Metformin reduces basal subpopulation and attenuates mammary epithelial cell stemness in FVB/N mice.

Metformin shows promise in breast cancer prevention, but its underlying mechanisms remain unclear. This study investigated the impact of metformin on the repopulation dynamics of mammary epithelial cells (MECs) and the signaling pathways in non-tumorigenic FVB/N mice. This study aimed to enhance our understanding of the role of metformin in reducing the susceptibility of MECs in premalignant tissues to oncogenic factors. In this study, female mice were administered 200 mg/kg/day of metformin via intraperitoneal (i.p.) injection from 8 to 18 weeks of age. After this treatment period, morphogenesis, flow cytometry, analyses of MEC stemness, and RNA sequencing were performed. The study findings indicated that metformin treatment in adult mice reduced mammary gland proliferation, as demonstrated by decreased Ki67+ cells and lateral bud formation. Additionally, metformin significantly reduced both basal and mammary repopulating unit subpopulations, indicating an impact on mammary epithelial cell repopulation. Mammosphere, colony-forming cell, and 3D culture assays revealed that metformin adversely affected mammary epithelial cell stemness. Furthermore, metformin downregulated signaling in key pathways including AMPK/mTOR, MAPK/Erk, PI3K/Akt, and ER, which contribute to its inhibitory effects on mammary proliferation and stemness. Transcriptome analysis with RNA sequencing indicated that metformin induced significant downregulation of genes involved in multiple critical pathways. KEGG-based pathway analysis indicated that genes in PI3K/Akt, focal adhesion, ECM-receptor, small cell lung cancer and immune-modulation pathways were among the top groups of differentially regulated genes. In summary, our research demonstrates that metformin inhibits MEC proliferation and stemness, accompanied by the downregulation of intrinsic signaling. These insights suggest that the regulatory effects of metformin on premalignant mammary tissues could potentially delay or prevent the onset of breast cancer, offering a promising avenue for developing new preventive strategies.

Atypical cell cycle regulation promotes mammary stem cell expansion during mammary development and tumourigenesis.

BACKGROUND: The cell cycle of mammary stem cells must be tightly regulated to ensure normal homeostasis of the mammary gland to prevent abnormal proliferation and susceptibility to tumorigenesis. The atypical cell cycle regulator, Spy1 can override cell cycle checkpoints, including those activated by the tumour suppressor p53 which mediates mammary stem cell homeostasis. Spy1 has also been shown to promote expansion of select stem cell populations in other developmental systems. Spy1 protein is elevated during proliferative stages of mammary gland development, is found at higher levels in human breast cancers, and promotes susceptibility to mammary tumourigenesis when combined with loss of p53. We hypothesized that Spy1 cooperates with loss of p53 to increase susceptibility to tumour initiation due to changes in susceptible mammary stem cell populations during development and drives the formation of more aggressive stem like tumours. METHODS: Using a transgenic mouse model driving expression of Spy1 within the mammary gland, mammary development and stemness were assessed. These mice were intercrossed with p53 null mice to study the tumourigenic properties of Spy1 driven p53 null tumours, as well as global changes in signaling via RNA sequencing analysis. RESULTS: We show that elevated levels of Spy1 leads to expansion of mammary stem cells, even in the presence of p53, and an increase in mammary tumour formation. Spy1-driven tumours have an increased cancer stem cell population, decreased checkpoint signaling, and demonstrate an increase in therapy resistance. Loss of Spy1 decreases tumor onset and reduces the cancer stem cell population. CONCLUSIONS: This data demonstrates the potential of Spy1 to expand mammary stem cell populations and contribute to the initiation and progression of aggressive, breast cancers with increased cancer stem cell populations.

Spatially distinct epithelial and mesenchymal cell subsets along progressive lineage restriction in the branching embryonic mammary gland.

How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors. Here, using single-cell RNA sequencing from E13.5 to birth, we produced an atlas of matched mouse mammary epithelium and mesenchyme and reconstructed the differentiation trajectories of MaSCs toward basal and luminal fate. We show that murine MaSCs exhibit lineage commitment just prior to the first sprouting events of mammary branching morphogenesis at E15.5. We identify early molecular markers for committed and multipotent MaSCs and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially restricted cell populations, and that dermal mesenchyme-produced FGF10 is essential for embryonic mammary branching morphogenesis. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent MaSCs, and uncover the signals from mesenchymal cells that guide mammary branching morphogenesis.

