensitivity to ET. We and others routinely use models that are highly enriched for ER+ cells to study the effect of ET, much less is known about ET efficiency for ERlow breast cancers. OBJECTIVES: The objective of this study is to identify ET-resistant cell populations specific to ERlow tumors and determine more effective therapeutic options to eradicate them. HYPOTHESIS: We hypothesize that we can identify and target ET-resistant cell populations that contribute to disease progression specifically in primary ERlow breast tumors using single cell and spatial RNA-seq, thus preventing tumor recurrence and improving patient outcome. METHODS: To study how ERlow breast cancer models respond to ET, we utilized patient-derived xenograft organoids (PDxOs). In addition to the already established PDxOs (HCI003 and HCI017), we developed another two models (UIC013 and UIC020) with low to moderate number of ER+ cells. We used single-cell RNA sequencing (scRNA-seq) to assess ET response among models and identify resistant populations. DREEP analysis was used to identify new therapies for ET-resistant cells. RESULTS: To understand whether PDxOs are sensitive to ET, we treated each model with fulvestrant or 4-hydroxytamoxifen and performed scRNA-seq. We found that some of the cell populations are lost with ET, suggesting that they are responsive to ET, while others showed no change or even grow with ET, suggesting that they are resistant to ET. Moreover, we found both common and model-specific ET-resistant populations. We suggested that molecular drivers derived from common across patients ET-resistant cell populations will pave the way for conventional therapies, while molecular drivers derived from patient-specific cell populations will guide personalized therapies. We next identified alternative therapeutic options for the ET-resistant cell populations using DREEP analysis, which predicts sensitivity to 450 drugs. To validate our findings, we choose to target common cell populations with mTOR inhibitor AZD-8055. All models showed similar response to the inhibitor, indicating its use as a conventional therapy. While targeting a UIC013-specific cell population with dasatinib significantly increased UIC013 sensitivity to the drug compared to other models, highlighting its potential as a personalized treatment. These findings showed that our analysis effectively predicts novel conventional and personalized treatments to target ET-resistant cell populations, and the selected drugs were successfully validated. CONCLUSIONS: Our findings indicate that stratifying breast cancer cell populations based on response to ET can lead to the identification of aggressive cell populations and novel therapies to target these populations, thereby improving outcomes for women with ERlow breast tumors.
AUTHOR: Keshav R. Gandhi
PolyG Genotyping to Infer Tumor Heterogeneity
ABSTRACT: BACKGROUND, PURPOSE, or OBJECTIVES: Microsatellites are short, repetitive units of DNA base pairs that are common across the span of an organism’s genome (e.g., TATATATATA). They are characterized by their high rates of insertions and deletions. As such, differences in the length of microsatellites across cells can be indicative of a tumor’s evolutionary history. Poly-guanine (polyG) sites are microsatellites that consist of multiple repeated guanine bases across the genome. They are particularly useful in analyzing cancer metastasis, tumor heterogeneity, and cell lineage due to their relatively high mutation rate, presence in many tissue types, and ability to remain agnostic to selective pressures that impact evolution (e.g., chemotherapy). However, the usefulness of polyG analysis can be limited by noise and sequencing errors; in particular, the polymerase chain reaction (PCR) process required for amplifying DNA to sequence genomes introduces additional insertions or deletions. These errors limit the ability of polyG sites when conducting downstream exploration into the unique evolutionary histories of tumors. HYPOTHESIS/AIMS: We create and validate a statistical model that corrects for errors in polyG lengths introduced by the PCR process. This reconstructs the most likely polyG genotype (i.e., pair of lengths) across two alleles. MATERIALS or METHODS: Our method makes use of the expectation-maximization (EM) algorithm, a gold standard in computational statistics and statistical inference. The algorithm works by maximizing the likelihood that we observe specific polyG lengths after PCR has occurred. RESULTS: We found that our model fit non-cancerous samples well and found the most-likely genotypes consistently across replicate samples from the same patient. Comparison to simulations of previous PCR error-correction models show the novelty, validity, and enhanced performance of our method. We also provide multiple proof-of-concept simulations. CONCLUSIONS: This model is currently being used to develop a method to infer tumor phylogenetic (evolutionary) histories from polyG sequencing data of colorectal cancers.
AUTHOR: Shruti Rodrigues
Mapping immuno-vascular signatures to characterize BBB disruption and tumor colonization in TNBC brain metastasis.
BACKGROUND, PURPOSE, or OBJECTIVES: Metastasis is the leading cause of breast cancerrelated deaths, with 6% of women in the US diagnosed with metastatic breast cancer at initial diagnosis. Brain metastasis (BrM) incidence in breast cancer (BC) patients varies by subtype, with higher rates in HER2-positive and triple-negative breast cancer (TNBC). TNBC patients with BrM face a poor prognosis, with a median survival of less than six months. The bloodbrain barrier (BBB) plays a critical role in BrM development, as its disruption facilitates tumor cell entry into the brain parenchyma (BP). However, the immune and vascular signatures driving BBB disruption and BrM establishment remain poorly understood. This study aims to investigate the vascular and immune mechanisms underlying BBB-to-blood-tumor barrier (BTB) transformation and identify potential therapeutic targets to disrupt BrM progression.
HYPOTHESIS/AIMS: Changes in the integrity of the BBB-characterized by a fenestrated vasculature with increased permeability-facilitate the entry of TNBC cells into the BP. However, the immune and vascular signatures driving this process are understudied. This study will investigate the transformation of the BBB into a BTB by identifying immunological and vascular responses associated with the TNBC-BrM microenvironment. We hypothesize that understanding the heterogeneity of the BTB, immune cell infiltration, and evolving tumor microenvironment (TME) will reveal how specific cell types promote or suppress BrM formation. This study aims to: 1. Define vascular signatures (adhesion and endothelial markers) associated with BBB disruption and BTB formation. 2. Identify brain-infiltrating and parenchymal cell types involved in immune invasion during BrM establishment. Our long-term goal is to identify novel immune and vascular signatures involved in BrM formation and uncover targetable constituents affecting BBB integrity in TNBC brain metastasis. MATERIALS or METHODS: Using the 4T1Br5 syngeneic Balb/c mouse model, which recapitulates the establishment of metastasis from breast tissue to the brain, we will study TNBC BrM in an immunocompetent system. Mice will be inoculated with 4T1-Br5 GFP-Luc cells via mammary fat pad with a control cohort receiving PBS. Tumor growth and metastasis will be monitored using bioluminescent imaging (BLI). Brain tissues will be harvested for analysis. Cutting-edge techniques, including spatial proteomics (CODEX imaging), CyTOF mass cytometry, spatial transcriptomics (Visium HD), and singlecell RNA sequencing, will map the evolving TME and BTB. CONCLUSIONS: This study will provide unprecedented insights into the cellular and molecular mechanisms driving BBB-toBTB transformation and BrM establishment in TNBC. By leveraging immunocompetent mouse models and advanced spatial and single-cell technologies, we aim to identify novel immune and vascular signatures that could serve as therapeutic targets to disrupt BrM progression and preserve BBB integrity. Comparative analysis with human data will further validate the clinical relevance of our findings, paving the way for improved treatment strategies for TNBC BrM.
AUTHOR: Haarisudhan Sureshkumar
Access Disparities in Uveal Melanoma Care: A Geographic Service
Area Analysis of Ocular Oncologists in the United States
Purpose: Uveal melanoma (UM) is the most common intraocular malignancy in adults and is known to have a poor prognosis. Although the genetic profiling of UM is well characterized, there is emerging evidence regarding other factors that can influence the presentation and prognostication of UM. Other factors, such as social determinants of health (SDOH) and geographic barriers, may play an important role in the presentation of UM. Our study aims to identify areas in the US with high unmet needs for UM care based on geographic distribution and potential confounding SDOHs that affect accessibility to care. Hypothesis: Geography, complicated by sociodemographic variables, lead to significant barriers to ocular oncologist care in the United States. Methods: Using multiple ophthalmological society lists, the addresses of various ocular oncologists were identified and used to generate a map of 15-, 30-, and 60minute radii of drive times from the nearest ocular oncologist office. A drive of time of less than 60 minutes was deemed as within accessible coverage. High unmet need was evaluated by cross evaluating areas with a drive time greater than 60 minutes to the closest ocular oncologist and areas of high population density. SDOH data from the US Census Bureau from census tracts within 60 minutes and outside 60 minutes of the closest ocular oncologist were analyzed and compared using Python. Results: 33.5% of the US population does not live within sixty minutes of an ocular oncologist. Furthermore, the census tracts that were outside of ocular oncologist coverage were found to have a lower proportion of individuals with a bachelor’s degree, a lower number of insured individuals, a lower median income, a higher proportion of impoverished households, and limited internet access. Lastly, 46% of patients living in rural areas were living in an area outside of ocular oncologist access. Conclusion: Our geographic service analysis revealed that a large portion of the US adult population has poor access to ocular oncologists, which may lead to delays in diagnosis and management. Individuals in regions with poor access are also more likely to be from socioeconomically disadvantaged backgrounds. Lastly, the large proportion of individuals living in rural areas without ocular oncology access underscores the need for ocular oncologists in these areas or the development of telemedicine solutions to provide remote ocular oncology care. Further study should assess the effects of this poor access, complicated by the aforementioned SDOHs, on the presentation and prognostication of uveal melanoma.
