1. The effects of a fibrotic
microenvironment on the
proliferation of prostate cancer cells.
Brad Christman
Macoska Lab
2. Background
• Prostate is a part of the male reproductive
system that aids in fertilization and surrounds
the upper portion of the urethra
• Prostate cancer occurs when uncontrolled
replication and metastasis of prostate cells
occur
• Cancer can occur from cells with damaged
cell-cycle checkpoints and adhesion
3. Background Cont…
• Cells become malignant and metastasize if they
can detach from the original tumor and invade
other tissues
• Cells divide in response to signals from other cells
know as “social control”
• Social control of the cell cycle is based on growth
factors
• Cancer cells can replicate in the absence of
growth factors because the cell cycle checkpoints
are damaged
5. Background Cont…
• Cytokines and growth factors regulate growth
of cells and contribute to the
microenvironment
• TGFb is an important growth factor previously
shown to cause phenoconversion of
mesenchymal fibroblasts to myofibroblasts
• Myofibroblasts synthesize collagen and alpha
smooth muscle actin
6. Aim
• To see if a fibrotic microenvironment has an
effect on the proliferation of adjacent cancer
cells.
Hypothesis:
-Conditioned media from TGFb treated
fibroblasts will increase prostate cancer cell
proliferation
7. I. WST Assay Protocol Standardization
• A colorimetric assay for quantification of cell
proliferation in response to growth factors
• WST Reagent breaks down in the presence of
cellular enzymes into a colored product that
you can measure
– More cells=More Enzyme=Darker Color=Higher V
8. Standardization WST Assay: N1 cells, Mean value at three time
points
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
5K cells
FoldoverSF
Expt 2: Reading at 24hrs
30min
60min
120min
10k cells
24 hr SF 5k HIE
5k
SF
10
HIE
10
30 min avg 0.2265 0.3199 0.6376 0.4664
stdev 0.03343 0.0229 0.1090 0.0790
fold 1 1.4119 1 0.7316
60 min avg 0.3764 0.5702 1.1360 0.8594
stdev 0.0591 0.0433
0.1504
8 0.1457
fold 1 1.5145 1 0.7564
120
min avg 0.6740 0.983 1.9451 1.4700
stdev 0.1874 0.0846 0.2430 0.2145
fold 1 1.4584 1 0.7557
11. II. Preparation of Conditioned Media
• N1 Fibroblasts cultured in small
dishes to 80% confluence
• Changed media to SF HIE for 24
hr
• Then treated the cells with
2ng/ml, 4ng/ml TGFB or Control
for 24 and 48hr
• Harvested the conditioned media
and froze it
12. III. Effect of Fibroblast conditioned
media on PC3 cancer cell proliferation
– Hemocytometer Count
– 700k cells/ml
– 1 ml cells +13 ml 10% RPMI 1:14 Dilution
– Cell suspension spun down and re-suspended in same
amount of conditioned media, SF HIE and 10% RPMI
– Plated 50k cells/well in a 96 well plate
– Incubated the cells for 24hr
– Performed WST assay
13. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
2ng/ml 4ng/ml 2ng/ml 4ng/ml 2ng/ml 4ng/ml
FoldoverUTConditionMedia
30min 60min 90min
24h
48h
Results: PC3 WST Assay with N1 condition media set 1
TGFb treated Condition Media
14. Percent Increase in WST Assay Normalize to UT 24h
-10.0%
-5.0%
0.0%
5.0%
10.0%
15.0%
20.0%
25.0%
30.0%
35.0%
UT 2ng/ml 4ng/ml
24 hr
48 hr
15. 5k cells #1 #3 #5 #7 #9 #11
SF RPMI 10% RPMI SF HIE Set 1: SF HIE condition media from N1 treated or not with TGFb
24hr treated 48hrhr treated
UT 2ng/ml 4ng/ml UT 2ng/ml 4ng/ml
Avg 0.172 0.3515 0.1249 0.1869 0.208 0.2015 0.1852 0.2369 0.2128
stdev 0.00513 0.04268 0.04096 0.0119 0.00901 0.0572 0.0154 0.0206 0.01465
Fold 1 2.03930 1 1.11237 1.0776 1 1.2794 1.1492
16. Control: Effect of TGFb on PC3 Cancer Cell Proliferation
• To make sure that the residual TGFb in the conditioned
media was not the cause of the increased proliferation of
PC3 cells in the prior experiment
– Grew PC3 Cancer Cells to 80% Confluence
– Trypsinize and count via Heamocytometer
– 700k cells/ml
– 1ml cells+13 ml of 10% RPMI 1:14 dilution
– 2ml of solution for 10%, 8ml for SF Control and TGFb treatment
– Plate control SF with 2 uL of Citric Acid, 2 ng/ml and 4ng/ml of
TGFb
19. Future Directions
• Both these experiments could be completed again!
• A scratch assay or well assay could be completed to
determine an increase in the motility of the cancer
cells with or without conditioned media!
• Focal Adhesion Kinases could be blocked in a PC3
conditioned media well or WST assay to see its role in
the cells motility and proliferation
• If (FAK) did not increase proliferation or motility a
protein lysate could be used to determine what type of
growth factors myofibroblasts synthesize into their
environment. Potentially IGF-1?