Sympathetic nerve signals: orchestrators of mammary development and stem cell vitality.

The mammary gland is a dynamic organ that undergoes significant changes at multiple stages of postnatal development. Although the roles of systemic hormones and microenvironmental cues in mammary homeostasis have been extensively studied, the influence of neural signals, particularly those from the sympathetic nervous system, remains poorly understood. Here, using a mouse mammary gland model, we delved into the regulatory role of sympathetic nervous signaling in the context of mammary stem cells and mammary development. Our findings revealed that depletion of sympathetic nerve signals results in defective mammary development during puberty, adulthood, and pregnancy, accompanied by a reduction in mammary stem cell number. Through in vitro three-dimensional culture and in vivo transplantation analyses, we demonstrated that the absence of sympathetic nerve signals hinders mammary stem cell self-renewal and regeneration, while activation of sympathetic nervous signaling promotes these capacities. Mechanistically, sympathetic nerve signals orchestrate mammary stem cell activity and mammary development through the ERK signaling pathway. Collectively, our study unveils the crucial roles of sympathetic nerve signals in sustaining mammary development and regulating mammary stem cell activity, offering a novel perspective on the involvement of the nervous system in modulating adult stem cell function and organ development.

Effects of 5-fluorouracil, thymoquinone, and mammary stem cells' exosomes on in vitro cultured breast cancer cells.

Background: 5-fluorouracil (5-FU) is an antimetabolic agent used for treating slowly growing solid tumors like breast and ovarian carcinoma. Thymoquinone (TQ) is the main biologically active constituent of Nigella sativa, it has been found to demonstrate anticancerous effects in several preclinical studies, and this is because TQ possesses multitarget nature. Stem cells-derived exosomes are in the spotlight of research and are promising tissue regenerative and anticancer cell-derived nanovesicles. Aim: Herein, we studied the antineoplastic effects of Exosomes derived from mammary stem cells (MaSCs-Exo) on breast cancer cells, alone or combined with TQ when compared to a breast cancer chemotherapeutic agent; 5-FU. Methods: Our approach included performing viability test and measuring the expression of pro-apoptotic gene (Bax), anti-apoptotic gene (BCL-2) and angiogenic gene (VEGF) on Human MCF-7 cells (breast adenocarcinoma cells), the MCF-7 cells were cultured and incubated with medium containing 5-FU (25 µg/ml), TQ (200 µg/ml), MaSCs-Exo (100 µg protein equivalent), a combination of TQ (200 µg/ml) and MaSCs-Exo (100 µg). Results: Our obtained results show that TQ and MaSCs-Exo each can effectively inhibit breast cancer cell line (MCF-7) proliferation and growth. Also, the results show that the combination of TQ and MaSCs-Exo had higher cytotoxic effects on MCF-7 breast cancer cells than TQ or 5-FU, alone. Conclusion: The present study shows a promising anticancer potential of exosomes isolated from mammary stem cells; this effect was potentiated by adding TQ with MaSCs-derived exosomes.

Gestational Intermittent Hypoxia Enhances Mammary Stem Cells and Alters Tumor Phenotype in Adult Female Offspring.

An adverse perinatal environment can increase long-term cancer risk, although the precise nature of associated perinatal triggers remain unknown. Sleep apnea is a common condition during pregnancy, characterized by recurrent cessations in breathing during sleep, and the potential consequences of sleep apnea during pregnancy as it relates to breast cancer risk in offspring have not been explored. To model sleep apnea, Sprague-Dawley dams were exposed during gestation to nightly intermittent hypoxia (GIH) or normoxia (GNx), and the mammary glands of female offspring were examined. GIH offspring demonstrated increased epithelial stem and progenitor cell populations, which are associated with diminished transforming growth factor beta (TGFß) activity. Elevations in adipose tissue stem cells in the mammary gland were also identified in GIH offspring. In aging females, mammary tumors formed in GIH offspring. These tumors displayed a dramatic increase in stroma compared to tumors from GNx offspring, as well as distinct patterns of expression of stem cell-related pathways. Together, these results suggest that exposure to sleep apnea during pregnancy leads to lasting changes in the mammary glands of female offspring. Increased stem and progenitor cell populations as a result of GIH exposure could enhance long-term breast cancer risk, as well as alter the clinical behavior of resulting breast tumors.

mTOR inhibition abrogates human mammary stem cells and early breast cancer progression markers.