AUTHOR: Hannah M Maluvac
Clinical Implications of HER3 Overexpression in a Diverse Patient Cohort with Prostate Cancer
Background: Prostate cancer (PCa) contributes to almost 15% of US cancer cases annually with significant racial disparities, where black men are more likely to develop and die from prostate cancer than any other cohort. Previously, we reported that HER3/ERBB3 overexpression (OE) was enriched in the tumors of Black/ African American patients and was correlated with a unique androgen receptor signature as well as worse clinical outcomes. Here, we evaluate an expanded and more mature patient cohort to understand the effect of HER3 OE on clinical outcomes. In vitro experiments further support the targeting of HER3 in prostate cancer disease. Hypothesis: We hypothesize the HER3 overexpressing prostate cancer is associated with poor prognosis in patients and that in vitro overexpression of HER3 will result in more aggressive phenotypes (increased cell growth, resistance to therapies), which may be rescued using HER3 targeted therapies. Methods: Chart review was performed on a diverse cohort of PCa patients from the University of Illinois Health (n=106). Tempus laboratories performed whole transcriptome RNA sequencing, and HER3/ERBB3 OE status was determined by Tempus as compared to a reference database. Patients were grouped into HER3 OE and wild-type groups, and clinical outcomes were analyzed between groups using students’ t-test and Kaplan Meier plots with the Gehan-Breslow-Wilcoxon test. LNCaP cells were virally transduced with HER3 overexpression and non-targeting control vectors and validated via Western blot. Cell growth was quantified via nuclear fluorescence object count using Incucyte instruments and software. Drug treatment efficacy was validated via Western blot. Results: The final cohort (n= 71 Black/AA, 19 white, and 5 other) demonstrated 42% of patients presenting with HER3 OE. A majority of patients presented with de novo metastases (55% of HER3 OE, 45% of HER3 WT). Of patients who initially presented with localized disease, time to metastasis was significantly faster in the HER3 OE group (p=0.04). HER3 OE was also associated with faster time to the development of resistance to first line therapy (castration resistance) (p=0.02). In preclinical prostate cancer models, HER3 OE cells grew significantly faster than control(p=<0.01), and were less sensitive to enzalutamide (p=<0.01). Additionally, we found that by inhibiting HER3 signaling (patritumab, erlotinib, trastuzumab, capivasertib) PCa cells become more sensitized to enzalutamide (p=<0.01), suggesting a role for dual AR and HER3 targeting in PCa treatment. Conclusions: Our clinical and translational data supports the role of HER3 overexpression as a novel and targetable prognostic marker in diverse patients with PCa. Future studies should further evaluate HER3 inhibition in combination with AR targeted therapies to improve therapy sensitivity and reduce development of resistant disease.
AUTHOR: Philana, V Phan
Adjuvant Decorated Dying Tumor Cells for Personalized Anti-tumor Immunity
BACKGROUND, PURPOSE, or OBJECTIVES: Cancer vaccines, harnessing the patients’ immune system to recognize and eliminate cancer cells, are a promising type of immunotherapy adaptive to various cancers. The potency of cancer vaccines require a tumor antigen as the core component in conjunction with the presence of additional immuno-activators to attain this effect. However, the efficacy of current investigative cancer vaccines remains limited, largely due to tumor heterogeneity and highly immunosuppressive tumor microenvironment. To generate a robust tumor-specific immune response, vaccine design must be tailored to incorporate primary antigens as along with customizable immune-stimulating adjuvants capable of eliciting a broad and durable anti-tumoral effect. HYPOTHESIS/AIMS: We introduce a novel tumor vaccine platform based on enucleated tumor cells integrated with combinatorial adjuvants for effective cancer immunotherapy. This approach leverages the immunogenic dying process of enucleated tumor cells for enhanced immune response induction, offering the potential for effective presentation of native tumor antigens in their physiologically relevant conformations for robust cancer immunotherapy. MATERIALS or METHODS: The removal of the cell nucleus via density gradient centrifugation produces enucleated cells, which serve as the central component of the vaccine system. Combinatorial adjuvants are encapsulated into polymeric nanoparticles, which are subsequently anchored to enucleated cell surface through metal-phenolic network formation, resulting in the assembly of a cell-based vaccine. The vaccine formulation was characterized through multiple in vitro assays and its therapeutic efficacy was evaluated in the B16F10 and MC38 tumor models in vivo. RESULTS: Enucleated tumor cells remained transiently lived for 48 hours, lost their proliferating capabilities, and showed increasing immunogenicity over time through the release of damage-associated molecular patterns. Analysis of the assembled vaccine formulation revealed non-uniform binding of adjuvant-loaded nanoparticles on the cell surface. By combining emulated tumor cells and Toll-like receptor (TLR) agonists into a single cell-based platform, we observed potent anti-tumor immune responses in the MC38 and B16F10 tumor models. In the MC38 model, when used in combination with the immune checkpoint inhibitor anti-PD-1 (aPD1), the enucleated tumor cell vaccine led to 83% of treated mice achieving complete tumor regression. Moreover, 80% of mice in the combination therapy group remained tumor-free following rechallenge, indicating the generation of strong and durable anti-tumor immune memory. CONCLUSIONS: Our vaccine design leverages biodegradable and biocompatible components, with selected adjuvants that modulate immune cell recognition and activation, creating an integrated, allin-one platform. Notably, our approach does not rely on the identification of a specific neoantigen, enhancing it potential translatability across diverse cancer types. The enucleated tumor cell vaccine technology can be a broadly effective approach for immunotherapy adaptable for various cancers.
AUTHOR: Olayinka G. David
Development of Genetically Defined Hepatocellular Carcinoma Models for Precision Medicine Testing
Objective: Liver cancer is the third leading cause of cancer-related mortality worldwide. Hepatocellular carcinoma (HCC), the most prevalent type of liver cancer, is an aggressive disease with poor response to existing therapies. Current treatment strategies are hindered by a ‘one-size-fits-all’ approach, failing to account for the distinct genetic and molecular alterations across different tumor subtypes. Available human HCC cell lines often accumulate random passenger mutations that complicate the identification of targeted therapies. To address this challenge, this study aimed to create in vitro models of HCC by inducing defined genetic alterations in primary human hepatocytes. These models will recapitulate diverse HCC molecular landscapes to facilitate the development of personalized treatment strategies. Materials and Methods: Cryopreserved normal primary human hepatocytes were commercially obtained and subjected to CRISPR knockout (KO) of three tumor suppressor genes with an oncogene overexpression (OE). CRISPR mediated gene KO was induced by lipid-based ribonucleoprotein transfection and c-MYC OE was achieved by lentiviral transduction. Transformed hepatocytes were propagated in vitro for 1-2 months to allow natural selection of cells with higher proliferative ability. Gene alteration combinations included: TP53KO; CDKN2AKO; c-MYCOE (TKM) and TP53KO; PTENKO; CDKN2AKO; c-MYCOE (TPKM). Single cell clones were isolated from each cell line. Gene alterations were validated by PCR, Sanger sequencing analysis, and Western blotting. The developed cell lines were injected subcutaneously into severe combined immunodeficient (SCID) mice to confirm their tumorigenicity and assess tumor histological features. Results: Two cell lines with 3 or 4 gene alterations (TKM and TPKM) were developed from primary human hepatocytes. Consistent with the anticipated CRISPR/Cas9 activity, sequencing analysis showed small insertions and deletions (indels) around predicted Cas9 cleavage sites for all targeted genes. PCR confirmed the integration of c-MYC construct in the cell pools and single cell clones for both cell lines. Western blotting revealed a complete loss of TP53, PTEN, P16 (one of the 2 products of CDKN2A), and c-MYC overexpression. P14 protein (the second CDKN2A product) was lost in TKM, but not TPKM, consistent with sequencing analysis. Mice injected with TPKM cell pool and single cell clones developed tumors within 6 weeks and were euthanized in 8-10 weeks. There was no tumor development in TKM-injected mice over a period of 4 months. TPKM xenograft mouse tumors are currently being analyzed to determine their histological features. Conclusion: Genetically defined tumorigenic cell lines were successfully developed by CRISPR editing of primary human hepatocytes. Altered genes are among the most frequently mutated in patient population. To our knowledge, these results provide the first proof of concept for the feasible generation of cancer cell lines and xenograft tumors with defined driver mutations by somatic gene editing of normal human hepatocytes. These cell lines will be valuable for developing in vivo models for preclinical evaluation of novel interventional therapies for precision medicine to improve patient outcomes.
