s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue

Mammary stem cells (MaSCs) play critical roles in normal development and perhaps tumorigenesis of the mammary gland. Using combined cell markers, adult MaSCs have been enriched in a basal cell population, but the exact identity of MaSCs remains unknown. We used the s-SHIP promoter to tag presumptive stem cells with GFP in the embryos of a transgenic mouse model. Here we show, in postnatal mammary gland development, that GFP⁺ cap cells in puberty and basal alveolar bud cells in pregnancy each exhibit self-renewal and regenerative capabilities for all mammary epithelial cells of a new functional mammary gland upon transplantation. Single GFP⁺ cells can regenerate the mammary epithelial network. GFP⁺ mammary epithelial cells are p63⁺, CD24^mod, CD49f^high, and CD29^high; are actively proliferating; and express s-SHIP mRNA. Overall, our results identify the activated MaSC population in vivo at the forefront of rapidly developing terminal end buds (puberty) and alveolar buds (pregnancy) in the mammary gland. In addition, GFP⁺ basal cells are expanded in MMTV-Wnt1 breast tumors but not in ErbB2 tumors. These results enable MaSC in situ identification and isolation via a consistent single parameter using a new mouse model with applications for further analyses of normal and potential cancer stem cells.     

Functional Characterization of Stem Cell Activity in the Mouse Mammary Gland

Any portion of the mouse mammary gland is capable of recapitulating a clonally derived complete and functional mammary tree upon transplantation into an epithelial divested mammary fat-pad of a recipient host. As such, it is an ideal model tissue for the study somatic stem cell function. This review will outline what is known regarding the function of stem/progenitor cells in the mouse mammary gland, including how progenitor populations can be functionally defined, the evidence for and potential role of selective DNA strand segregation, and the role of the niche in maintaining and controlling stem cell function.     

Transplanted mammary epithelium grows in association with host stroma: aging of serially transplanted mammary gland is intrinsic to epithelial cells

Mouse mammary gland displays an irreversible decline in growth rate when propagated by serial transplantation in gland-free mammary fat pads of isogeneic mice. Because transplanted fragments of gland contain both mammary epithelial and stromal elements, the present study was undertaken to distinguish between two possibilities: (1) stromal cells in the implants proliferate in coordination with epithelium as the mammary ductal tree regenerates at each passage, or (2) transplanted epithelial tissue interacts exclusively with host stroma. Mammary xenografts from 18-week-old virgin Sprague-Dawley rats were implanted into gland-free mammary fat pads of athymic Balb/cNu/Nu mice. These rat xenografts regenerated chimeric mammary ductal outgrowths. When sectioned and stained with Hoechst dye 33258, a procedure that provides for unambiguous identification of mouse cell nuclei, rat mammary epithelium was found to be associated with mouse stromal cells; only at the site of transplantation were occasional rat stromal nuclei observed. This indicates that as mouse epithelial tissue becomes progressively aged during serial transfer in young mice, the stromal components are refreshed during each passage. The primary lesion underlying the mammary aging phenomenon must therefore be intrinsic to the epithelial cells.     

Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis

The epithelium of the mammary gland exists in a highly dynamic state, undergoing dramatic morphogenetic changes during puberty, pregnancy, lactation, and regression. The recent identification of stem and progenitor populations in mouse and human mammary tissue has provided evidence that the mammary epithelium is organized in a hierarchical manner. Characterization of these normal epithelial subtypes is an important step toward understanding which cells are predisposed to oncogenesis. This review summarizes progress in the field toward defining constituent cells and key molecular regulators of the mammary epithelial hierarchy. Potential relationships between normal epithelial populations and breast tumor subtypes are discussed, with implications for understanding the cellular etiology underpinning breast tumor heterogeneity.     

