Although malignant epithelial cells are the origin of breast cancer and the main focus of research, evidence is increasing that the tumour microenvironment plays an important role in disease progression. Cellular interactions within the breast cancer microenvironment promote tumour growth, invasion, metastasis and resistance to therapy. Breast tumour stroma consists of various cell types including immunocytes, pericytes, endothelial cells and carcinoma associated fibroblasts. Stromal cells are the predominant cell type in the tumour microenvironment. Tumour stromal cells actively secrete growth factors, chemokines and cytokines that support tumourigenesis. Although the tumour promoting effect of stromal-epithelial interactions is recognized, the precise mechanisms involved are poorly understood. Further characterisation of tumour stromal cells will facilitate elucidation of these interactions.

1. Discussion
Analysis of primary breast tumour stromal cells and their potential role in disease progression
Marion C Hartmann, Roisin M Dwyer, Michael J Kerin
Department of Surgery, National University of Ireland Galway, Ireland
Methods I Primary culture
Introduction
Aim
Results I
Although malignant epithelial cells are the origin of breast cancer and the main focus of research,
evidence is increasing that the tumour microenvironment plays an important role in disease
progression. Cellular interactions within the breast cancer microenvironment promote tumour
growth, invasion, metastasis and resistance to therapy. Breast tumour stroma consists of various cell
types including immunocytes, pericytes, endothelial cells and carcinoma associated fibroblasts.
Stromal cells are the predominant cell type in the tumour microenvironment. Tumour stromal cells
actively secrete growth factors, chemokines and cytokines that support tumourigenesis. Although the
tumour promoting effect of stromal-epithelial interactions is recognized, the precise mechanisms
involved are poorly understood. Further characterisation of tumour stromal cells will facilitate
elucidation of these interactions.
The aim of this study was to isolate primary tumour stromal cells from breast cancer specimens and
investigate their potential mode of action in the breast tumour microenvironment, based on
expression of genes associated with disease progression.
Breast tissue specimen
Finely minced with scalpels
Digested overnight in mild collagenase
Differential centrifugation
Epithelial cell fractionOrganoid fraction Stromal cell fraction
Primary cell culture
700 rpm x 2min 1000 rpm x 4 min
Stromal cells isolated from tissue harvested at reduction mammoplasty served as normal controls.
Tumour associated normal (TAN) tissue refers to tissue harvested from the tumour bearing breast at least
2cm from the primary tumour site.
Tan StroTum StroNorm Stro
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Log10relativeMMP3expression
MMP3 ex pr ession in st r omal cells
Fig 4: (A) There was a trend towards increased expression of MMP3 in primary tumour compared
to normal stromal cells. (B) Expression of VEGF remained unchanged across the groups analysed.
(C) There was a significant positive correlation between MMP3 and VEGF expression detected.
R=0.29 P<0.05
Fig 3: There was no significant difference in (A) TGFβ1 and (B) TGFβR2 gene expression in
tumour compared to normal stromal cells. (C) A significant positive correlation between TGFβ1
(ligand) and its receptor TGFβR2 was detected across all samples.
A trend towards increased expression of the invasion associated MMP3 gene was observed in tumour
compared to normal, and tumour-associated normal (TAN) stromal cells. Significant positive
correlations were found between expression of TGFβ1 and its principle receptor TGFβR2, and also
between MMP3 and VEGF. Tumour stromal cells may support breast cancer progression through
promotion of angiogenesis and invasion mediated by expression of factors such as VEGF and MMP3.
Further characterisation of stromal cells and their impact on epithelial cell genotype and phenotype is
critical to identify targets to inhibit initiation of the metastatic cascade.
Fig. 1 Method for isolation of primary stromal cells from fresh breast tissue specimens
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Relativelevelofgeneexpression
Gene expression of TGFb 1 in stromal cells
Correlation TGFβ1 and TGFβR2
0.00
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Primary stromal cells n=40
RelativeTGFβ1andTGFβR2
expressionlevel
TGFβ1
TGFβR2
R=0.65 P<0.01
Stromal cells were isolated from breast tumour specimens using differential centrifugation (Fig.1)
followed by culture in selective media.
Methods II Gene expression analysis
RNA was extracted from primary stromal cells at passage 4, reverse transcribed and relative
quantitative PCR performed (Fig. 2) using primers targeting vascular endothelial growth factor
(VEGF), matrix metalloproteinase 3 (MMP3), transforming growth factor beta 1 (TGFβ1),
transforming growth factor beta receptor 2 (TGFβ R2) and fibroblast activation protein (FAP).
Results were expressed relative to the endogenous control gene PPIA.
Results II
RNA Extraction
cDNA synthesis
Real time
quantitative PCR
Tan StroTum StroNorm Stro
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Relativegeneexpressionlevel
Gene expression of VEGF in stromal cells
A B
C
A B
C
Fig. 2 Gene expression analysis
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Relativegeneexpressionlevel
Gene expression of TGFbR2 in stromal cells
400 rpm x 1 min