AUTHOR: Trieu Phong
Identification of Novel Cell-Penetrating Peptides Derived from Bacterial Signal Sequences
Identification of Novel Cell-Penetrating Peptides Derived from Bacterial Signal Sequences
PRESENTING AUTHOR: Trieu Phong1 CO-AUTHORS: Duy Binh Tran1, Jediael Chintha2, and Tohru Yamada1, 3 1Department of Surgery, Division of Surgical Oncology, University of Illinois College of Medicine, Chicago, IL 60612, USA 2Illinois Mathematics and Science Academy 3Richard & Loan Hill Department of Biomedical Engineering, University of Illinois College of Engineering, Chicago, IL 60607, USA ABSTRACT BACKGROUND Targeted drug delivery has been a major focus in biomedical research to improve therapeutic efficacy while minimizing off-target effects. One promising strategy involves cell-penetrating peptides (CPPs), which facilitate the intracellular transport of diverse molecular cargoes, including small molecules, proteins, RNA, and DNA. Their ability to traverse biological membranes makes them valuable tools in drug delivery, diagnostics, and biomedical applications. Among the various sources of CPPs, bacterial proteins and their derivative peptides have emerged as promising candidates. Notably, azurin, a bacterial protein, has attracted attention for its ability to enter human cells and exhibit anticancer activity. Our previous studies have identified a fragment of azurin, p28, as a CPP with preferential tumor penetration, and have advanced into clinical trials. Building on these findings, we aim to identify new CPPs from novel sources. In this study, we focused on signal sequences derived from bacterial protein azurin. As natural signal sequences (aka signal peptides) can direct proteins to specific locations within a cell, we hypothesized that they could offer a promising avenue for CPP discovery. This research evaluates the cellular uptake efficiency of azurin-derived signal sequences in human cancer cells, investigating their potential as novel CPPs for therapeutic applications. METHODS Signal sequences from the azurin protein were retrieved from the NCBI protein database and analyzed using CellPPD tool for CPP prediction. Selected candidates were chemically synthesized. To evaluate their ability to enter cells, each peptide was conjugated to the fluorescent dye Alexa Fluor 568. Human cancer cell lines-including melanoma, glioblastoma, breast, prostate, and ovarian cancers-were incubated with the labeled peptides (20 µM) for 2 hours. Uptake was assessed using confocal microscopy (Zeiss LSM 710) and quantified via flow cytometry. Highly cationic octaarginine peptide (Arg8, 5 µM) serves as the positive control. RESULTS Total of 51 unique azurin signal sequences were identified among 31,031 azurin-related protein sequences. Based on prediction scores, isoelectric points, and molecular weights, five potential peptides (named Kk_20, Pp_20, Rm_21, Pf_21, and Pa_29) were selected for synthesis at >92% purity. Confocal microscopy and flow cytometry analyses showed that all five azurin-derived signal peptides could penetrate various cancer cell lines. Notably, Pf_21 exhibited the broadest uptake, successfully entering breast, prostate, and glioblastoma cancer cells. Kk_20 showed selectivity for prostate and ovarian cancer cells. In contrast, Rm_21, Pp_20, and Pa_29 displayed more limited penetration, with uptake observed in prostate cancer cells.
CONCLUSIONS Our findings highlight the potential of azurin-derived signal peptides as novel CPPs with intracellular penetration in several types of human cancer cell lines. These results provide valuable insights for developing new CPPs by leveraging naturally occurring signal peptide motifs for improved intracellular delivery strategies.
AUTHOR: Nusrat I Khan
Genetic Modification of Hepatocellular Carcinoma Cells to Model Tumor Aggressiveness Related to Creatine Metabolism
ABSTRACT: BACKGROUND: Liver cancer is a major global health burden, representing the sixth most diagnosed cancer and the third most common cause of cancer death. Recent reports have described the contribution of creatine (Cr) metabolism to hepatocellular carcinoma (HCC) development, aggressiveness, and prognosis. However, there is a lack of preclinical models that allow investigating the effects of Cr metabolism on tumor imaging. Cr is metabolized by mitochondrial Cr kinase (MtCK), which is encoded by the CKMT1 gene. MtCK converts ATP and Cr to ADP and phosphocreatine, which diffuses through the cytosol where cytosolic creatine kinases (CKs) regenerate Cr and ATP to meet energy demands. The goal of this study was to develop HCC preclinical models with varying Cr levels to serve as a platform for developing novel diagnostic molecular imaging strategies based on Cr metabolism. METHODS: A genetically engineered porcine model, the Oncopig, was used in the study due to the similarity of pigs to humans in size, anatomy, genetics, and metabolism. Oncopigs harbor Cre recombinase inducible KRASG12D and TP53R167H transgenes. Porcine HCC cells were developed by exposing Oncopig hepatocytes to an adenoviral vector encoding Cre recombinase. To investigate the functional effects of CKMT1 in the porcine HCC cells, the CKMT1 gene was overexpressed (OE) using a lentiviral vector or CRISPR Cas9 for gene knock out. Single cell isolation led to the generation of two CKMT1KO cell lines. Cr levels in these cells were measured by a colorimetric assay. Porcine HCC cells (parental, CKMT1 OE, and CKMT1 KO) were injected into immune deficient mice to assess the tumor growth rate in vivo. RESULTS: Cr metabolite profile in the CKMT1 KO clones demonstrated increased Cr levels (1.6- and 1.9fold increase respectively) compared to the parental line. Additionally, these cells showed reduced migratory and proliferation rates. In contrast, cells with CKMT1 OE demonstrated a decreased Cr level (0.4-fold less as compared to parental). However, CKMT1 upregulation did not affect cell proliferation and migration rates. Comparison of tumor growth rate over time revealed that tumors with CKMT1 overexpression had faster growth rate when compared to parental cells (p< 0.001). Tumors with CKMT1 KO grew at rates comparable to those of the parental group. CONCLUSIONS: This study demonstrates the feasibility of the development of genetically defined Oncopig HCC cells with varying Cr levels. This serves as a foundation for the development of in vivo porcine models with varying HCC tumor metabolism enabling the development of non-invasive in vivo molecular and metabolic imaging methods to improve HCC diagnostic performance, risk stratification, and treatment response monitoring.
AUTHOR: Shreya Pattisapu
Impact of Immunosuppression on Oncopig Lung Tumor Growth and Regression
Background: While the Oncopig shows promise for improving translation of novel therapeutics into clinical practice, observed spontaneous tumor regression limits its applicability for longterm efficacy studies. Aims: This study aimed to evaluate immunosuppression (cyclosporine (CSA)) for elimination of spontaneous tumor regression in the Oncopig lung cancer model. Methods: Oral CSA dosing (15 mg/kg 2x/day) was initiated >1-week prior to tumor induction and maintained for either 4- (n=3) or 10-weeks (n=7) post tumor induction. Two control Oncopigs were not immunosuppressed. Lung tumors were induced through bronchoscopeguided intrapulmonary injection of adenoviral vector encoding Cre recombinase (AdCre; n=6 sites/Oncopig). Therapeutic CSA trough levels (400-2000 ng/mL) were assessed biweekly, while serum C-reactive protein (CRP) levels were assessed weekly. Weekly CT scans were performed to track tumor growth and regression. Tissue samples were stained for H&E and KRASG12D to confirm the presence of neoplastic cells. Results: Tumor formation was observed in all Oncopigs. An average of 4 tumors/pig was observed for non-immunosuppressed Oncopigs (range 0.5-3.2 cm), 8 tumors/pig for Oncopigs immunosuppressed for 4-weeks (range 0.4-5.7 cm), and 6 tumors/pig for Oncopigs immunosuppressed for 10-weeks (range 0.4-6.4 cm). Tumor sizes peaked at 2-weeks post tumor induction for non-immunosuppressed Oncopigs, followed by tumor regression and resolution by 4-5 weeks. In contrast, sustained tumor formation was observed in immunosuppressed Oncopigs for the duration of the immunosuppression timeframe, followed by tumor regression within 2-weeks of CSA removal. Tumor burden and CRP levels were highly correlated for non-immunosuppressed Oncopigs and Oncopigs immunosuppressed for 4-weeks (Correlation Coefficient range 0.578-1), but not for Oncopigs immunosuppressed for 10-weeks (Correlation Coefficient -0.336 to 0.660) due to inconsistent tumor burden calculations related to lung collapse. Histopathological results confirmed the presence of neoplastic cells in 20% of lung tumors in non-immunosuppressed Oncopigs, 28.6% of lung tumors in Oncopigs immunosuppressed for 4-weeks, and 71.7% of lung tumors in Oncopigs immunosuppressed for 10-weeks. Conclusion: This study confirmed the ability of CSA to modulate Oncopig lung tumor growth and regression, indicating Oncopig tumor regression is a result of T cell mediated tumor cell killing. Immunosuppressed Oncopig models therefore have the potential to be effective tools for long-term efficacy studies.