BACKGROUND: Mammary physiology is distinguished in containing adult stem/progenitor cells that are actively amending the breast tissue throughout the reproductive lifespan of women. Despite their importance in both mammary gland development, physiological maintenance, and reproduction, the exact role of mammary stem/progenitor cells in mammary tumorigenesis has not been fully elucidated in humans or animal models. The implications of modulating adult stem/progenitor cells in women could lead to a better understanding of not only their function, but also toward possible breast cancer prevention led us to evaluate the efficacy of rapamycin in reducing mammary stem/progenitor cell activity and malignant progression markers. METHODS: We analyzed a large number of human breast tissues for their basal and luminal cell composition with flow cytometry and their stem and progenitor cell function with sphere formation assay with respect to age and menopausal status in connection with a clinical study (NCT02642094) involving a low-dose (2 mg/day) and short-term (5-7 days) treatment of the mTOR inhibitor sirolimus. The expression of biomarkers in biopsies and surgical breast samples were measured with quantitative analysis of immunohistochemistry. RESULTS: Sirolimus treatment significantly abrogated mammary stem cell activity, particularly in postmenopausal patients. It did not affect the frequency of luminal progenitors but decreased their self-renewal capacity. While sirolimus had no effect on basal cell population, it decreased luminal cell population, particularly in postmenopausal patients. It also significantly diminished prognostic biomarkers associated with breast cancer progression from ductal carcinoma in situ to invasive breast cancer including p16INK4A, COX-2, and Ki67, as well as markers of the senescence-associated secretary phenotype, thereby possibly functioning in preventing early breast cancer progression. CONCLUSION: Overall, these findings indicate a link from mTOR signaling to mammary stem and progenitor cell activity and cancer progression. Trial registration This study involves a clinical trial registered under the ClinicalTrials.gov identifier NCT02642094 registered December 30, 2015.

Research progress of Claudin-low breast cancer.

Claudin-low breast cancer (CLBC) is a subgroup of breast cancer discovered at the molecular level in 2007. Claudin is one of the primary proteins that make up tight junctions, and it plays crucial roles in anti-inflammatory and antitumor responses as well as the maintenance of water and electrolyte balance. Decreased expression of claudin results in the disruption of tight junction structures and the activation of downstream signaling pathways, which can lead to tumor formation. The origin of Claudin-low breast cancer is still in dispute. Claudin-low breast cancer is characterized by low expression of Claudin3, 4, 7, E-cadherin, and HER2 and high expression of Vimentin, Snai 1/2, Twist 1/2, Zeb 1/2, and ALDH1, as well as stem cell characteristics. The clinical onset of claudin-low breast cancer is at menopause age, and its histological grade is higher. This subtype of breast cancer is more likely to spread to lymph nodes than other subtypes. Claudin-low breast cancer is frequently accompanied by increased invasiveness and a poor prognosis. According to a clinical retrospective analysis, claudin-low breast cancer can achieve low pathological complete remission. At present, although several therapeutic targets of claudin-low breast cancer have been identified, the effective treatment remains in basic research stages, and no animal studies or clinical trials have been designed. The origin, molecular biological characteristics, pathological characteristics, treatment, and prognosis of CLBC are extensively discussed in this article. This will contribute to a comprehensive understanding of CLBC and serve as the foundation for the individualization of breast cancer treatment.

Activated breast stromal fibroblasts exhibit myoepithelial and mammary stem cells features.

BACKGROUND: Active breast cancer-associated fibroblasts (CAFs) promote tumor growth and spread, and like tumor cells they are also heterogeneous with various molecular sub-types and different pro-tumorigenic capacities. METHODS: We have used immunoblotting as well as quantitative RT-PCR to assess the expression of various epithelial/mesenchymal as well as stemness markers in breast stromal fibroblasts. Immunofluorescence was utilized to assess the level of different myoepithelial and luminal markers at the cellular level. Flow cytometry allowed to determine the proportion of CD44- and ALDH1-positive breast fibroblasts, while sphere formation assay was used to test the ability of these cells to form mammospheres. RESULTS: We have shown here that IL-6-dependent activation of breast and skin fibroblasts promotes mesenchymal-to-epithelial transition and stemness in a STAT3- and p16-dependent manner. Interestingly, most primary CAFs isolated from breast cancer patients exhibited such transition and expressed lower levels of the mesenchymal markers N-cadherin and vimentin as compared to their adjacent normal fibroblasts (TCFs) isolated from the same patients. We have also shown that some CAFs and IL-6-activated fibroblasts express high levels of the myoepithelial markers cytokeratin 14 and CD10. Interestingly, 12 CAFs isolated from breast tumors showed higher proportions of CD24low/CD44high and ALDHhigh cells, compared to their corresponding TCF cells. These CD44high cells have higher abilities to form mammospheres and to enhance cell proliferation of breast cancer cells in a paracrine manner relative to their corresponding CD44low cells. CONCLUSION: Together, the present findings show novel characteristics of active breast stromal fibroblasts, which exhibit additional myoepithelial/progenitor features.

