Alveolar progenitor cells develop in mouse mammary glands independent of pregnancy and lactation

We have previously described pluripotent, parity-induced mammary epithelial cells (PI-MEC) marked by Rosa26-lacZ expression in the mammary glands of parous females. PI-MEC act as lobule-limited epithelial stem/progenitor cells. To determine whether parity is necessary to generate PI-MEC, we incubated mammary explant cultures from virgin mice in vitro with insulin alone (I), hydrocortisone alone (H), prolactin alone (Prl), or a combination of these lactogenic hormones (IHPrl). Insulin alone activated the WAP-Cre gene. Hydrocortisone and prolactin alone did not. Any combination of hormones that included insulin was effective. Only I, H and Prl together were able to induce secretory differentiation and milk protein synthesis. In addition, EGF, IGF-2 and IGF-1 added individually produced activated (lacZ(+)) PI-MEC in explant cultures. Neither estrogen nor progesterone induced WAP-Cre expression in the explants. None of these positive initiators of WAP-Cre expression in PI-MEC were effective in mammospheres or two-dimensional cultures of mammary epithelium, indicating the indispensability of epithelial-stromal interaction in PI-MEC activation. Like PI-MEC, lacZ(+) cells from virgin explants proliferated and contributed progeny to mammospheres in vitro and to epithelial outgrowths in vivo after transplantation. LacZ(+) cells induced in virgin mouse mammary explants were multipotent (like PI-MEC) in impregnated hosts producing lacZ(+) mammary alveolar structures comprised of both myoepithelial and luminal progeny. These data demonstrate PI-MEC, a mammary epithelial sub-population of lobule-limited progenitor cells, are present in nulliparous female mice before parity and, like the PI-MEC observed following parity, are capable of proliferation, self-renewal and the capacity to produce progeny of diverse epithelial cell fates.     

Mammary stem cells come of age, prospectively

Two recent reports have contributed direct evidence for the existence of a pluripotent mouse mammary epithelial stem cell. In both reports, the investigators have prospectively isolated an enriched fraction of mammary stem cells using fluorescence-activated cell sorting from freshly dispersed epithelial cells. This fraction of cells, upon transplantation in limiting dilution (in some cases as a single cell), produces complete mammary development within the host mammary fat pad. These studies extend and confirm earlier work that demonstrated that retroviral-tagged mammary fragments produce complete functional mammary glands comprising their clonal progeny upon fat-pad transplantation. This technical advance opens the possibility to use similar methodologies to isolate and characterize human breast epithelial stem cells, and elucidate their role in regeneration and neoplasia.     

Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells

Parity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24(+)/CD49f(high) mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland.     

An adjunct mammary epithelial cell population in parous females: its role in functional adaptation and tissue renewal

Mammary gland biologists have long assumed that differentiated secretory epithelial cells undergo programmed cell death at the end of lactation and that the alveolar compartment is reconstituted from undifferentiated precursor cells in subsequent pregnancies. It is generally agreed that the remodeled gland in a parous animal resembles that of a mature virgin at the morphological level. However, several physiological differences have been noted in comparing the responses of mammary epithelia from nulliparous versus parous females to hormonal stimulation and carcinogenic agents. We present genetic evidence that an involuted mammary gland is fundamentally different from a virgin gland, despite its close morphological resemblance. This difference results from the formation of a new mammary epithelial cell population that originates from differentiating cells during pregnancy. In contrast to the majority of fully committed alveolar cells, this epithelial population does not undergo cell death during involution or remodeling after lactation. We show that these cells can function as alveolar progenitors in subsequent pregnancies and that they can play an important role in functional adaptation in genetically engineered mice, which exhibit a reversion of a lactation-deficient phenotype in multiparous animals. In transplantation studies, this parity-induced epithelial population shows the capacity for self-renewal and contributes significantly to the reconstitution of the resulting mammary outgrowth (i.e. ductal morphogenesis and lobulogenesis). We propose that this parity-induced population contributes importantly to the biological differences between the mammary glands of parous and nulliparous females.     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG^−/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.     