AUTHOR: Chih-Jia Chao
Antigen capturing nanoparticle boosted cDC1 therapy for in situ cancer immunization
Background: Cancer immunotherapy, which stimulates the immune system to induce antigenspecific immune responses against cancers, has emerged as a promising approach for cancer intervention. However, following the initial striking results observed in trials of checkpoint blockade and chimeric antigen receptor (CAR) T cell therapy for certain cancers, progress of immune therapy has been modest. Induction of effective antitumor immune responses is hindered by the scarcity of appropriate antigen-presenting cells and their impaired function within the immunosuppressive tumor microenvironment. This weakens the presentation of tumor antigens to immune cells in the lymph nodes and reduces the ability to eliminate tumors. To improve the success of cancer immunotherapy, developing a robust immunotherapeutic technology capable of both modulating the suppressive tumor microenvironment and facilitating tumor antigen presentation remains an urgent unmet need. Aims: Our goal is to develop a novel anti-tumor immunotherapy that overcomes the suppressive tumor microenvironment and facilitates antigen presentation to achieve efficacious and enduring therapeutic effects. Methods: We developed a novel strategy, named Antigen Capturing nanoparticle Transformed Dendritic Cell therapy (ACT-DC), incorporating two key components: antigen-capturing nanoparticles (AC-NPs) and a unique subtype of dendritic cells known as CD103+ type 1 conventional dendritic cells (cDC1s). AC-NPs are engineered to capture antigens directly from the tumor and facilitate their delivery to adoptively transferred cDC1s. With the efficient migration ability and potent antigen presentation capacity of cDC1s, ACT-DC therapy reshapes the tumor microenvironment and generates a robust anti-tumor immune response. Results: Our findings demonstrate that ACT-DC significantly improves tumor antigen collection and increases antigen delivery to the tumor draining lymph nodes. Additionally, ACT-DC could trigger a strong systemic immune response and transform the tumor environment into a more immunogenic state with more activated dendritic cells and cytotoxic T cells. Across multiple tumor models, including colon cancer, melanoma, and glioma, ACT-DC combined with immune checkpoint inhibitors led to 50-100% tumor-free survival and successfully prevented tumor recurrence after rechallenges. Conclusions: This study highlights the potential of ACT-DC to drive systemic tumor eradication. By harnessing intratumoral antigen, modulating local tumor microenvironment, and improving antigen presentation, ACT-DC elicits efficient anti-cancer immunity and durable tumor rejection. Our findings suggest that ACT-DC could serve as a broadly effective strategy for in situ cancer immunization, with the potential to treat multiple types of cancer.
AUTHOR: Rade R. Jibawi Rivera
SGLT-2 Inhibitors in Cancer Care: A Translational Review of Preventative Therapies in Anthracycline Induced Cardiomyopathy
Background, Purpose, or Objectives: Anthracycline-based chemotherapeutic agents remain cornerstone treatments in a variety of hematologic and solid malignancies due to their potent antineoplastic efficacy. However, their clinical utility has been shown to be limited by dose-dependent, cumulative cardiotoxicity, primarily manifesting as non-ischemic, dilated cardiomyopathy with resultant heart failure. This cardiotoxicity is thought to arise from a combination of oxidative stress, mitochondrial dysfunction, and topoisomerase II_-mediated DNA damage in cardiomyocytes, underscoring the need for effective cardioprotective strategies that do not compromise oncologic outcomes. While angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) have shown cardioprotective effects in chemotherapy-induced cardiomyopathy, the role of newer heart failure therapies remains poorly defined. Since sodium-glucose cotransporter-2 (SGLT-2) inhibitors were designated as Class I therapy for heart failure with reduced ejection fraction (HFrEF) in the 2022 AHA/ACC/HFSA guidelines, robust data from cardiovascular outcomes trials have demonstrated their cardioprotective benefits. However, their utility in the prevention or mitigation of anthracycline-induced cardiotoxicity has yet to be comprehensively evaluated. Hypothesis/Aims: We hypothesized that SGLT2 inhibitors may confer superior cardioprotection relative to standard therapies in individuals exposed to anthracyclines. This study aimed to systematically evaluate the existing preclinical and clinical evidence supporting their role in cardio-oncology and to identify knowledge gaps warranting further investigation. Materials or Methods: A systematic review was conducted according to PRISMA 2020 guidelines. Comprehensive searches of biomedical databases and clinical trial registries were performed through March 2025 to identify studies evaluating the cardioprotective effects of SGLT2 inhibitors, beta-blockers, or ACE inhibitors in adult patients undergoing anthracycline chemotherapy or in relevant animal models. Primary endpoints included changes in left ventricular ejection fraction (LVEF), global longitudinal strain (GLS), and incidence of heart failure. Studies were excluded if they involved patients with preexisting heart failure or cardiotoxicity unrelated to anthracycline exposure. Results: Preclinical studies (n=4) consistently demonstrated that SGLT2 inhibitors attenuate anthracycline-induced cardiac injury, including reductions in cardiomyocyte apoptosis, myocardial fibrosis, and oxidative stress. These effects translated into improved preservation of cardiac function. In one representative study, animals receiving SGLT2 inhibitors in conjunction with anthracyclines exhibited significantly higher LVEF (61.3% ± 11%) compared to controls receiving anthracyclines alone (49.2% ± 8%, p = 0.007). Histopathologic analyses corroborated reduced myocardial damage and improved tissue integrity. No clinical trials to date have directly assessed SGLT2 inhibitors in cancer populations. However, large cardiovascular trials-such as EMPA-REG OUTCOME and DECLARE-TIMI 58-have demonstrated reduced heart failure events in highrisk patients outside of oncology. In contrast, ACE inhibitors and beta-blockers have yielded variable results in patients undergoing chemotherapy, with moderate-quality evidence and inconsistent trial outcomes. Conclusions: SGLT2 inhibitors represent a compelling candidate class for cardioprotection in patients receiving anthracyclines. Their favorable effects in preclinical cancer models, coupled with wellestablished benefits in non-oncologic cardiovascular populations, support the rationale for dedicated clinical trials in cardio-oncology. Validation of their efficacy could significantly reduce the burden of chemotherapy-induced heart failure, with transformative implications for survivorship care.
AUTHOR: Samira R Cheruku
Brush Biopsy miRNA-Based Classifier for Oral Squamous Cell Carcinoma Diagnosis
BACKGROUND: RNA-based diagnosis and prognostic methods for oral squamous cell carcinoma (OSCC) have been slow in clinical adoption, despite early diagnosis being crucial in improving patient prognosis. Current diagnostic techniques with 90% accuracy often require a referral to an oral surgeon for a scalpel biopsy and histopathology. We have explored analysis of RNA from brush biopsy of oral lesions to detect and diagnose OSCC versus a benign look alike lesion, oral lichen planus (OLP), that can be done by a primary care clinician. An initial pilot study demonstrated 87% accuracy in differentiating OSCC from control samples collected by a single clinician. AIMS: The primary aim is to expand the number of samples so to better train an accurate miRNA-based classifier for OSCC and OLP. Additionally, we aim to evaluate the accuracy and reproducibility of miRNA profiles across samples collected by multiple clinicians and develop a machine learning-based classifier that utilizes identified miRNAs. METHODS: Brush biopsy samples were collected from patients with confirmed OLP and OSCC by four clinicians. Total RNA was extracted and analyzed for miRNA expression via polyadenylation RT-PCR over a panel of 360 miRNAs. Differential expression analysis was performed to identify miRNAs associated with each condition and simple machine learning was utilized to produce a classifier to differentiate OSCC within the new study group. RESULTS: Usage of Support Vector Machines (SVM) produced a classifier which with K-fold cross validation (kCV) showed 100% sensitivity and 62.5% specificity with the sample set collected almost exclusively by a single clinician. This data set was expanded to 55 samples, by collecting 21 additional samples from 3 clinicians. When SVM was used to generate a classifier, kCV revealed 89% sensitivity and 80% specificity. A random forest classifier generated with kCV could differentiate OLP from OSCC, with 94% sensitivity and 75% specificity.
CONCLUSIONS: The usage of four different clinicians to perform brush biopsies produced similar predictive accuracy based on miRNA profiles as the earlier classifier with 90% of samples from one clinician. Although differences in N in the two datasets can contribute to the results, both methods showed fairly similar accuracy overall. To create a more accurate miRNA classifier we will acquire more brush biopsy samples from additional patients. Based on the miRNA profiles of the samples collected so far, 78 samples would be required to generate a classifier that achieves a tolerance of 0.1; essentially, the average values of the resulting classifier would be within 10% of a classifier developed with unlimited sample size.
