Gene expression signature of primary breast tumour stromal

cells

MC Hartmann, RM Dwyer and MJ KerinDepartment of Surgery, National University of Ireland, Galway

Breast cancer microenvironment

• The importance of the breast cancer microenvironment in tumour progression is well recognized

• Breast tumour stroma consists of various cell types such as immunocytes, pericytes and fibroblasts

• Cellular interactions within the breast cancer microenvironment promote tumour growth, invasion, metastasis and resistance to therapy

Lorusso et al, Histochem Cell Biol (2008)

CAF: carcinoma associated fibroblasts

ECM: extracellular matrix

TC: tumour cell

BMDC: bone marrow derived cell

PC: pericyte

MSC: mesenchymal stem cell

LV: lymphatic vessel

• Stromal cells are the predominant cell type within the tumour microenvironment

• Stromal-epithelial cell cross talk facilitated by paracrine factors and direct cell-cell contact

• Tumour stromal cells are actively secreting factors (growth factors, chemokines, cytokines) supporting tumourigenesis

Stromal cell Tumour cell

Tumour stromal-epithelial interaction

GF

ECM

CK

The stromal-epithelial interaction in the tumour microenvironment

GF : Growth factors

ECM : Extracellular matrix

CK: Cytokines

Aim

Characterise stromal cells derived from primary breast tumours and potentially elucidate mechanisms through

which they exert their actions

Primary cell culture

Stromal cell fraction

Breast tumour specimen

Finely minced with scalpels

Digested overnight

Differential centrifugation

Epithelial cell fraction Organoid fraction

Analysis of gene expression

Primary stromal cells isolated, cultured and harvested

RNA Extraction

Targets of interest

•Vascular endothelial growth factor (VEGF)

• Matrix metalloproteinase 3 (MMP3)

• Transforming growth factor beta 1 (TGFβ1)

• Transforming growth factor beta receptor 2 (TGFβ R2)

•Fibroblast activation protein (FAP)

cDNA synthesis

Real time quantitative PCR

MMP3 expression in stromal cells

Tan StroTum StroNorm Stro

5

4

3

2

1

Log1

0 re

lati

ve M

MP3

exp

ress

ion

MMP3 expr ession in st r omal cel ls

N=4 N=24 N=12

FAP, VEGF, TGFβ1, TGFβR2 expression in stromal cells

Tan StroTum StroNorm Stro

20

15

10

5

0

rela

tive

FA

P ex

pres

sion

FAP expression in st romal cel ls

Tan StroTum StroNorm Stro

16

14

12

10

8

6

4

2

0R

elat

ive

VEG

F ex

pres

sion

VEGF expression in st r omal cel ls

Tan stroTum StroNorm stro

12

10

8

6

4

2

0

Rel

ativ

e TG

Fb1

expr

essi

on

TGFb1 expression in st romal cel ls

Tan StroTum StroNorm Stro

35

30

25

20

15

10

5

0TG

FbR

2 ex

pres

sion

leve

l

TGFbR2 expression in st romal cel ls

Correlation of TGFβ 1 and TGFβ R2 expression

Correlation of TGFβ1 and TGFβ R2

0

2

4

6

8

10

12

14

16

18

Primary stromal cells n=40

Rel

ativ

e T

GF

β1 a

nd

TG

FβR

2 e

xpre

ssio

n le

vel

TGFβ1

TGFβR2

R=0.65 P<0.01

Correlation of VEGF and MMP3 expression

Correlation of VEGF and MMP3

0

2

4

6

8

10

12

14

16

18

Primary stromal cells n=40

Rel

ativ

e le

vel o

f V

EG

F a

nd

M

MP

3 ex

pre

ssio

n

MMP3VEGF

R=0.29 P<0.05

Correlation of FAP and TGFβR2 expression

Correlation of FAP and TGFβR2

0

5

10

15

20

25

30

35

Primary stromal cells n=40

Rel

ativ

e le

vel

of

FA

P a

nd

TG

FβR

2

FAP

TGFBR2

R=0.28 P<0.05

Summary

• Trend towards increased expression of MMP3 in tumour compared to normal stromal cells

• Significant positive Pearson correlation found between • TGFβ1 and TGFβR2 • MMP3 and VEGF• FAP and TGFβR2

Conclusion

Tumour stromal cells secrete proangiogenic and prometastatic factors, such as MMP3 and may support

breast cancer progression through promotion of tumour angiogenesis and invasion