Stem cells and breast cancer

Proliferation in continuously renewing tissues, including the mammary gland, is hierarchically organized with a small number of slowly dividing stem cells and a greater number of more rapidly proliferating 'transit amplifying' cells. Mammary stem cells have been recently identified and purified based on their surface antigen expression. The recognition of mammary epithelial stem cells had led to the hypothesis that these may be at the root of breast cancer. In support of this, a highly tumorigenic subpopulation of cancer cells – cancer stem cells – has recently been identified in primary and metastatic breast cancer samples and in a number of established breast cancer cell lines. The existence of cancer stem cells would explain why only a small minority of cancer cells is capable of extensive proliferation and transferral of the tumour. In this article we aim to review the evidence in support of the existence of both normal mammary stem cells and breast cancer stem cells, and provide further insight into how taking this subpopulation of cells into account may affect the way we treat epithelial cancers in the future.     

Ovarian steroids and the human breast: regulation of stem cells and cell proliferation

Ovarian steroidal control of mammary gland proliferation and differentiation is not well defined in the human. We therefore developed the athymic nude mouse model in which intact normal human breast tissue is xenografted subcutaneously and treated with human physiological serum levels of oestrogen (E) and/or progesterone (P). We showed that: (i) E, and not P, is the major steroid hormone inducing proliferation of epithelial cells in the adult non-pregnant, non-lactating breast; (ii) E induces progesterone receptor (PR) expression; and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E was required to induce proliferation. Using double label immuno-fluorescence, we demonstrated that cells expressing the oestrogen receptor- (ER) invariably contained the PR but that steroid receptor expression and cell proliferation (Ki67 antigen) were dissociated. Recently, we have demonstrated that some ER/PR-positive epithelial cells are quiescent breast stem cells suggesting that they act as “steroid hormone sensors” that secrete paracrine factors to regulate the proliferative activity of adjacent ER/PR-negative epithelial cells. The dissociation between steroid receptor expression and cell proliferation in normal epithelium was lost at an early stage in ER/PR-positive breast tumour formation perhaps indicating that they arise from deregulation of the normally quiescent breast stem cells.     

Wnt Signaling, Stem Cells, and the Cellular Origin of Breast Cancer

The breast epithelium comprises cells at different stages of differentiation, including stem cells, progenitor cells, and more differentiated epithelial and myoepithelial cells. Wnt signaling plays a critical role in regulating stem/progenitor cells in the mammary gland as well as other tissue compartments. Furthermore, there is strong evidence suggesting that aberrant activation of Wnt signaling induces mammary tumors from stem/progenitor cells, and that Wnt exerts its oncogenic effects through LRP5/6-mediated activation of β-catenin and mTOR pathways. Recent studies using avian retrovirus-mediated introduction of oncogenes into a small subset of somatic mammary cells suggest that polyoma middle T antigen (PyMT) may also preferentially transform stem/progenitor cells. These observations suggest that stem/progenitor cells in the mammary gland may be especially susceptible to oncogenic transformation. Whether more differentiated cells may also be transformed by particular oncogenes is actively debated; it is presently unclear whether stem cells or differentiated mammary cells are more susceptible to transformation by individual oncogenes. Better stem cell and progenitor cell markers as well as the ability to specifically target oncogenes into different mammary cell types will be needed to determine the spectrum of oncogene transformation for stem cells versus more differentiated cells.     

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell‐intrinsic and ‐extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP‐based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time‐consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell‐intrinsic and ‐extrinsic factors. Although the latter can be achieved by cleared fat‐pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild‐type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat‐pad transplantations with MMECs isolated from K14cre;Cdh1^F/F; Trp53^F/F mice expressing Cre recombinase under control of the cytokeratin‐14 promoter and carrying conditional null alleles for p53 and E‐cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell‐intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Adipose tissue: a vital in vivo role in mammary gland development but not differentiation.