Understanding, Status, and Therapeutic Potentials of Stem Cells in Goat.

The utility of animal stem cells finds implications in enhancing milk, meat, and fiber production and serving animal models for human diseases. Stem cells are involved in tissue development, growth, and repair, and in regenerative therapy. Caprine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and other tissue-specific adult stem cells (ASCs) have tremendous potential for their use in regenerative medicine. The application of goat ESCs, iPSCs, mammary stem cells (MaSC), mesenchymal stem cells (MSCs), spermatogonial stem cells (SSCs) and others can find their implication in increasing caprine production potential and human disease model. The onset of the disease and therapeutic effects of stem cells of many human diseases like sub-fertility, joint conditions, intervertebral disc defects, osteoarthritis, and chondrogenesis can be well studied in goats. Increasing evidence of MSCs and their secreted factors have drawn the attention of animal scientists in regenerative medicine. This review summarizes a comprehensive overview of research made on caprine stem cells and illustrates some potential applications of stem cells in caprine regenerative medicine and their utility as a model animal in understanding human diseases.

microRNAs transcriptional profiling of mammary stem cells isolated by PKH26 staining and FACS sorting.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small (18-22 nucleotides) noncoding RNAs involved in the regulation of a variety of cellular and developmental processes. MiRNA expression is frequently altered in human cancers compared to normal tissues, potentially contributing to tumorigenesis. Generally, high-throughput profiles of miRNA expression levels are generated using bulk samples, from both normal and cancer tissues. However, cancer tissues are quite heterogeneous and might contain subpopulations critical for tumor development, i.e., cancer stem cells (CSCs) or tumor-initiating cells (TICs) with aberrant stem-like features, such as unlimited self-renewal potential. The isolation of these aberrant subpopulations from solid tumors is a relatively recent achievement, with breast cancer being one of the first solid human cancers in which CSCs have been identified and biologically characterized. Here, we describe a new methodology that can overcome the main challenge in dealing with rare cells such as SCs/CSCs, represented by the paucity of the starting material. Based on previously published protocols, used by both our and other research groups, we used the FACS-sorting approach to isolate mammary normal and cancer stem cells based on the amount of PKH26 fluorescent dye they retained. Depending on the number of SCs/CSCs isolated, we established two different protocols for the reliable and analytically sensitive detection of up to 384 miRNAs using the Taqman Low Density Array (TLDA) platform.

Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492.

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

Asymmetric cell division of mammary stem cells.

Somatic stem cells are distinguished by their capacity to regenerate themselves and also to produce daughter cells that will differentiate. Self-renewal is achieved through the process of asymmetric cell division which helps to sustain tissue morphogenesis as well as maintain homeostasis. Asymmetric cell division results in the development of two daughter cells with different fates after a single mitosis. Only one daughter cell maintains "stemness" while the other differentiates and achieves a non-stem cell fate. Stem cells also have the capacity to undergo symmetric division of cells that results in the development of two daughter cells which are identical. Symmetric division results in the expansion of the stem cell population. Imbalances and deregulations in these processes can result in diseases such as cancer. Adult mammary stem cells (MaSCs) are a group of cells that play a critical role in the expansion of the mammary gland during puberty and any subsequent pregnancies. Furthermore, given the relatively long lifespans and their capability to undergo self-renewal, adult stem cells have been suggested as ideal candidates for transformation events that lead to the development of cancer. With the possibility that MaSCs can act as the source cells for distinct breast cancer types; understanding their regulation is an important field of research. In this review, we discuss asymmetric cell division in breast/mammary stem cells and implications on further research. We focus on the background history of asymmetric cell division, asymmetric cell division monitoring techniques, identified molecular mechanisms of asymmetric stem cell division, and the role asymmetric cell division may play in breast cancer.

