This document describes a study that aimed to: 1) Characterize dissociated tumor (DT) cells derived from estrogen receptor positive and negative breast cancers. 2) Determine a protein marker that can distinguish cancer-associated fibroblasts (CAFs). 3) Examine the effect of CAFs on tumor formation of epithelial cells. The study found that DT fibroblasts overexpressed vimentin and FAP compared to controls, confirming their fibroblastic identity. FAP was determined to be a reliable CAF marker. Co-culture experiments showed that several DT CAF lines, particularly DT 23, increased tumor formation of epithelial cells.

1. CHARACTERIZATION OF
DISSOCIATED BREAST CANCER
TUMOR FIBROBLASTS DERIVED
FROM ESTROGEN RECEPTOR
POSITIVE AND NEGATIVE
BREAST CARCINOMAS
Alana Steinberg Pine Crest School, Fort Lauderdale
University of Miami

2. Background
DT (CAF) 18 Fibroblasts
 Cells involved in the wound healing
process of the innate immune
response
 When active, release growth factors
that stimulate cells to grow and
proliferate
 “Turn off ” once wound healing
process is complete
 CAFs have a “perpetually active”
phenotype
 Fibroblasts are a prospective target
for cancer therapy
 Dissociated Tumor (DT) cells
 Fibroblasts are ER- in nature, no
matter what kind of cell they are
derived from

3. Purpose
 Characterize a series of Dissociated Tumor (DT)
cells,
 Determine a protein marker that can be used to
distinguish Cancer-Associated Fibroblasts (CAFs)
 Study the effect that the CAFs have on DT epithelial
cells.

4. Methods
 Western Blots: determine protein expression across a
group of cells under specific conditions
 Soft Agar Assay: determine the effect on
tumorigenisity that the CAFs have on established
epithelial tumor cells

5. CAF Markers in DT Cells
DT CAFs Control Fibroblasts Control Epithelial Cells
DT 6 DT 14 DT 18 DT 19 DT 21 DT 23 HMF MCF7 MDA RAF HCO
231 14C Pool C
Viniculin
FAP
Vimentin
Estrogen
Receptor

6. Analysis of CAF Markers in DT Cells
 Overexpression of both Vimentin and FAP as
compared to HMF, no expression of Vimentin or
FAP in epithelial cells
 Confirms fibroblastic properties
 Band on ER blot at lower KD weight
 Either nonspecific binding or isoform  strip cells of
Estrogen to see if protein expression changes (if protein
expression changes, than ER+ and not fibroblasts)

7. Stripping ER+ Derived CAFs from Estrogen and Testing
Viability of FAP as a CAF Marker
DT CAFs Control Fibroblasts Control Epithelial Cells
DT 6 E- DT 6 DT 14 DT MDA DT 18 DT HMF MC RAF
E- 14 231 E- 18 F7 14C
FAP
Vimentin
• Comparing Vimentin and FAP as CAF Markers
• Performed under Estrogen-Stripped and normal conditions
• No FAP or Vimentin in epithelial cells
• FAP is more consistent and produces a stronger signal

8. MDA 231 and Co-Cultures in FBS
MDA 231 Alone (5,000) MDA 231 (5,000) + DT 18 MDA 231 (5,000) + DT 19
(15,000) (15,000)
MDA 231 (5,000) + DT 21 MDA 231 (5,000) + DT 23
(10,000) (15,000)

9. Soft Agar Assay Results
Average colony count in FBS Soft Agar
300
250 207 205 227
196
200 151 160
129 128 133 126
150 103 90
100
50 0 0 0
0
-50 MCF 7 MDA 231 MCF 7 (5)MDA 231 DT 18 DT 21 DT 23 DT 18 DT 18 DT 19 DT 19 DT 21 DT 21 DT 23 DT 23
(20) (20) (5) (20) (15) + (15) + (10) + (10) + (15) + (15) + (15) + (15) +
MCF 7 (5) MDA 231 MCF 7 (5) MDA 231 MCF 7 (5) MDA 231 MCF 7 (5) MDA 231
(5) (5) (5) (5)
150 136
Average colony count in CFBS Soft Agar
100 86
72
62
50 32 34
10 2 3 1
1 0 0 0 0
0
MCF 7 MDA MCF 7 MDA DT 18 DT 21 DT 23 DT 18 DT 18 DT 19 DT 19 DT 21 DT 21 DT 23 DT 23
-50 (20) 231 (20) (5) 231 (5) (20) (20) (20) (15) + (15) + (10) + (10) + (15) + (15) + (15) + (15) +
MCF 7 MDA MCF 7 MDA MCF 7 MDA MCF 7 MDA
(5) 231 (5) (5) 231 (5) (5) 231 (5) (5) 231 (5)
Vertical Axis: Number of Colonies | Horizontal Axis: Type and Number of Cells Cultured (in thousands)

10. Analysis of Soft Agar Assay Results
 In general, the fibroblasts increased the colony count
of the epithelial cells, implying that they do in fact
play a role in tumorigenesis
 DT 23 increased the colony count of both epithelial
cells nearly uniformly, suggesting that it is the most
efficient and reliable of the DT CAFs

11. Discussion and Conclusion
 Confirmed that all presumed DT CAFs were in fact
CAFs
 High expression of both FAP and Vimentin as compared to
HMF
 No ER
 FAP is best marker for CAFs
 Expression does not change in absence of estrogen
 Flow Cytometry
 DT 21 and DT 23 have most effect on tumorigenisity of
epithelial cells in FBS agar
 DT 23 will be used for co-culture experiment with epithelial
DT tumor cell lines

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