Development and differentiation of the mammary gland occurs by means of critical stromal-epithelial interactions. Although many studies have attempted to understand these complex interactions, it has been difficult to demonstrate the essential role of adipose tissue in the development and function of the mammary gland. By using the A-ZIP/F-1 transgenic mice lacking in white adipose tissue (WAT), we have studied the role of adipocytes in mammary gland development and differentiation. In the absence of WAT, rudimentary mammary anlagen form but are unable to grow and branch normally, resulting in a few, short, severely distended ducts. However, during pregnancy, a tremendous amount of epithelial cell division and alveolar cell formation occurs even in the absence of adipocytes, illustrating that adipose tissue is not required for mammary gland differentiation. Mammary gland transplantation revealed that epithelial cells from these transgenic mice possess the potential for normal growth and differentiation when placed into a normal stromal environment. These experiments clearly demonstrate that the absence of adipocytes in the mammary gland results in disruption of stromal-epithelial interactions that prevent normal mammary gland development. The rudimentary epithelial anlage, however, contain mammary stem cells, which are fully capable of alveolar differentiation.     

Stem cells in mammary development and carcinogenesis

Recently, substantial progress has been made in the identification and characterization of stem and progenitor cells in the mouse and human mammary gland. Furthermore, there is increasing evidence that a variety of neoplasms, including breast cancer, may result from transformation of normal stem and progenitor cells. Consistent with this model of carcinogenesis, a breast cancer stem cell population, with the phenotype CD24-CD44+ lineage, was recently identified utilizing flow-cytometry based cell sorting and nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografts. As few as 200 cells of this cancer stem cell population were capable of generating tumors in animals, whereas the bulk of the tumor population was tumorigenic only when implanted in high numbers. Like their normal counterparts, the cancer stem cells have the ability to self-renew, driving tumorigenicity and possibly recurrence and metastasis, and have the ability to differentiate, generating the heterogeneity of the tumors. This stem cell model of carcinogenesis has important implications for understanding the basic biology of breast cancer, as well as other cancers. Furthermore, the concept of cancer as a disease of stem and progenitor cells has profound implications for the development of new strategies for cancer prevention and therapy.     

Stem Cells and the Stem Cell Niche in the Breast: An Integrated Hormonal and Developmental Perspective

The mammary gland is a unique organ in that it undergoes most of its development after birth under the control of systemic hormones. Whereas in most other organs stem cells divide in response to local stimuli, to replace lost cells, in the mammary gland large numbers of cells need to be generated at specific times during puberty, estrous cycles and pregnancy to generate new tissue structures. This puts special demands on the mammary stem cells and requires coordination of local events with systemic needs. Our aim is to understand how the female reproductive hormones control mammary gland development and influence tumorigenesis. We have shown that steroid hormones act in a paracrine fashion in the mammary gland delegating different functions to locally produced factors. These in turn, affect cell–cell interactions that result in changes of cell behavior required for morphogenesis and differentiation. Here, we discuss how these hormonally regulated paracrine interactions may impinge on stem cells and the stem cell niche and how this integration of signals adds extra levels of complexity to current mammary stem cell models. We propose a model whereby the stem cell niches change depending on the developmental stages and the hormonal milieu. According to this model, repeated hormone stimulation of stem cells and their niches in the course of menstrual cycles may be an important early event in breast carcinogenesis and may explain the conundrum why breast cancer risk increases with the number of menstrual cycles experienced prior to a first pregnancy.     

Developmental stage‐specific contribution of LGR5+ cells to basal and luminal epithelial lineages in the postnatal mammary gland

The leucine‐rich repeat‐containing heterotrimeric guanine nucleotide‐binding protein‐coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks mammary epithelial stem cells, we performed in situ lineage‐tracing studies and mammary gland reconstitutions with LGR5‐expressing mammary epithelial cells. Interestingly, the LGR5 progeny population in mammary epithelium switches from the luminal to the myoepithelial compartment during the first 12 days of postnatal development, likely reflecting local changes in Wnt signalling. Together, our findings point to a stage‐specific contribution of LGR5‐expressing cells to luminal and basal epithelial lineages during postnatal mammary gland development. Copyright © 2012 Pathological Society of Great Britain and Ireland.     

