Understanding the impact of UTI-induced systemic signals on mammary epithelial cell organoids Carol
1 Wang ,
1Donald
Samantha Cyrill,
2 PhD. ,
and Camila dos Santos,
and Barbara Zucker School of Medicine at Hofstra/Northwell, 2Cold Spring Harbor Laboratory
Results
PB
0 .1
0 .2
0 .3
39.7
34.4
8.5
28.0
5.7
2.7
B
EP + TMS
20.7
3.4
0.2
0.6
11.2
0.2
0.6
A
C
58.8
Inhibition of Matrix Metalloproteinases
Signaling pathways involved in pro-survival activities of TIMP-1 independent of MMPinhibition
D
L27 CC
L16 CC
F5 -C S M
F3
CL 14 CX
EP
GCS
P
+ P EP
Organoid Size (pixels^2)
EP + UTI
TI M P12
EP
S
P
TI
0.5
Caspase 3/7 activity 60
U +
Col1: Type I collagen
20
0
te ea nt r
K8: luminal K5: basal
COL1
A
Conclusions •
•
PIGR
0.0452
0
3
Fold Change (2^-ddCt)
2 1
1.5
2
1
0.0318
•
1.0
•
0.5
0.0
0 ea te d co nt in uo ep us p w ith dr aw n ep p
nt r U
te d co nt in uo ep us p w ith dr aw n
ea nt r
ep p
U
3
0
Untreated
0.0744
<0.0001
<0.0001
Fold Change (2^-ddCt)
Fold Change (2^-ddCt)
50000
0.0010
<0.0001
4
100000
0.0031
<0.0001
<0.0001
150000
There may be increased, and more disorganized, collagen deposition in MEC organoids treated with pregnancy hormones and UTI plasma Involution can be simulated transcriptionally by withdrawing pregnancy hormones Treating pregnancy-simulated organoids with Timp1 may reduce apoptosis even after pregnancy hormone withdrawal
Future Directions
<0.0001
EPP continuous Condition
Caspase 3/7 activity
EPP withdrawn
0.0011 0.9994 0.0012
50 40
% of cells
20 10
References
P
nt r
ea
te d co nt in uo EP us P w ith dr aw n
0
Cell Permeability (LIVE/DEAD Violet)
•
Quantify collagen from whole-mount IFs and try using Z-stacks to better observe how collagen is distributed throughout the organoid Measure expression/transcription of other markers of involution to continue optimizing involution model (i.e. MMP2 and MMP9 involved in ECM remodeling) Assess changes in collagen induced by treating organoids with Timp1 and withdrawing pregnancy hormones
30
U
Caspase 3/7 activity (Magic Red)
Figure 3. What is the mechanism by which UTI-induced, systemic signals delay involution? (A) Experimental design. MEC organoids were harvested from nulliparous C57BL/6J mice and treated in 5 conditions (B) Schematic showing functions of Timp1. (C) Plasma from UTI-bearing mice had higher levels of Timp1 compared to controls. (D) Magic Red assay of caspase 3/7 activity indicates decreased apoptosis in the condition with EPP withdrawn but supplemented by Timp1.
•
C
D
EP
P
MERGED
40
d co nt in uo us EP P EP w ith P dr w ith aw dr n aw n + Ti Ti m m p1 p1 co nt in uo us
EP
EP
P
P
+
% of cells
TI
S
P
U
PB
EP
TI
S
d te ea nt r
EPP + UTI
II. Can we simulate an involution phenotype in MEC organoids?
B
• Organoids are a culture system that allows us to harvest biological samples and culture them in a gel that allows the cell to organize into 3D structures and interact with the ECM components in the gel • The organoids were treated with pregnancy hormones (estrogen, progesterone, and prolactin) to induce a pregnancy and lactation state
B
1.0
EPP + PBS
0.1604
(2 days)
1.5
U
EPP
Csn2
(45 minutes)
0.7
Untreated
0.0071
2.0
Figure 1. What are the effects of UTI-induced systemic signals on MECs? (A) Experimental design. Nulliparous C57BL/6J mice had either PBS (control) or UTI89 (urogenic E. coli strain) injected into their bladder. Plasma was then collected via cardiac puncture. Harvested MEC organoids from nulliparous C57BL/6J mice both with and without pregnancy hormones were then treated with the plasma. (B) Brightfield images taken over the time course of the experiment at t = baseline, 24h, 48h, 72h, and 96h after treatment. (C) Sizing quantification comparison at 72h and 96h timepoints. (D) Wholemount immunofluorescence staining demonstrating differences in collagen I accumulation and organization.
Methods
C57BL/6J Nulliparous mice
0.3
EP
• UTI-derived systemic signals (inflammatory signals that travel through circulation) have a direct impact on mammary epithelial cells (MECs). • UTIs may induce pregnancy-associated breast cancer by delaying involution.
23.2
0.0
0
D
1.1
0.0
Fold Change (2^-ddCt)
Hypothesis
1
0.6
0.5
nt r
• Involution is a crucial process for reverting mammary glands to tissue homeostasis post-pregnancy • The processes that occur in involution harbor a pro-tumorigenic environment • Mutations that cause delayed involution have been linked to pregnancyassociated breast cancer (PABC), which has worse patient outcomes than non-PABC • Pregnancy is a risk factor for developing urinary tract infections (UTIs) • Previous experiments show that UTI-bearing mice, supplemented with pregnancy hormones, exhibit signs of delayed involution
2
0
3.6
Placebo
U
Organoid Size (pixels^2)
72h
B
3
0.2
32.3
1.0
U
ea
PB
te
S
d
0.0
0.4
1.5
CL 13 1
0.5
0.6
CX
1.0
C
2.0
TI
1.5
A
U
2.0
Untreated
+
2.5
PB
Organoid Size (pixels^2)
96h
0.0006
U
C
Organoid Size (pixels^2)
A
0.0004
III. What is the mechanism by which UTI-induced, systemic signals delay involution? 0 .4
I. What are the effects of UTI-induced, systemic signals on MECs?
0 .5
Background
(2 days)
2 PhD.
Figure 2. Can we simulate an involution phenotype in MEC organoids? (A) Experimental design. MEC organoids were harvested from nulliparous C57BL/6J mice and treated in 3 conditions (B) Brightfield images taken over the time course of the experiment at t = baseline, 24h, 48h, 72h, and 96h after treatment. (C) RT-qPCR analysis of pregnancy and lactation related genes demonstrates decreased transcription in the simulated involution condition. (D) Magic Red assay of caspase 3/7 activity indicates increased apoptosis in both EPP conditions compared to untreated.
1. Sumbal, J., Chiche, A., Charifou, E., Koledova, Z., & Li, H. (2020). Primary Mammary Organoid Model of Lactation and Involution. Frontiers in cell and developmental biology, 8, 68. https://doi.org/10.3389/fcell.2020.00068 2. Ciccone, M. F., Trousdell, M. C., & Dos Santos, C. O. (2020). Characterization of Organoid Cultures to Study the Effects of Pregnancy Hormones on the Epigenome and Transcriptional Output of Mammary Epithelial Cells. Journal of mammary gland biology and neoplasia, 25(4), 351–366. https://doi.org/10.1007/s10911-020-09465-0 3. Hughes, K., & Watson, C. J. (2018). The Multifaceted Role of STAT3 in Mammary Gland Involution and Breast Cancer. International journal of molecular sciences, 19(6), 1695. https://doi.org/10.3390/ijms19061695