AUTHOR: Arian Karimi
Metastatic Parathyroid Carcinoma with Refractory Hypercalcemia and Multimodal Imaging Findings: A Case Review
BACKGROUND/ Purpose/Objectives: Parathyroid carcinoma is a rare type of cancer and one of the causes of primary hyperparathyroidism (PHPT). It often presents with clinical symptoms including but not limited to hypercalcemia, neck mass, bone disease, etc. Hence, given the rarity, conditions such as parathyroid adenoma and primary parathyroid hyperplasia are often considered first on differential diagnosis. Ultimately, the diagnosis involves a multidisciplinary group of healthcare fields, from genetics and oncology to radiology and surgery specialties. HYPOTHESIS/AIMS: The study aims to review the longitudinal imaging findings, pitfalls in imaging, the importance of nuclear medicine imaging, and multidisciplinary management of a rare case of metastatic parathyroid carcinoma. MATERIALS/METHODS: A 55-year-old female patient was diagnosed with parathyroid carcinoma after undergoing total thyroidectomy. Serial CT, ultrasound, sestamibi-based SPECT/CT parathyroid scans, and FDG PET/CT studies were used to evaluate disease recurrence, metastatic progression, and treatment response. Imaging findings were correlated with clinical parameters and treatments. RESULTS: Initial imaging demonstrated findings suggesting a parathyroid adenoma and thyroid nodule with FNA cellular analysis suggesting a thyroid malignancy. Surgical pathology indicated parathyroid carcinoma. Post-thyroidectomy surveillance imaging (2019-2021) showed no recurrence. In 2022, CT and PET identified multiple pulmonary nodules and a neck lesion, with subsequent growth and new nodules observed in follow-up studies. However, serial imaging studies did not detect definitive recurrent metastatic disease. Serial CT studies documented interval growth of treated lung nodules, and PET/CT studies revealed nonspecific hypermetabolic activity in these nodules. However, the most recent NM parathyroid SPECT/CT demonstrated increased uptake in cervical/thoracic lymph nodes and pulmonary nodules, indicating metastatic recurrent disease. Imaging findings paralleled rising PTH and hypercalcemia, underscoring the progression of metastatic activity. CONCLUSIONS: This case highlights the critical role of multimodal imaging in managing metastatic parathyroid carcinoma. It also demonstrates the complexity of evaluating treatment response, including the potential pitfalls of surveillance imaging and the importance of multidisciplinary management.
AUTHOR: Edidiong Udofa
i-Bind: In Situ Red Blood Cell (RBC) Hitchhiking Nanoparticles for Targeted Treatment of Lung Metastasis
BACKGROUND: Lung metastasis is a common and often fatal condition among patients with metastatic cancer. It occurs in 40-50% of these patients and remains a leading cause of cancerrelated death. Effective treatment of lung metastasis is hindered by the poor accumulation of therapeutics to metastatic lesions due to the presence of various biological barriers. Here, we report a new nanoparticle (NP)-based delivery technology capable of lung-specific deposition, which we refer to as i-Bind NPs, for effective treatment of lung metastasis. i-Bind NPs are biodegradable polymeric NPs surface coated with polyphenols. We demonstrate that i-Bind NPs spontaneously hitchhike onto red blood cells (RBCs) in the bloodstream, mechanically shear off, and deposit in lung capillaries to achieve lung-specific delivery. We also demonstrate that when loaded with a STING agonist, i-Bind NPs enable effective modulation of the immune environment in the lungs and improve and improve lung metastasis treatment. AIM: To develop and characterize polyphenol-functionalized PLGA nanoparticles (i-Bind NPs) for enhanced RBC hitchhiking in the bloodstream, enabling targeted lung accumulation and improved drug delivery to metastatic sites. METHODS: Poly(lactic-co-glycolic acid) (PLGA) NPs were synthesized using a double emulsion technique. Surface modification of NPs was carried out using a biocompatible polyphenol coating consisting of tannic acid (TA) and ferric ion complex, resulting in the in-situ hitchhiking (i-Bind) NPs. Surface functionalization of NPs was confirmed by Fourier-transform infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), and Energy Dispersive X-ray Spectroscopy (EDS). The RBC binding affinity of i-Bind NPs was evaluated in vitro, and the lung targeting ability was also assessed in vivo in a B16F10 lung metastases model. RESULTS: Size characterization of i-Bind NPs indicated the presence of a coating, as evidenced by a slight increase in particle size (190.12 ± 1.28 nm) compared to uncoated NPs (179.33 ± 1.84 nm). TEM imaging of i-Bind NPs further confirmed this coating, revealing an approximate thickness of ~20 nm, which was also supported by corresponding TA peaks in FTIR analysis. The strong binding affinity of the phenolic motifs present on i-Bind NPs resulted in a 6-fold increase in RBC binding in whole blood samples compared to uncoated PLGA NPs. This surface functionalization also resulted in a 23-fold decrease in the liver-to-lung ratio of NPs compared to uncoated PLGA NPs, indicating reduced clearance and enhanced accumulation in the lung. In a B16F10 murine lung metastasis model, i-Bind NPs effectively targeted various immune cell populations in metastatic lungs, including dendritic cells, macrophages, and neutrophils. Delivering diABZI STING agonist with the i-Bind platform significantly decreased disease progression and tumor burden by recruiting more dendritic cells, particularly CD103+ conventional dendritic cells (cDC1s), and cytotoxic (CD8+) T-cells.
CONCLUSION: Collectively, our results demonstrate that the i-Bind technology offers a simple but efficient approach for spontaneous RBC hitchhiking, increasing drug accumulation in the lungs, and targeting specific immune cell populations for lung metastasis treatment.
AUTHOR: Rachel Campoli
Role of FTO in EGFR Tyrosine Kinase Inhibitor Resistance and Non-Small Cell Lung Cancer Tumorigenicity
BACKGROUND, PURPOSE, or OBJECTIVES: The US will have 226,650 new lung cancer cases diagnosed in 2025. Many patients become resistant to EGFR-tyrosine kinase inhibitor (TKI) erlotinib, after 6-19 months of treatment. Fat mass and obesity-associated gene (FTO), linked with obesity, has been shown to have a role in tumor development. In this study, we aimed to identify a possible link between FTO, EGFR -TKI resistance and NSCLC tumorigenicity. HYPOTHESIS/AIMS: We hypothesize that FTO is modulated in NSCLC tumorigenicity, survival and EGFR- TKI resistance. MATERIALS or METHODS: In order to study FTO gene expression, parental and EGFR TKI resistant cells were plated in 60mm dishes, followed by RNA isolation and RT-qPCR. To study protein levels, parental and EGFR TKI resistant cells were plated and treated with EGF in the presence and absence of EGFR-TKI before collection of lysates, followed by Western blotting. To study the localization of FTO and its expression levels within cells, cells were fixed for immunofluorescence when they reached 80% confluency. MTT assay was performed to determine the half-maximal inhibitory concentration (IC50) of Dac51 and its effect on cell viability and erlotinib efficacy. To study the migratory capacities of cells, wound healing assays were performed for which cells were grown to 100% confluency, followed by creating a scratch and treatment with erlotinib and or Dac51 for 48 hours. Migration assays were also performed. To study the ATP levels, luciferase assays were performed. RESULTS: FTO gene expression was found to be upregulated in resistant cells by 2.2-fold (H358 ER), 2.9-fold (H2170 ER), 1.7-fold (PC9 ER), and 1.9-fold (H1975 ER), compared to their parental cells. FTO protein was upregulated by 1.3-1.6 fold as seen by western blotting. IF analysis showed FTO was mainly found in the nucleus of cells, and its expression was upregulated 2.6-fold, 1.75-fold, and 1.7-fold in the H1975 ER, H2170 ER and PC9 ER cells, respectively, when compared to parental cells. IHC analysis of 64 early-stage and 51 late-stage lung tumor samples showed greater FTO expression in late-stage non-small cell lung cancer (NSCLC) patients compared to early-stage p<0.01. NSCLC patients with higher expression of FTO had a shorter median survival time (14 months) compared to those with low expression (27 months). CONCLUSIONS: In conclusion, FTO plays a role in EGFR-TKI resistance. It is highly expressed in late-stage NSCLC patients and patients with upregulated FTO have shorter survival times.
AUTHOR: Arman H. Sharifi
Single-Stranded DNA-Binding Protein from the Human Tumor Microbiome Suppresses Prostate Cancer Cell Proliferation
BACKGROUND, PURPOSE, or OBJECTIVES: In 2025, approximately 313,780 men will be diagnosed with prostate cancer and, unfortunately, 35,770 will succumb to the disease. Although current treatment regimens involving androgen deprivation therapy, which are designated for high-risk patients with disseminated disease, can prolong patient survival for three to four years, the cancer cells develop resistance to the androgen deprivation therapy, better known as Castration-Resistant Prostate Cancer (CRPC). Resistance to treatment remains a significant challenge, despite advancements in the therapeutic landscape for CRPC. There is an urgent need for novel therapeutic strategies that can overcome resistance, enhance efficacy, and reduce side effects. HYPOTHESIS/AIMS: Microbial interactions with cancer have long been studied, and bacterial proteins have demonstrated tumor-suppressing properties. Our previous work showed that Pseudomonas aerugisa secretes azurin in response to cancer cells, selectively entering them, inducing apoptosis, and stabilizing tumor suppressor p53. Based on this we hypothesized that other bacteria may also secrete proteins with anti-tumor activity, especially CRPC. MATERIALS or METHODS: Through a cancer-bacteria co-culture approach, we identified a single-strand DNA-binding protein secreted exclusively by Massilia sp., which was isolated from tumors of human patients, in the presence of cancer cells. Since SSB is secreted in response to cancer cells, we anticipated that SSB may have a biological function(s) against cancer cells. It should be noted that Massilia sp. SSB has not previously been identified as playing a role in cancer treatment, making its investigation especially groundbreaking. SSBs are a group of nonspecific DNA binding proteins that play a key role in DNA metabolism by modulating DNA repair, replication, and recombination. These proteins are being explored as a potential marker as well as therapeutic target. SSB proteins targets DNA gaps which are essential to DNA repairs, thereby sensitizing cancer cells to chemotherapeutic agents RESULTS: Our results show that purified SSB can selectively kill CRPC cells. Notably, it can penetrate and induce DNA damage in these cells, suggesting that SSB may inhibit cell proliferation by triggering DNA damage. This research promises to provide crucial knowledge for developing a new class of therapies for CRPC cancer, addressing the current therapeutic challenges and improving patient outcomes. CONCLUSIONS: In conclusion, the development of SSB proteinbased therapy represents a promising new strategy for treating CRPC cancer. By specifically targeting and disrupting cancer cell DNA repair mechanisms, this approach could offer an effective and less toxic alternative to current treatments, ultimately leading to better survival rates and quality of life for patients.