Fonction catabolique de l'épithélium mammaire

Summary The histochemical (iron, lipopigments, acid phosphatase, leucine aminopeptidase) and cytologic (lysosomes) changes occuring during pregnancy, lactation and involution of mouse, rat, rabbit, guinea-pig mammary glands are studied by light microscopy and electron microscopy.In all the animals examined, the mammary epithelium has an intracellular digestive system which is adapted to subserve two functions. The first one is the segregation of cytoplasmic components which often precedes cellular involution. The second one is the regulation of secretory processes in the non lactating glands. This digestion of endogenous materials results in the formation of various lytic bodies: dense bodies sometimes containing ferritin, vacuolated dense bodies with membranous residues, autophagic vacuoles. The lysosomes can give large complex dense bodies like lipofuscin pigments with or without ferritin.Leucine aminopeptidase which always disappears in the mouse mammary epithelium during lactation is not present in rat, rabbit, guinea-pig mammary epithelium. In these species only the vascular tissue contains the enzyme. This observation indicates that leucine aminopeptidase does not take care of the overproduction of secretory products in the non-lactating glands.Acid phosphatase is concentrated in secretory granules and in lytic bodies: multivesicular bodies, dense bodies with ferritin, vacuolated dense bodies, lipopigments. This enzyme constitutes probably a mechanism for controlling and triggering the destruction of the secretory material with no active elimination.The iron of the mammary epithelium appears in virgin mice older than 30 weeks and in mice, rats, rabbits, guinea-pigs during glandular cells involution. This is a catabolic iron located in lysosomes. Its amount depends upon the iron content of the milk and upon the competitive secretory and catabolic activities of the glandular cells. An explanation of iron disappearance during a second pregnancy and lactation is discussed.     

EGF controls the in vivo developmental potential of a mammary epithelial cell line possessing progenitor properties

The bilayered mammary epithelium comprises a luminal layer of secretory cells and a basal layer of myoepithelial cells. Numerous data suggest the existence of self-renewing, pluripotent mammary stem cells; however, their molecular characteristics and differentiation pathways are largely unknown. BC44 mammary epithelial cells in culture, display phenotypic characteristics of basal epithelium, i.e., express basal cytokeratins 5 and 14 and P-cadherin, but no smooth muscle markers. In vivo, after injection into the cleared mammary fat pad, these cells gave rise to bilayered, hollow, alveolus-like structures comprising basal cells expressing cytokeratin 5 and luminal cells positive for cytokeratin 8 and secreting beta-casein in a polarized manner into the lumen. The persistent stimulation of EGF receptor signaling pathway in BC44 cells in culture resulted in the loss of the in vivo morphogenetic potential and led to the induction of active MMP2, thereby triggering cell scattering and motility on laminin 5. These data (a) suggest that BC44 cells are capable of asymmetric division for self-renewal and the generation of a differentiated progeny restricted to the luminal lineage; (b) clarify the function of EGF in the control of the BC44 cell phenotypic plasticity; and (c) suggest a role for this phenomenon in the mammary gland development.     

Isolation of mouse mammary epithelial progenitor cells with basal characteristics from the Comma-Dbeta cell line

A mouse mammary epithelial cell line with morphogenetic properties in vivo, Comma-Dbeta, was used to isolate and to characterize mammary progenitor cells. We found that a homogeneous cell population expressing high surface levels of stem cell antigen 1 (Sca-1) was able to give rise in vivo to ductal and alveolar structures comprising luminal secretory and basal myoepithelial cells. Unlike the Sca-1(high), the Sca-1(neg/low) cell population displayed a reduced morphogenetic potential. The Sca-1(high) cells presented moderate CD24, high CD44 and alpha6 integrin surface levels, expressed basal cell markers p63, keratins 5 and 14, but no luminal and myoepithelial lineage markers. In culture, the Sca-1(high) cells generated identical daughter cells that retained their in vivo developmental potential, indicating that these cells were maintained by self-renewal. Plated at clonogenic density in Matrigel, Sca-1(high) cells formed spheroids that included luminal and myoepithelial cells. Thus, the isolated Sca-1(high) basal cells possess several features of stem/progenitor cells, including specific markers, self-renewal capacity, and the ability to generate the two major mammary lineages, luminal and myoepithelial. These data provide evidence for the existence of basal-type mouse mammary progenitors able to participate in the morphogenetic processes characteristic of mammary gland development.     

