Most breast cancers progress from morphologically distinct pre-invasive lesions (DCIS) to invasive carcinomas. Often, sites of transition from DCIS to invasive carcinoma are characterized by a breakdown of the basement membrane and an increased number of stromal cells and cytokines. We have sought to mimic in vitro the in vivo tumor microenvironment through organotypic co-cultures in which DCIS cells MCF10.DCIS.com are grown in a three dimensional (3D) co-culture of reconstituted basement membrane (rBM) with either normal human breast fibroblasts, or human breast myoepithelial cells. We also analyzed the effects of co-culture with HGF over-secreting fibroblasts in order to mimic the elevated levels of HGF found as DCIS progresses to invasion. Along with these contact co-cultures, conditioned media and recombinant HGF were used to evaluate effects on DCIS morphology, growth, progression to invasion and proteases thought to be involved in ECM turnover. HGF and conditioned media from HGF-secreting fibroblasts activated the HGF receptor c-Met, stimulated the secretion of proteases uPA, the formation of invasive foci, and the degradation of rBM. By adding myoepithelial cells, we can reciprocate in vivo morphology in which the myoepithelial cells form a single layer surrounding luminal epithelial cells. Myoepithelial co-culture with DCIS cells reversed or delayed DCIS progression by inhibiting disorganized growth and the onset of invasive outgrowths. A novel confocal microscope based proteolysis assay showed that both HGF and conditioned media from HGF-secreting fibroblasts increased the degradation of type IV collagen. Employing cysteine cathepsin selective activity-based probes we are able to co-localize cysteine cathepsin activity with intracellular cleavage products of type IV and type I collagen. We have created several in vitro 3D organotypic models that mimic the changing microenvironments (myoepithelial contact, normal fibroblasts and HGF-secreting fibroblasts) of DCIS in vivo and can be used to study novel therapeutics aimed at microenvironmental targets.