AUTHOR: Pariksha Thapa
Utilizing Surface Plasmon Resonance to identify small molecule binders of BiP for Prostate Cancer
Background: Prostate cancer is the second most diagnosed cancer in men in the United States and is often driven by androgen receptor signaling. Over time, prostate cancer becomes resistant to standard anti-androgen therapies due to the expression of active AR splice variants like AR-V7. AR-V7 lacks the ligand-binding domain and drives prostate cancer progression. Our recent studies have shown that _-mangostin, a xanthone from Garcinia mangostana, promotes AR and AR-V7 protein degradation via the activation of molecular chaperone-binding immunoglobulin protein (BiP, also known as GRP78). Interestingly, compounds that bind and enhance BiP expression can shift the balance from cell protection to apoptosis, promoting cancer cell death. However, the mechanism of _-mangostin and BiP interaction is not known.
Aim: In this study, we aim to identify novel binding sites of _-mangostin and potential small molecules to BiP that promote AR and AR-V7 degradation. Methods: We developed a Surface Plasmon Resonance (SPR) assay to identify allosteric binders of BiP. We prepared fractions of Garcinia mangostana using flash prep chromatography to separate naturally occurring xanthones. These fractions were analyzed using our SPR assay to determine if “hits” can be identified from complex plant fractions. We then utilized SPR to study the molecular interaction between _-mangostin and BiP using 8 other purified xanthones. Next, we developed a high throughput screening (HTS) SPR assay in 384-well format to screen 5,360 drugs or drug-like compounds that bind with BiP. Results: We identified _-mangostin from complex plant fractions as an allosteric binder of BiP. Our results demonstrated that _-mangostin directly binds to BiP in a dose-dependent manner. From this data, we were able to establish a structure-activity relationship (SAR) of xanthones binding to BiP. Further, to identify the specific binding site of _-mangostin and potential small molecules to BiP, we expressed six different constructs of BiP (BiP-FL, BiP-N, BiP-C, BiP-C41A, BiP-C420A and BiP-No KDEL) in E.coli BL21 cells and purified using Ni-NTA column. Our data shows that _-mangostin directly binds to the N-terminal domain of BiP. Next, we screened 5,360 drugs or drug-like compounds and identified 132 compounds that bind with BiP. We further plan to conduct a secondary screening of these hits using HiBiT- AR and AR-V7 22Rv1 cells to evaluate their ability to induce AR and AR-V7 degradation. _-Mangostin and eight purified xanthones were used to study AR and AR-V7 degradation using HiBiT- AR and AR-V7 22Rv1 cells. Conclusions: This study reveals a novel mechanism of BiP-associated AR and AR-V7 degradation and develops an HTS SPR assay to identify novel allosteric binders of BiP for overcoming drug-resistant in prostate cancer
AUTHOR: Farah Yousef
Streamlined sample multiplexing to scale spatial transcriptomics
BACKGROUND: The tumor microenvironment (TME) plays a critical role in tumor biology through heterotypic cellular interactions. In addition to single cell analysis, methods accounting for the spatial arrangement of the cells within tissue, such as spatial transcriptomics, are of great value. However, many assays available to date are costly and workflows are time consuming, limiting scalability of spatial transcriptomics analysis. Here, we evaluate a multiplexing approach to parallelize capture area based spatial transcriptomics, and pairing with single cell RNA sequencing (scRNA-seq). AIMS: To leverage conventional histology techniques to optimize tissue microarray (TMA) construction for streamlining spatial transcriptomics workflows of formalin fixed paraffin embedded (FFPE) tissue samples.
METHODS: Tissue samples were obtained from a carcinogen induced murine model of oral cancer. After obtaining initial H&E sections from the FFPE blocks, a board certified pathologist reviewed and graded the lesions according to dysplasia severity. Following pathology review, we selected representative tissue from all grades by each time point to construct TMAs with an optimized workflow for spatial transcriptomics. Tissue cores were extracted from the donor blocks and re-embedded for a total of 9 tissues per TMA block to maximize allowable RNA capture in the 10x Genomics Visium spatial transcriptomics workflow. 5 µm sections were processed in the Visium assay, and the remaining tissue in the block was used for cell isolation followed by the 10x Genomics FLEX scRNA-seq assay. RESULTS: Samples were used to construct TMAs which allowed for an optimized Visium spatial transcriptomics workflow to assess multiple tissues in a parallelized and scalable manner. Multiplexed samples maintained tissue integrity and spatial and single cell sequencing outputs met standard quality metrics criteria. CONCLUSIONS: Multiplexing tissue samples in the form of TMAs is an efficient way to streamline sample preparation procedures for spatial transcriptomics. It allows for direct comparisons of multiple samples within one capture area, minimizing batch effects across sample to sample comparison. This reduces costs, and increases efficiency, reliability and reproducibility of assays. In addition, this workflow allows for pairing with the FLEX scRNAseq assay, which improves the resolution of the generated Visium data, facilitating a more comprehensive analysis of the TME to generate a multiomic data set for cell-cell interaction analysis.
AUTHOR: SUBARANJANA SARAVANAGURU VASANTHI
Enhancing Awareness and Access to Low-Dose Computed Tomography Screening for Early Lung Cancer Detection in Rural and Minority Communities of Northern Illinois.
Background: Lung cancer poses a significant public health threat, with 226,650 new cases and 124,730 deaths expected in 2025. Lung cancer’s high mortality rate is partially due to the fact that approximately 75% of new cases are diagnosed in late stages. Causes of Lung Cancer include smoking, radon exposure, second-hand smoke, and environmental toxins such as asbestos. Lung cancer age-adjusted mortality rate (2018-2022) in Winnebago County was 22.2% higher than the national rate. In the National Lung Screening trial (NLST), 90.9% of participants were white, 4.5% were African American, and 1.8% were Hispanic. A clear need thus exists in our county and the nation for more widespread implementation of early-stage lung cancer screening. Low-dose computed tomography (LDCT) is a valuable lung screening technique that utilizes 90% less ionizing radiation than a conventional chest CT scan. The U.S. Preventive Services Task Force (USPSTF) and ACS have issued new guidelines that recommend LDCT screening for 20 pack-year smokers within 50-80 years of age who currently smoke or who have quit within the last 15 years. Hypothesis: Increasing promotion of LDCT screening in minority communities in Northern Illinois would lead to increased detection of lung cancer at its early stages and decreased mortality rates attributed to the disease. Study Design: Our group implemented a multifaceted approach to promote Low-Dose Computed Tomography (LDCT) in four primary counties in Illinois, namely Winnebago, Boone, Ogle, and Stephenson. We engaged in community outreach by hosting booths at minority-centered events, educating community members and physicians on the LDCT guidelines set by the US Preventive Services Task Force (USPSTF) and ACS. Ethnicity of the community members and their zip codes were noted at each event. To assess the impact of our efforts, we analyzed hospital data from local hospitals, spanning from June 2015 to December 2024, to determine the number of LDCT screenings and lung cancer detections in the area. Results: 25 seminars and 65 public awareness booths targeting an estimated 332 physicians and 1,132 attendees were conducted to promote LDCT. From February 2015 - March 2025, our outreach work was able to reach 18.5% of Hispanics and 32.5% of African Americans out of 10,412 screening. Data were collected from local hospitals from June 2015 - December 2024 to determine the number of screenings and detection of lung cancer cases in the area. Total LDCT screening data was obtained from local hospitals, including Javon Bea Mercy Health, UW Health Swedish American Hospital, and OSF Health Care. There were 10,412 LDCT screenings conducted, detecting 145 lung cancer cases, with 86 being early-stage cases. Approximately 86% of the population who underwent LDCT screenings were White, 7% were African American, 1% were Hispanic, and 1% were Asians and the rest 5% were other populations. Conclusion: Rural and minority communities are not well represented in screenings for lung cancer. Thus, continued efforts to spread lung cancer screening and awareness in rural and minority communities may help drive smokers to get screened and raise the rate of early detection of lung cancer which is the focus of our study.