Stat3 Is Required to Maintain the Full Differentiation Potential of Mammary Stem Cells and the Proliferative Potential of Mammary Luminal Progenitors

Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during post-lactational regression. On the other hand, Stat3 is required for the self-renewal of embryonic stem cells and is sufficient for the induction of a naïve pluripotent state in epiblast stem cells. Mammary stem cells (MaSCs) have a high capacity for self-renewal and can grow robustly in transplantation experiments in vivo. However, a role for Stat3 in MaSCs has not been investigated. Here we show that depletion of Stat3 from basal cells results in reduced primary transplantation efficiency and diminishes the potential to generate ductal, but not alveolar, outgrowths. In addition, Stat3 is required for maximal proliferation of luminal progenitors.     

P190A RhoGAP is required for mammary gland development

P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5 day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.     

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub‐populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi‐potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell‐enriched population showed the most pronounced deregulation of migration‐associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.     

Cellular localization of human secretory beta globulin in normal and tumor tissues]

Distribution of secretory beta-globulin (S beta G) which possesses affinity for steroids was investigated immunohistochemically. Tissue specificity of S beta G, produced in adult secretory epithelial cells of the seminal vesicles, salivary glands, prostate, bronchi and mammary gland was discovered. The protein was not detected in fetal and embryonal tissues. S beta G synthesis is abnormal in neoplasms: its expression partly preserves in breast cancer cells and increases in epithelium of mammary ducts near the focus of malignancy. In lung cancer and bronchial glands cells near the focus of neoplastic transformation S beta G positive reaction was not observed.     

Generation of lobuloalveolar development from isolated rat mammary ducts and end buds

Implantation of excised bud-free ductal fragments (DUCTS), terminal end buds (TEBs), or alveolar buds (ABs) from virgin mammary glands of Wistar-Furth rats into interscapular fat pads of syngeneic female rats produces, after 16 weeks, complete ductal outgrowths including TEBs and ABs. Treatment of the recipient rats with perphenazine for 1 day or mating them after 12 weeks and then isolating the resultant outgrowths after 16 weeks produces significantly larger outgrowths than those from untreated hosts. The outgrowths consist of distended ducts and lobules or distended ducts and alveoli, respectively. Histochemical and immunocytochemical staining of the outgrowths with reagents that depict epithelial, myoepithelial, and lactating alveolar cells (peanut lectin alone, monoclonal and polyclonal antibodies to rat caseins) indicate similar cell compositions and arrangements for all outgrowths irrespective of their source; these are also similar to the mammary glands of the perphenazine-stimulated or lactating hosts. There is one major difference: the degree of staining of peanut lectin alone and the anti-caseins is greater for outgrowths produced by the ABs and TEBs than for those produced by the DUCTs. DUCT implants left for 1 year after cessation of lactation of the hosts are still stained appreciably by peanut lectin alone and by the anti-caseins, particularly the luminal secretions. Therefore, the complete morphogenetic and cell differentiating ability for generating mammary glands is present in bud-free ducts, but this ability can be enhanced in TEBs/ABs or abnormally expressed at ectopic sites.     

Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands

It has been postulated that the stem cells of somatic tissues protect themselves from mutation and cancer risk by selective segregation of their template DNA strands. Self-renewing mammary epithelial stem cells that were originated during allometric growth of the mammary ducts in pubertal females were labeled using [3H]-thymidine (3HTdR). After a prolonged chase during which much of the branching duct morphogenesis was completed, 3HTdR-label retaining epithelial cells (LREC) were detected among the epithelium of the maturing glands. Labeling newly synthesized DNA in these glands with a different marker, 5-bromodeoxyuridine (5BrdU), resulted in the appearance of doubly labeled nuclei in a large percentage of the LREC. By contrast, label-retaining cells within the stroma did not incorporate 5BrdU during the pulse, indicating that they were not traversing the cell cycle. Upon chase, the second label (5BrdU) was distributed from the double-labeled LREC to unlabeled mammary cells while 3HTdR was retained. These results demonstrate that mammary LREC selectively retain their 3HTdR-labeled template DNA strands and pass newly synthesized 5BrdU-labeled DNA to their progeny during asymmetric divisions. Similar results were obtained in mammary transplants containing self-renewing, lacZ-positive epithelial cells suggesting that cells capable of expansive self-renewal may repopulate new mammary stem cell niches during the allometric growth of new mammary ducts.     

Small Molecule Antagonists of the Wnt/Beta-Catenin Signaling Pathway Target Breast Tumor-Initiating Cells in a Her2/Neu Mouse Model of Breast Cancer

Background

Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure.

Methods/Findings

To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118–310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118–310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118–310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118–310 treated mice were unable to seed the growth of secondary tumors after transplant.

Conclusions

These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.