41Dissection of a stem cell hierarchy in the human breast

The human breast is a unique organ in that it undergoes most of its development after birth under the control of systemic hormones. The adult human breast undergoes impressive cycles of cell proliferation and apoptosis during pregnancy and with the menstrual cycle many times throughout the reproductive life of women. These highly dynamic changes are thought to rely on the presence of a mammary stem cell population. Recently, we have succeeded in defining a stem cell hierarchy as well as a stem cell zone located in ducts in the normal human mammary gland. In the present study we have utilized multiparameter cell sorting to enable us to isolate two hitherto uncharacterized progenitors present in the luminal compartment of the mammary gland. One population defines a lineage-restricted progenitor with the capacity of expressing endocrine receptors. The other compartment is enriched for cells with stem cell activity, including the ability to form TDLU-like structures in laminin-rich extracellular matrix. Further characterization of these populations included testing for the expression of markers for stem cell activity, lineage-differentiation, anti-apoptotic pathways as well as defining optimal culture conditions for propagating these cells in vitro . In conclusion, characterization of these cells may lead to identification of those long-term breast resident(s) that accumulate enough genetic hits for clonal expansion and tumor development, i.e. the cellular origin(s) of breast cancer.     

In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells

Although the existence of mammary stem cells has been suggested by serial transplantation studies in mice, their identification has been hindered by the lack of specific surface markers, and by the absence of suitable in vitro assays for testing stem cell properties: self-renewal and ability to generate differentiated progeny. We have developed an in vitro cultivation system that allows for propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, based on their ability to proliferate in suspension, as nonadherent mammospheres. We demonstrate that nonadherent mammospheres are enriched in early progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems. Gene expression analysis of cells isolated from nonadherent mammospheres revealed overlapping genetic programs with other stem and progenitor cells and identified new markers that may be useful in the identification of mammary stem cells. The isolation and characterization of these stem cells should help elucidate the molecular pathways that govern normal mammary development and carcinogenesis.     

Prospective Isolation and Functional Analysis of Stem and Differentiated Cells from the Mouse Mammary Gland

Prospective isolation and in vitro and in vivo analysis of primary mouse mammary epithelial cells has been used to separate cell subpopulations and identify stem, progenitor and differentiated cell compartments. Progress has been made from cell separation strategies based on a single marker of the luminal epithelial or myoepithelial compartments to use of markers that allow simultaneous isolation of non-epithelial, basal/myoepithelial and luminal epithelial cells. Transplant analysis has shown that mammary stem cells are found in the basal/myoepithelial compartment, whereas in vitro colony progenitors are found in the luminal compartment. A basal population enriched for stem cell activity can be purified from the myoepithelial cells and the most recent data shows that the luminal population can now be prospectively split into estrogen receptor positive and estrogen receptor negative cells. Future work aims to molecularly characterise these populations to identify new drug targets, which can be used to specifically kill breast cancer stem cells.     

Hormonal regulation of the expression of la antigens on mammary gland epithelium

Guinea pig and mouse mammary gland epithelial cells express Ia antigen-like molecules that react specifically with a rabbit anti-la antigen antiserum. The murine Ia antigen-like molecules were shown to share alloantigenic determinants with regular spleen cell Ia antigens. The expression of the mammary gland Ia antigens is under hormonal control, i.e. the Ia antigen expression is induced by pregnancy and lactation and can also be induced by the exogenous administration of lactotropic hormones.     

Morphological characteristics of mucinous cancer of the breast containing endocrine cells

It was established that mucus-forming mammary gland carcinoma containing endocrine cells may have a structure of an ordinary colloid mammary gland carcinoma or carcinoid carcinoma with mucus formation. The presence of amyloid in the stroma of colloid carcinoma containing endocrine cells may serve as an additional sign of the endocrine cell differentiation. Simultaneous observation in the tumour cells of both mucous inclusions and endocrine granules may be regarded as a proof of an origin of epithelial and endocrine cells of the mammary gland from a pluripotent stem cell.