AUTHOR: Hitesh Kapoor
MLKs Inhibitor CEP-1347
Suppresses Tumorigenic Potential and Survival of Colon Cancer Cells
BACKGROUND: Somatic mutations in an MLK family member, Mixed Lineage Kinase 4 (MLK4), have been reported to activate its kinase activity in colorectal cancers (CRCs). The TCGA (The Cancer Genome Atlas) data analysis revealed that increased MLK4 expression correlates with poor overall survival in colon cancers. Novel MLK4 inhibitors are being developed using various computational techniques and in vitro models to target different types of cancers. AIMS: This study evaluates the effects of CEP-1347, a pan-MLK inhibitor, on MLK4 expression, apoptosis, and tumorigenesis in colon cancer cells. METHODS: Colon cancer cell line, HCT 116 cells were treated with CEP-1347 (500nM) every 24 hours for 2 days. Cell extracts were primarily incubated with MLK4 antibody, and western blot analysis was performed. Apoptosis was assessed with Bax, Bcl-2, PARP, and caspase-3 expression. A wound healing assay was conducted on CEP-1347-treated HCT 116 cells, with migration evaluated every 24 hours for six days. RESULTS: MLK4 expression was reduced in the cells treated with CEP-1347. The Apoptosis assay demonstrated an increased Bax/Bcl-2 ratio and other apoptotic markers like cleaved PARP and caspase-3. Migration assay showed delayed wound closure in CEP-1347-treated cells, with control cells achieving full closure by day 5, whereas treated cells retained ~10% of the initial wound area by day 6. CONCLUSIONS: CEP-1347 effectively inhibits MLKs/MLK4 kinase activity in colon cancer cells, leading to increased apoptosis and reduced migratory potential. These findings suggest that MLK4 inhibition may be a promising therapeutic strategy for treating intractable colon cancers unresponsive to current therapeutic approaches.
AUTHOR: Didi Zha
Modeling Fallopian Tube Epithelium Cellular Signaling Reprogramming Driven by Extracellular Vesicles to Define the Ovarian Cancer Precancerous Landscape
BACKGROUND, PURPOSE, or OBJECTIVES: Epithelial ovarian cancer (EOC) remains the leading cause of death from gynecological malignancy. The most common and lethal subtype, high-grade serous ovarian cancer (HGSOC), tends to originate from the fallopian tube epithelium (FTE). Serous tubal intraepithelial carcinoma (STIC) is a precursor lesion of HGSOC in FTE. Mathematical modeling suggests an average of 6.5 years between STIC and the development of HGSOC. However, the mechanism of how STIC disseminates and contaminate the adjacent healthy tissue is less understood. We are particularly interested in the role of extracellular vesicles (EVs) in facilitating this process. Cancer cells commonly secrete more EVs than healthy cells and shift their EV cargo to promote tumorigenesis. HYPOTHESIS/AIMS: We hypothesized that extracellular vesicles cargo secreted by tumor cells drives the cell signaling reprogramming in the FTE to favor tumorigenesis. MATERIALS or METHODS: We optimized a microfluidic model using a 3D dynamic culture system termed PREDICT-Multi-Organ-System to enable the direct interaction of FTE with EVs and the long-term culture of the fallopian tube explant. We are interested to 1) test the role of EVs to drive cell signaling reprogramming of FTE and 2) to test whether tumor-derived EVs shift EV cargos from FTE after long-term exposure. The hFTE cultured on PREDICT-MOS is exposed to cancer cell-derived EVs and embedded for transcriptomic profiling utilizing GeoMx Digital Spatial Profiler to identify differentially expressed genes in the epithelium. RESULTS: Using spatial transcriptomics, we show that the short-term 1-day exposure to ovarian cancer-derived sEVs alters expression of 61 transcripts in secretory cells, the progenitor of HGSOC, notably upregulating immune-related mRNA, including CXCL family chemokines, VCAM1, and pro-inflammatory mediators (NFKB1, IL1B, IFNA7/17). Additionally, we observed the long-term 14-day exposure to sEVs alters the expression of 7 transcripts and 25 EV cargo proteins of fallopian tube derived EVs (“secondary release EVs”) following stimulus from cancer EVs. Together, tissue transcriptomics and tissuederived EV proteomics indicate that ovarian cancer derived sEVs rewire target cell signaling to modify the tubal immune landscape. CONCLUSIONS: This study provides insights into the early molecular changes associated with the pathogenesis of ovarian cancer in its tissue of origin, providing a platform to study EV-tissue interactions and identify how sEVs drive cell signaling reprogramming in hFTE.
AUTHOR: Adriana Duraki
Vitamin D deficiency leads to a proinflammatory microenvironment in the prostate that supports carcinogenesis
Background: Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men, with African American (AA) men experiencing double the mortality rate of their European American (EA) counterparts. Melanin-related vitamin D (vitD) deficiency in AA men may contribute to their increased risk of aggressive and lethal PCa. VitD is essential for various physiological processes, including immune regulation and cellular differentiation. The prostate is influenced by vitD, with all three of its primary cell types-stromal, immune, and epithelialdirectly regulated transcriptionally to impact prostate homeostasis and disease progression. Given the crucial role of immune infiltration in PCa surveillance, vitD deficiency may shift immune cell subpopulations, fostering an inflamed, potentially immunosuppressive environment. The prostate stroma, through secretion of growth factors, chemokines, and cytokines, influences epithelial transformation and tumor progression. Here, we examine how vitD deficiency affects the stromal and immune compartments of the prostate, potentially fostering an environment that supports carcinogenesis. Hypothesis: We hypothesize that vitD deficiency alters the prostate microenvironment to support carcinogenesis. Methods: Male C57BL/6 mice were fed a vitDdeficient or control diet for 6 months. Dorsal and lateral (dorsolateral) prostate lobes were isolated for single-cell RNA sequencing (scRNAseq). Gene expression and cell populations were analyzed in R. Differentially expressed genes (DEGs) were validated by treating patient-derived prostate stromal cells with or without vitD and assessing gene expression via quantitative PCR. A multicolor flow cytometry panel was optimized to quantify CD8+ T cell subtypes (naïve, central memory, and effector) in the mouse dorsolateral prostate lobe. The OT1 transgenic mouse model was used to culture CD8+ T cells ex vivo, with or without vitD, to study its effects on T cell gene expression and secretome profiles. Results: Multiple types of immune cells and stroma were identified in the scRNAseq analysis, with distinct transcriptional shifts in vitD-deficient prostates. Several DEGs within the stroma are associated with inflammation, paracrine signaling to the epithelium, and tumor microenvironment remodeling. Preliminary validation in patientderived stromal cells highlights vitD’s role in modulating stromal gene expression. VitD deficiency also shifted immune infiltration, notably reducing central memory T cells essential for cancer surveillance and increasing activated T cell markers, potentially fostering an immunosuppressive environment. Flow cytometry confirmed distinct CD8+ T cell subtypes in the dorsolateral prostate. Ex vivo, vitD altered the response of activated CD8+ T cells, affecting differentiation profiles and cytokine expression. Conclusions: These data suggest that maintaining adequate vitD levels may be essential for regulating the prostate microenvironment. Future work will assess how this may contribute to aggressive PCa. VitD deficiency may lead to heightened inflammation and impaired immune surveillance. As vitD is easily supplemented, our studies offer a rationale for preventing vitD deficiency to support prostate health and reduce the risk of aggressive PCa. Given the vulnerability of AA men to vitD deficiency and lethal PCa, this research aims to mitigate this disparity by addressing underlying factors. Our findings highlight shifts in immune infiltrates due to vitD deficiency, suggesting that maintaining optimal vitD levels could improve treatment outcomes for AA patients facing PCa.
AUTHOR: Joohyun Im
PAX8-TGF-_ Axis Modulates
the Tumor M0icroenvironment in High-Grade Serous Ovarian Cancer
BACKGROUND, PURPOSE, or OBJECTIVES: High-grade serous cancer (HGSC) is the most aggressive and lethal subtype of ovarian cancer. PAX8 is a transcription factor that is commonly found in HGSC, promoting tumor growth. Although PAX8 is also present in normal tissues like the fallopian tubes, deletion in non-tumorigenic cell lines did not impact cell viability. This makes PAX8 an attractive target for cancer treatment, with the potential to stop tumor growth without harming normal cells. This study explores how PAX8 increases cancer progression, especially by examining its connection with TGF-_, a molecule involved in tissue repair, immune response, and angiogenesis.
