An organotypic assay for the quantification and characterization of regenerative primary human mammary epithelial cells

Breast cancer is a heterogeneous disease with a high degree of intra- and intertumoral diversity, which impedes accurate patient stratification, prognosis and optimal treatment. The mammary gland consists of a complex network of epithelial ducts which end in clusters of alveoli, called terminal ductal lobular units (TDLUs) which are the functional units of the mammary gland. Postnatal mammary gland development and homeostasis require an enormous regenerative output, suggesting the existence of tissue stem/progenitor cells and a high degree of cellular plasticity to ensure functional robustness, i.e. the production and secretion of milk during lactation. Therefore, the observed heterogeneity in breast cancer is likely the result of normal mammary gland architecture and functionality. Unfortunately, the identification and characterization of human stem/progenitor cells and the analysis of cellular plasticity are hampered by the limited applicability of currently used murine in vivo assays and the failure of 2D and 3D in vitro assays to recapitulate the histological architecture and functionality of the human mammary gland. Organoid assays span the bridge between standard in vitro culture and in vivo studies by mimicking the stem cell niche during self-renewal or repair in 3D culture. Thereby, organoid assays support the formation of organoids which recapitulate the architecture and functionality of the original tissue. In this study, a novel organotypic assay was developed for the human mammary gland, in which single, freshly isolated human mammary epithelial cells from healthy donors cultured in floating collagen gels generate spheres and branched ductal structures that resemble TDLUs. The TDLU-like organoids were shown to form alveolar buds, express lineage markers at correct positions, and display functionality by contraction. In line with recent literature from transplantation studies, it was found that TDLU-like structure formation is enriched in a CD49fhi/EpCAM− population, commonly referred to as basal, while CD49f+/EpCAM+ LP cells predominantly formed spheres. In addition, by performing limiting dilution analysis, the metalloendopeptidase CD10 was revealed as marker to enrich for TDLU-like structure forming cells within the CD49fhi/EpCAM− population. The use of CD10 further led to identification of stromal cells present within the CD49fhi/EpCAM− population. As expression of CD10 was not restricted to the CD49fhi/EpCAM− cell population, it was shown that a combination of all three markers is required to optimally enrich for TDLU-like structure forming cells. Importantly, the formation of TDLU-like structures was observed only in floating/compliant, but not in attached/rigid collagen gels and required actin-myosin-based cellular contraction. Structures generated in attached collagen gels exhibited aberrant marker expression and invasive phenotype, emphasizing the importance of physical parameters in directing differentiation of the mammary gland and suggesting a role in tumorigenesis. In summary this study describes a defined in vitro assay system to quantify mammary epithelial cells with regenerative potential by limiting dilution, to analyze cellular plasticity, differentiation potential and functionality and to systematically investigate interactions with the physical environment at distinct steps of morphogenesis and the effect on cell fate decisions.