Characterisation of primary tumour stromal cells and their role in the breast tumour microenvironment

Marion C Hartmann, Roisin M Dwyer, Michael J KerinDepartment of Surgery, National University of Ireland, Galway

Although malignant epithelial cells are the origin of breast cancer and the main focus of research, evidence is increasing that the breast cancer microenvironment plays an important role in breast cancer 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 factors (growth factors, chemokines, cytokines) supporting tumourigenesis.Although the tumour promoting effect of stromal-epithelial interactions is recognized, the precise mechanisms involved are poorly understood.

Results

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Introduction

Aim

Methods

Discussion

Fig 2: Trend towards increased expression of MMP3 in primary tumour stromal compared to normal stromal cells

Fig 3: Significant positive correlation between TGFβ1 (ligand) and its receptor TGFβ R2

Fig 4 Significant positive correlation between expression of VEGF and MMP3 in stromal cells

Fig 5 Significant positive correlation between expression of FAP and TGFβR2 in stromal cells

Breast tissue specimen

Finely minced with scalpels

Digested overnight

Differential centrifugation

Epithelial cell fractionOrganoid fraction Stromal cell fraction

Primary cell culture

400 rpm x 1 min

700 rpm x 2min 1000 rpm x 4 min

Fig 1 Method for isolation of primary stromal cells from fresh breast tissue specimen.

Stromal cells isolated from tissue harvested at reduction mammoplasty served as normal control. Tumour associated normal (TAN) tissue specimen refers to tissue harvested from the tumour bearing breast at least 2cm from the primary tumour site. RNA was extracted from primary stromal cells at passage number 4, reverse transcribed and relative quantitative PCR performed using primers targeting vascular endothelial growth factor (VEGF), matrix metalloproteinase 3 (MMP3), transforming growth factor beta 1 (TGF1), transforming growth factor beta receptor 2 (TGF R2), fibroblast activation protein (FAP) Results were expressed relative to endogenous control gene PPIA

Correlation of FAP and TGFβR2

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Primary stromal cells n=40

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TGFBR2

R=0.65 P<0.01

Correlation of VEGF and MMP3

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R=0.29 P<0.05

Correlation of FAP and TGFβR2

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R=0.28 P<0.05

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 cancer progression.

A trend towards increased expression of the invasion associated MMP3 was observed in tumour versus normal and TAN tissue. Tumour stromal cells might support cancer invasion and metastasis by expression of invasion supporting factors such as MMP3. Significant positive correlations were found between expression of MMP3 and VEGF. A link between angiogenic VEGF expression and MMP3 expression could possibly represent a mechanism in the stromal epithelial interaction that supports tumour growth/angiogenesis and invasion. The positive correlation between FAP and TGFR2 points towards a connection between the TGF pathway and fibroblast activation protein, which is involved in tissue remodelling, thus supporting tumour progression.

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MMP3 expression in stromal cells

N=4 N=24 N=12