Novel multicellular organotypic models of normal and malignant breast: tools for dissecting the role of the microenvironment in breast cancer progression

INTRODUCTION: There is increasing recognition of the role of the microenvironment in the control of both normal and tumour cell behaviour. In the breast, myoepithelial cells and fibroblasts can influence tumour cell behaviour, with myoepithelial cells exhibiting a broad tumour-suppressor activity while fibroblasts frequently promote tumour growth and invasion. This study describes the development of physiologically relevant three-dimensional heterotypic culture systems containing mixed normal or tumour-derived breast populations and shows how such models can be used to dissect the interactions that influence cell behaviour. METHODS: Populations of luminal cells, myoepithelial cells and fibroblasts were isolated from normal and malignant breast tissue, characterised and compared with immortalised cell lines. Co-localisation of normal and malignant luminal cells with myoepithelial cells alone or with either normal or tumour-derived fibroblasts was studied. Cultures were grown for seven days, and then gels were fixed and whole gel immunofluorescence carried out to assess co-localisation and polarisation. The potential role of matrix metalloproteinases (MMP) or hepatocyte growth factor(HGF)-c-met signalling in disrupting cellular organisation was investigated by incorporating inhibitors into cultures either alone or in combination. RESULTS: Over a culture period of seven days, myoepithelial cells organised themselves around luminal cell populations forming dual-cell co-units. Characterisation of co-units showed established basal polarity and differentiation analogous to their in vivo counterparts. Tumour cell co-units revealed subtle differences to normal co-units including disruption of basement membrane and loss of β4-integrin, as described in ductal carcinoma in situ (DCIS) in vivo. Inclusion of normal fibroblasts had no influence on co-unit formation; however, inclusion of tumour-associated fibroblasts lead to disruption of co-unit organisation, and this was significantly inhibited in the presence of MMP and/or c-met inhibitors. CONCLUSIONS: To the best of the authors' knowledge, this study describes for the first time a co-culture model comprising three major components of normal and malignant breast: luminal cells, myoepithelial cells and stromal fibroblasts. These cells organise into structures recapitulating normal and DCIS breast, with homing of myoepithelial cells around the luminal population. Importantly, differences are exhibited between these systems reflecting those described in tissues, including a central role for tumour-associated fibroblasts and MMPs in mediating disruption of normal structures. These findings support the value of these models in dissecting normal and tumour cell behaviour in an appropriate microenvironment.