analysis of primary breast tumour stromal cells and their potential role in disease progression

Discussion

Analysis of primary breast tumour stromal cells and their potential role in disease progressionMarion C Hartmann, Roisin M Dwyer, Michael J Kerin

Department of Surgery, National University of Ireland Galway, Ireland

Methods I Primary culture

Introduction

AimResults 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 fraction

Organoid 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.

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MMP3 expression in stromal 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.

Correlation of VEGF and MMP3

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Fig 3: There was no significant difference in (A) TGF1 and (B) TGFR2 gene expression in tumour compared to normal stromal cells. (C) A significant positive correlation between TGF1 (ligand) and its receptor TGFR2 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 TGF1 and its principle receptor TGFR2, 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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Correlation TGFβ1 and TGFβR2

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Stromal cells were isolated from breast tumour specimens using differential centrifugation (Fig.1) followed by culture in selective media.

Methods II Gene expression analysisRNA 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 (TGF1), transforming growth factor beta receptor 2 (TGF R2) and fibroblast activation protein (FAP). Results were expressed relative to the endogenous control gene PPIA.

Results II

RNA ExtractioncDNA synthesis

Real time quantitative

PCR


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Gene expression of VEGF in stromal cells

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Fig. 2 Gene expression analysis


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Gene expression of TGFbR2 in stromal cells

400 rpm x 1 min