Abstract P1-06-02: A force-sensitive organoid assay to quantify regenerative potential of single primary human mammary cells

Abstract

Abstract We have developed an organoid regeneration assay to quantify the ability of freshly isolated, single human mammary epithelial cells to generate complex branched ductal structures with basal and luminal features. For this purpose, cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix. In both conditions, single luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts in multicellular structures generated by basal cells develop a lumen and alveoli at their tips. Importantly, branched structures generated by single basal cells express basal and luminal markers at correct positions, thereby demonstrating bi-potential. Functionally, basal cells in branched structures display cellular contractility, which we reveal to be required for alveologenesis. In conclusion, branched structures generated by single basal cells in compliant collagen gels resemble terminal ductal-lobular units (TDLU), the functional units of the mammary gland. To prospectively isolate basal cells with regenerative potential, we added the membrane metallo-endopeptidase CD10 as a cell surface marker to existing sorting protocols, thereby enriching for TDLU-formation and enabling rigorous quantification of regenerative potential by extreme limiting dilution assay (ELDA). Moreover, the use of CD10 as a cell surface marker reveals the presence of stromal cells with endothelial qualities within the CD49fhi/EpCAM– population, previously labeled basal. In summary, we describe a defined in vitro assay system to quantify primary human mammary epithelial cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. Citation Format: Scheel CH, Linnemann JR, Miura H, Meixner LK, Irmler M, Kloos UJ, Hirschi B, Bartsch HS, Sass S, Beckers J, Theis FJ, Gabka C, Sotlar K. A force-sensitive organoid assay to quantify regenerative potential of single primary human mammary cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-06-02.