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.
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 beta4-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.