Transplantation assays which measure the potential of cells

Transplantation assays, which measure the potential of lrrk2 under investigation, traditionally were used as the gold standard for characterizing tissue stem cells. However, recent studies in epithelial tissues based on pulse-chase lineage-tracing approaches, which measure the activity of primary cells in their native habitats, often revealed a discrepancy in the phenotype of an epithelial stem cell population when different assays were used (Choi et al., 2012; Van Keymeulen et al., 2011; Wang et al., 2013). In the mammary gland (MG), lineage-tracing studies based on MEC lineage-specific CreER (Cre-estrogen receptor fusion) mice demonstrated that adult luminal and basal lineages are largely self-sustained (Prater et al., 2014; van Amerongen et al., 2012; Van Keymeulen et al., 2011); in particular, in unperturbed tissues, the luminal lineage appears to be maintained largely by its own lineage-restricted luminal stem cells (LuSCs), rather than by basal MaSCs (Van Keymeulen et al., 2011). However, more recent lineage-tracing studies provided new evidence of the existence of both bipotent basal MaSCs and distinct long-lived LPs to support homeostasis of the luminal lineage in the physiological setting (Rios et al., 2014; Wang et al., 2015). Collectively, these studies reflect the intrinsic complexity of the MEC hierarchy that operates in vivo and a need to further dissect the MEC hierarchy in the physiological setting. Furthermore, since most promoters used in previous lineage-tracing studies either target a broad range of MECs or are subject to developmental stage-dependent regulation, it is difficult to use these genetic tools to directly determine the cell of origin of breast cancer and how the cellular origin contributes to breast cancer heterogeneity. In this study, we analyzed a narrower MEC lineage (i.e., the alveolar luminal sub-lineage) defined based on genetic marking by Cre expression controlled by the same Wap promoter (Wagner et al., 1997), a MEC-specific promoter frequently used in breast cancer mouse models. We provide evidence for a long-lived luminal MEC subpopulation enriched with alveolar-committed progenitors that may serve as the cell of origin of multiple breast cancer subtypes.
Results
Discussion Whether the luminal MEC lineage in adults is sustained by lineage-restricted LuSCs (Van Keymeulen et al., 2011) or by bipotent basal stem cells (Rios et al., 2014; Wang et al., 2015) in the physiological setting has become a topic of intense debate. Nevertheless, one common observation that started to emerge was evidence for long-lived LuSCs/LPs that could sustain the luminal lineage in vivo, yet their identity and role in breast cancer remained largely elusive. Our study uncovered that (Ad-)Wap-Cre-marked MECs in virgin females (i.e., WVs) are luminal MECs enriched with a pre-existing population of long-lived LPs committed to the alveolar luminal sub-lineage. In contrast to the previous finding in parous females for PI-MECs, we found that, during pregnancy and lactation, these WVs only produced ALs, but not myoepithelial cells, in their native habitat. This observation is consistent with a recent clonal analysis study based on K5-rtTA/TetO-Cre and a multicolor Cre-reporter, which demonstrated different cellular origins of luminal and myoepithelial cells within individual alveoli (Rios et al., 2014). WVs appear to be the target of recently revealed paracrine basal-to-luminal cell signaling controlled by p63 (Forster et al., 2014), and they overlap with LPs defined by either Elf5-based (Rios et al., 2014) or Notch1-based lineage tracing (Rodilla et al., 2015). WVs may represent the classic APs that work together with the two novel MEC subpopulations revealed by Notch2-based genetic marking (i.e., S [small] and L [large] cells) (Šale et al., 2013), for the formation of alveolar clusters in response to alveologenic signals. Furthermore, due to the self-sustained, long-lived nature of WVs, our study provides an alternative model to explain the developmental origin of PI-MECs. Rather than derived from de-differentiation of secretory alveolar cells that survived involution, PI-MECs detected via Wap-Cre might represent a combination of equipotent APs, either marked by Wap-Cre at the virgin stage (i.e., WVs) or freshly marked by Wap-Cre during late gestation (i.e., the second wave of genetic marking when the Wap promoter activity is dramatically upregulated; Figure 7A). These APs may be intrinsically resistant to involution. As there should be a lot more of them freshly labeled by Wap-Cre during late gestation (due to much higher Wap activity at this stage) than those pre-labeled at the virgin stage, it would appear as if a larger population of Wap-Cre-marked long-lived MECs emerged only after parity (Wagner et al., 2002). This model is supported by a recent study characterizing parous PI-MECs using Wap-Cre mice (Chang et al., 2014). Lastly, the following also should be pointed out: (1) our model does not exclude the possibility that PI-MECs also can be derived from de-differentiation of mature secretory ALs that survive involution; (2) our model does not suggest that nulliparous WVs and parous PI-MECs are identical, and, in fact, parity is expected to have a profound effect on this progenitor cell lineage (in support of this, it was shown recently that, depending on the timing of acquiring premalignant lesions and of pregnancy, the Wap-marked alveolar cell population was the target cell mediating the dual roles of pregnancy in increasing or reducing breast cancer risks [Haricharan et al., 2013, 2014]); and (3) our model does not rule out a possibility that WVs may include LuSCs that can give rise to both ductal luminal cells and APs.