Background Breast cancer arises within specific regions in the human breast referred to as the terminal duct lobular units (TDLUs). These are relatively dynamic structures characterized by sex hormone driven cyclic epithelial turnover. TDLUs consist of unique parenchymal entities embedded within a fibroblast-rich lobular stroma. Here, we establish and characterize a new human breast lobular fibroblast cell line against its interlobular counterpart with a view to assessing the role of region-specific stromal cues in the control of TDLU dynamics.
Methods Primary lobular- and interlobular fibroblasts are transduced to express human telomerase reverse transcriptase (hTERT). Differentiation of the cell lines along lobular- and interlobular pathways is determined by immunocytochemical staining and genome-wide RNA sequencing. Their functional properties are further characterized by analysis of mesenchymal stem cell (MSC) differentiation repertoire in culture and in vivo . The cells´ physiological relevance for parenchymal differentiation is examined in heterotypic co-culture with fluorescence-activated cell sorting (FACS)-purified normal breast primary luminal- or myoepithelial progenitors. The co-cultures are immunostained for quantitative assessment of epithelial branching morphogenesis, polarization, growth and luminal epithelial maturation. In extension, myoepithelial progenitors are tested for luminal differentiation capacity in culture and in mouse xenografts. To unravel the significance of transforming growth factor-beta (TGF-β)-mediated crosstalk in TDLU-like morphogenesis and differentiation, fibroblasts are incubated with SB431542 prior to heterotypic co-culture with luminal cells.
Results hTERT immortalized fibroblast cell lines retain critical phenotypic traits in culture and links to primary fibroblasts. Cell culture assays and transplantation to mice show that the origin of fibroblasts determines TDLU-like and ductal-like differentiation of epithelial progenitors. Whereas lobular fibroblasts support a high level of branching morphogenesis by luminal cells, interlobular fibroblasts support ductal-like myoepithelial characteristics. TDLU-like morphogenesis, at least in part, relies on intact TGF-β signaling.
Conclusions The significance of the most prominent cell type in normal breast stroma, the fibroblast, in directing epithelial differentiation is largely unknown. Through establishment of lobular and interlobular fibroblast cell lines we here demonstrate that epithelial progenitors are submitted to stromal cues for site-specific differentiation. Our findings lend credence to considering stromal subtleties of crucial importance in the development of normal breast and, in turn, breast cancer.