HYPOTHESIS/AIMS: We hypothesize that PAX8 interacts with different protein partners in non-tumorigenic fallopian tube cells versus HGSC and that these differences allow PAX8 to promote tumor growth and reshape the environment around the tumor. Based on early findings linking PAX8 to TGF-_ secretion, we hypothesize that PAX8 encourages cancer progression by modulating TGF-_-mediated signaling pathways that control angiogenesis, tissue structure, and immune cell activity. MATERIALS or METHODS: We used two mouse cell models (MOE PTENshRNA KRASG12V and STOSE) and reduced the expression of PAX8 using PAX8-targeting shRNA. ELISA was used to quantify the reduction in TGF-_ levels following PAX8 knockdown. To better understand this pathway, we performed western blot analysis and treated the cells with either TGF-_ or a drug that blocks TGF-_ activity. These experiments helped identify SOX17-a transcription factor implicated in angiogenesisas a downstream target of both TGF-_ signaling and PAX8. Additional testing using antibody arrays showed that PAX8 influences the secretion of several molecules related to angiogenesis. To understand how these changes impact tumor growth and the surrounding environment, we injected the modified cells into mice with healthy immune systems and used 3D imaging to study the resulting tumors. RESULTS: Loss of PAX8 lowered the levels of TGF-_ and SOX17 and led to increased production of molecules that block angiogenesis, while reducing those that promote it. In mice, tumors that lacked PAX8 took longer to form, grew more slowly, and were associated with longer survival. The tissue around these tumors was less dense and more immune cells were found inside the tumors compared to controls. CONCLUSIONS: Our study shows that PAX8 plays a key role in the progression of HGSC by influencing the secretion of key molecules, angiogenesis, and the structure of tissues surrounding tumors. Targeting PAX8 could offer a new way to treat this aggressive cancer by cutting off its support system. These findings provide a foundation for future studies to characterize specific immune cell populations affected by PAX8 loss and better understand their roles in tumor suppression. In addition, identifying other molecular targets within the PAX8-TGF-_ signaling axis may uncover novel combinatorial strategies to enhance treatment efficacy and improve clinical outcomes for patients with HGSC.
AUTHOR: Kavya Sudhir
Centering Community Voices: Addressing Barriers and Building Engagement in Pancreatic Cancer Care
BACKGROUND: Stark racial disparities exist in pancreatic cancer incidence and mortality. To inform interventions, we explored knowledge of pancreatic cancer care among diverse Black communities in Illinois. Cook and Peoria Counties represent contrasting urban settings, differing in transit and safety-net hospital availability. Gender also influences health behaviors, including preventive care engagement. Understanding geographic and gender-based differences can guide more tailored outreach strategies. HYPOTHESIS/AIMS: We aim to assess baseline knowledge of pancreatic cancer and explore differences by gender and geography. METHODS: A survey was developed focusing on demographics, exposure to, and knowledge of pancreatic cancer. Surveys were distributed at community gatherings in Peoria and Cook County. Multivariate logistic regression analyses were conducted to examine gender and geography differences, after adjusting for demographic and social factors. RESULTS: Of 377 respondents, 313 identified as Black/African American and were included in the analysis. 68% were males and 32% were female. Most participants were from Cook County (82%) vs. Peoria (18%), and 4064 years old (54%). Education levels varied: 31% had a high school diploma or less, while 33% held a bachelor’s degree or higher. Insurance coverage included private (51%), public (31%), and uninsured (15%). Most had a primary care provider (79%) and lived experience with cancer (73%), but denied prior community event participation (63%) and clinical trial participation (86%). Despite this, 71% expressed interest in pancreatic cancer engagement events, preferably through virtual/observational formats (42%). Barriers to events included transportation (27%), work (20%), and childcare (8%). Males expressed less interest in events relative to females (64% vs. 84%; OR = 0.30, 95% CI: 0.14-0.63, p = 0.002). There were no significant gender differences in pancreatic cancer (23% vs 16%; OR=1.25, 95% CI:0.59-2.64, p=0.56), endoscopy (36% vs 17%; OR=2.32, 95% CI:0.99-5.46, p=0.05) or biopsy knowledge (52% vs 36%; OR=1.57, 95% CI:0.763.26, p=0.23). General knowledge of pancreatic cancer was similar between Cook County (21%) and Peoria (20%), as was knowledge of endoscopies (27% vs. 41%) and biopsies (49% vs. 52%), with no statistically significant differences. Conclusion: Although most participants had primary care providers, private insurance, and reported lived experience with cancer, knowledge of pancreatic cancer remained low. While the majority expressed interest in educational events, barriers such as transportation and work obligations may limit attendance. Incorporating pancreatic cancer education into primary care visits leverages existing healthcare while reducing the need for separate events. No significant differences in knowledge were found by gender or geography, although further research is needed in rural areas. With visual learning modalities being participants’ most preferred learning method, and a higher interest in health education events among women, future educational efforts should focus on being visually engaging and gender-responsive. Finally, because this sample had relatively high access to healthcare, it may not be representative of broader communities. Further research is needed to more comprehensively examine disparities related to socioeconomic status, insurance coverage, and healthcare access.
AUTHOR: Alireza Rahnama
ENDOTHELIAL CELLS REDUCE THERAPEUTIC SENSITIVITY IN 3D LUNG CANCER MODELS
The tumor microenvironment (TME) is a complex and dynamic network consisting of cancer cells, stromal cells, immune cells, extracellular matrix (ECM), and signaling molecules. These components collectively influence tumor progression, metastatic potential, and therapeutic response. The TME fosters an immunosuppressive and drug-resistant niche, limiting the efficacy of conventional and targeted therapies. Among the key players in the TME, endothelial cells form the tumor vasculature and contribute significantly to therapy resistance. Beyond restricting drug delivery due to impaired blood flow, endothelial cells actively modulate the microenvironment. They secrete pro-survival cytokines, regulate immune cell infiltration, and activate tumor-promoting signaling pathways that enhance cancer cell adaptability. These interactions promote tumor survival, angiogenesis, and metastasis, underscoring the importance of targeting endothelial-mediated resistance mechanisms to improve therapeutic outcomes. In this work, using established 3D models, the impact of endothelial signaling, on non-small lung cancer (NSCLC) spheroids is investigated. Two separate 3D models, previously established by this group, were implemented in culture and treatment of NSCLC spheroids. These models were both previously validated in culture of spheroids and patient derived organoids (PDO’s) with high viability and size uniformity. Said models include an agarose microarray formed inside conventional microwell plates and a microfluidic chip that allows the culture of two tissue types at the same time. Spheroid culture duration was optimized to achieve spheroids of desired size with consistency. In order to focus on the signaling from endothelial cells and its impact on cancer spheroids sensitivity to treatment, HUVEC cells were cultured separately, and supernatant was collected from them to be introduced to the spheroids in the presence of Osimertinib treatment ranging from 0 (control) to 2 µM. Two cell lines were studied, H1975 which is susceptible to Osimertinib, and A549 which is resistant, as control. After treatment, spheroids were stained with live, dead, and nuclei fluorescent markers, and imaged using a fluorescence microscope. Collected images were then analyzed via ImageJ to quantify viability of spheroids. Studies in both models demonstrate a decreased sensitivity to Osimertinib in H1975 spheroids, with increase in viability up to 50% (observed at 2 µM Osimertinib concentration) suggesting the activation of signaling pathways from HUVEC cells that promote a therapeutic resistance in NSCLC cells. This finding is promising and encourages further pursuit of this study at the molecular level to identify such specific pathways for the derived therapeutic resistance, which can subsequently be targeted as a treatment strategy.
AUTHOR: Jane Miglo
Microfluidic approach shows luteal phase promotes pro-tumorigenic changes in preneoplastic fallopian tube epithelium
BACKGROUND/PURPOSE/OBJECTIVES: Ovulation has been linked to an increased risk of high-grade serous ovarian cancer (HGSOC), the most prevalent and lethal histotype of ovarian cancer. The molecular mechanisms through which ovulation promotes ovarian cancer formation are largely unknown because this dynamic process occurs deep within the abdominal cavity and requires invasive procedures for monitoring. Thus, our collaborative team has developed a physiologically accurate menstrual cycle model using the PREDICT Multi-Organ System, a microfluidic (organ-on-chip) device that enables us to recapitulate the process of ovulation and capture secretions from the ovary in an abbreviated 14-day cycle. HYPOTHESIS/AIMS: The aim of this project was to evaluate the transcriptional and phenotypical changes that occur in a pre-cancerous fallopian tube cell model treated with ovarian secretions from three phases of the menstrual cycle: follicular, ovulation, and luteal. We hypothesize that the use of a microfluidic system will yield insight into the mechanisms underlying the initiation of HGSOC in the fallopian tube epithelium (FTE) in response to ovulation. MATERIALS/METHODS: Our lab engineered a preneoplastic FTE cell models that represents the earliest known aberration in HGSOC: Pax2-null secretory cell outgrowth (SCOUT) and P53-signatures. We surgically isolated mouse ovaries and performed the ovulation protocol on the PREDICT microfluidic platform to collect secretions from the follicular, ovulation, and luteal phases of the menstrual cycle. We then performed viability, spheroid, and invasion assays. We also conducted mRNA sequencing and proteomics analysis on FTE cells treated with secretions from the ovary. RESULTS: In this study, we demonstrate that luteal phase secretions enhance the proliferation, invasion, and spheroid formation of preneoplastic fallopian tube epithelial (FTE) cell lines. Transcriptomic analysis identified upregulated pro-proliferative transcripts, including Wfdc2, Stat1, Cdkn1a, Mt1, and Mt2, in FTE cells exposed to luteal phase secretions. Additionally, proteomic analysis revealed an enrichment of heat shock proteins (HSPs), which further promote proliferation in our models. CONCLUSIONS: These findings provide new insights into the phase-dependent mechanisms that may contribute to HGSOC initiation in the fallopian tube in response to ovarian secretions during the luteal phase.