Final Report Summary - LGR6MAMMARYSC (The role of Lgr6 as a marker of progenitor cells and cancer-initiating cells in the mammary gland) The LGR6 receptor has recently been described to be involved in Wnt/beta catenin signaling. Two Wnt-pathway members, LGR5 and Axin2, were shown to mark subsets of mammary gland stem cells. We studied the contribution of Lgr6 expressing mammary epithelial cells to murine mammary gland development and tumorigenesis, as well as human breast cancer. We could elucidate the role of LGR6+ cells within the mammary gland stem cell/progenitor cell hierarchy and, most importantly, uncover the role of LGR6 expressing cells in mammary gland tumorigenesis in vivo.We employed different reporter mouse strains to detect and isolate Lgr6+ mammary epithelial cells and study their contribution to mammary gland development in vivo. By using laser scanning confocal microscopy and multicolor flow cytometry on freshly isolated mammary gland cells we analyzed their distribution throughout mammary gland development and identified different populations of LGR6 expressing mammary gland cells. To further characterize them we tracked the fate of LGR6 expressing mammary epithelial cells at different stages of mammary gland development and pregnancy and assessed the contribution of their cellular progeny to the developing mammary gland. Our genetic lineage tracing studies demonstrated that the LGR6+ cell populations contributed to mammary gland organogenesis in distinctive ways. Similarly to Axin2 expressing mammary gland cells, the developmental timepoint and stage influence the capability of LGR6+ mammary gland cells to contribute to mammary gland development and function. Currently, two cellular hierarchy models of mammary gland development are discussed. One model proposes that only embryonic mammary gland stem cells are bipotential, whereas postnatal progenitor cells are unipotential and 2 independent progenitor cell populations maintain the basal and luminal lineage, respectively. The second model, in contrast, claims the existence of a bipotential stem cell even in the adult mammary gland. Our observations add information to the underlying stem cell/progenitor cell hierarchy and can be integrated into both proposed models, suggesting that mammary gland stem and progenitor cells can behave according to both models in a dynamic fashion. Next, we elucidated the role of LGR6 expressing cells in 2 different murine models of human breast tumorigenesis (representing basaloid and luminal breast tumors). We applied genetic lineage tracing to follow the progeny of theses cells and assessed their contribution to the initiation and progression of mammary tumors. Such, we could decipher the role of LGR6 expressing cells not only in normal mammary gland development but also in mammary tumorigenesis. By employing basal and luminal tumor models, we could gain insight into the cells underlying these specific tumor types and added valuable information about the behavior of described mammary cancer stem cells. Finally, we screened commercially available breast cancer tissue microarrays for expression of LGR6. Indeed, LGR6 is expressed to various degrees in app. 50% of the screened tumors. We are currently extending our studies regarding human breast cancer by screening a larger cohort of tumor cases. Our studies of normal mammary gland development gave insight into the dynamics of stem cells and progenitor cells involved in murine mammary gland development and mammary tumorigenesis. We strongly believe that our results provide opportunities to study the cell types underlying different mammary and breast tumors in more detail. Furthermore, this knowledge can be exploited to study the effects that cancer therapeutics exert on tumor cell behavior. Even more so, we will be able to study the mechanisms with which breast tumors evade therapeutic regimens and develop therapy resistance. Considering the fact that 1 in 8 women are confronted with breast cancer throughout their lifetime such studies could provide valuable knowledge for future strategies targeting different types of breast cancer.