New insights into lineage restriction of mammary gland epithelium using parity-identified mammary epithelial cells

Abstract

IntroductionParity-identified mammary epithelial cells (PI-MECs) are an interesting cellular subset because they survive involution and are a presumptive target for transformation by human epidermal growth factor receptor 2 (HER2)/neu in mammary tumors. Depending on the type of assay, PI-MECs have been designated lobule-restricted progenitors or multipotent stem/progenitor cells. PI-MECs were reported to be part of the basal population of mammary epithelium based on flow cytometry. We investigated the cellular identity and lineage potential of PI-MECs in intact mammary glands.MethodsWe performed a quantitative and qualitative analysis of the contribution of PI-MECs to mammary epithelial cell lineages in pregnant and involuted mammary glands by immunohistochemistry, fluorescence-activated cells sorting (FACS), and quantitative polymerase chain reaction. PI-MECs were labeled by the activation of Whey Acidic Protein (WAP)-Cre during pregnancy that results in permanent expression of yellow fluorescent protein.ResultsAfter involution, PI-MECs are present exclusively in the luminal layer of mammary ducts. During pregnancy, PI-MECs contribute to the luminal layer but not the basal layer of alveolar lobules. Strikingly, whereas all luminal estrogen receptor (ER)-negative cells in an alveolus can be derived from PI-MECs, the alveolar ER-positive cells are unlabeled and reminiscent of Notch2-traced L cells. Notably, we observed a significant population of unlabeled alveolar progenitors that resemble PI-MECs based on transcriptional and histological analysis.ConclusionsOur demonstration that PI-MECs are luminal cells underscores that not only basal cells display multi-lineage potential in transplantation assays. However, the lineage potential of PI-MECs in unperturbed mammary glands is remarkably restricted to luminal ER-negative cells of the secretory alveolar lineage. The identification of an unlabeled but functionally similar population of luminal alveolar progenitor cells raises the question of whether PI-MECs are a unique population or the result of stochastic labeling. Interestingly, even when all luminal ER-negative cells of an alveolus are PI-MEC-derived, the basal cells and hormone-sensing cells are derived from a different source, indicating that cooperative outgrowth of cells from different lineages is common in alveologenesis.

Bringing androgens up a NOTCH in breast cancer

“…Initial evidence indicates a luminal, HS-MEC identity of these cells but clearly demonstrates, at least in the case of NOTCH2, that such cells are restricted to mammary ductal patterning during development and the coordination of lobule growth associated with pregnancy. While these recent findings demonstrate that a lobule-restricted progenitor may indeed exist, it appears to be more lineage-restricted than the lobule progenitor originally posited by Smith (1996), and rather than serving as a 'stem cell' for milk-producing lobules, it is involved in the co-ordinated action and integration of all three MEC lineages that develop together as part of lobule growth during pregnancy , Lain et al 2013, Chang et al 2014 and that in mouse results in an expansion of basal MECs in early pregnancy (as identified by FACS markers (Fig. 3)).…”

“…Ductal branching, lobular growth and alveolar differentiation during the menstrual cycle or at pregnancy are thought to be principally directed by the actions of progesterone, though oestrogen signalling is required for progesterone action, which induces reorganisation and expansion of the mammary epithelium at ductal branch-points (Sampayo et al (2013); reviewed in Macias & Hinck (2012)). Recent studies have shown that this growth process involves MECs from both basal and luminal cell lineages (Lain et al 2013, Chang et al 2014, rather than the activity of a common stem/progenitor cell population that gives rise to all cells within the newly developed lobules. Therefore, coordinated cell-cell signalling multiple MEC lineages is critical to the normal growth and differentiation of the adult mammary epithelium.…”

Mammary gland development: cell fate specification, stem cells and the microenvironment

“…A different study found that progeny of secretory alveolar cells, marked according to whey acidic protein promoter (Wap) expression, persist through multiple cycles of pregnancy and involution. These parity-induced epithelial cells are found in the lumens between pregnancy and in the secretory alveoli during pregnancy; they express markers of luminal cells, including Elf5, and appear to not express hormone receptors (Chang et al, 2014).…”

“…The more recent use of multi-color labeling schemes makes this less likely, as the probability of two cells being labeled with the same color is related to the probability of labeling squared times the probability of the two having the same color (1/16 for fourcolor labeling), but it is still possible that some single-color patches were not clonal. Although lineage tracing studies appear to be superior for identifying the normal developmental potential of various mammary gland stem and progenitor cell populations, further studies are needed to reconcile the relationship between the K5 + , K8 + , K14 + , Lgr5 + , Axin2 + , CD1d + and parity-induced mammary epithelial cell populations (Chang et al, 2014;dos Santos et al, 2013;Plaks et al, 2013;Šale et al, 2013;Van Keymeulen et al, 2011;van Amerongen et al, 2012;Wagner et al, 2002).…”

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“…In contrast, Wap-mRNA is detected in primary mouse estrogen receptor knockout mammary epithelial cells preparations at day 7 of first pregnancy. 47 In addition, Jhappan et al 48 showed that the Wap promoter is minimally active in mammary glands of virgin and seven-day pregnant mice. In 1995, Kordon et al 49 showed that the Wap promoter is active in mammary glands of Wap-LACZ mice during estrus.…”