Progesterone receptor antagonists reverse stem cell expansion and the paracrine effectors of progesterone action in the mouse mammary gland.

BACKGROUND: The ovarian hormones estrogen and progesterone (EP) are implicated in breast cancer causation. A specific consequence of progesterone exposure is the expansion of the mammary stem cell (MSC) and luminal progenitor (LP) compartments. We hypothesized that this effect, and its molecular facilitators, could be abrogated by progesterone receptor (PR) antagonists administered in a mouse model. METHODS: Ovariectomized FVB mice were randomized to 14 days of treatment: sham, EP, EP + telapristone (EP + TPA), EP + mifepristone (EP + MFP). Mice were then sacrificed, mammary glands harvested, and mammary epithelial cell lineages separated by flow cytometry using cell surface markers. RNA from each lineage was sequenced and differential gene expression was analyzed using DESeq. Quantitative PCR was performed to confirm the candidate genes discovered in RNA seq. ANOVA with Tukey post hoc analysis was performed to compare relative expression. Alternative splicing events were examined using the rMATs multivariate analysis tool. RESULTS: Significant increases in the MSC and luminal mature (LM) cell fractions were observed following EP treatment compared to control (p < 0.01 and p < 0.05, respectively), whereas the LP fraction was significantly reduced (p < 0.05). These hormone-induced effects were reversed upon exposure to TPA and MFP (p < 0.01 for both). Gene Ontology analysis of RNA-sequencing data showed EP-induced enrichment of several pathways, with the largest effect on Wnt signaling in MSC, significantly repressed by PR inhibitors. In LP cells, significant induction of Wnt4 and Rankl, and Wnt pathway intermediates Lrp2 and Axin2 (confirmed by qRTPCR) were reversed by TPA and MFP (p < 0.0001). Downstream signaling intermediates of these pathways (Lrp5, Mmp7) showed similar effects. Expression of markers of epithelial-mesenchymal transition (Cdh1, Cdh3) and the induction of EMT regulators (Zeb1, Zeb2, Gli3, Snai1, and Ptch2) were significantly responsive to progesterone. EP treatment was associated with large-scale alternative splicing events, with an enrichment of motifs associated with Srsf, Esrp, and Rbfox families. Exon skipping was observed in Cdh1, Enah, and Brd4. CONCLUSIONS: PR inhibition reverses known tumorigenic pathways in the mammary gland and suppresses a previously unknown effect of progesterone on RNA splicing events. In total, our results strengthen the case for reconsideration of PR inhibitors for breast cancer prevention.

Wnt signaling and mammary stem cells.

Wnt signaling is an important morphogenetic signaling pathway best known for its essential role in determining embryonic cell fates; it is often activated to re-specify fetal cells or to maintain the lineage flexibility of somatic stem cells. In this review, we consider the role of this pathway in the remarkable process of differentiation, growth and morphogenesis of the mammary gland during embryogenesis, ductal outgrowth and pregnancy. Specifically, mammary stem cells are compared with stem cells from other tissues, to identify commonalities and differences. Wnt signaling is known to be required to maintain the bipotent basal stem cell present in adult mammary ductal trees, however, the absence of this stem cell has little effect on growth or morphogenesis, and Wnt signaling is not induced during the ductal/alveolar expansion during pregnancy. The evidence for pre-determined hierarchies of mammary epithelial cells is reviewed, together with the role of signaling between mixtures of specified mammary epithelial cells in the maintenance of Wnt-dependent clonagenic stem cells. The dazzling variety of Wnt signaling components expressed by mammary epithelial cells is presented, along with some potential stromal sources of Wnt proteins that may be important starting points for the induction of plasticity in the epithelium.

Integrated Single-Cell Transcriptomics and Chromatin Accessibility Analysis Reveals Regulators of Mammary Epithelial Cell Identity.

The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal cells, maintained by a lineage of stem and progenitor populations. Here, we used integrated single-cell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define differentiation states within the secretory type of luminal cells, which forms a continuous spectrum of general luminal progenitor and lactation-committed progenitor cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identify cis- and trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides a resource to reveal cis/trans-regulatory elements associated with MEC identity and differentiation that will serve as a reference to determine how the chromatin accessibility landscape changes during breast cancer.

Integrating single-cell RNA-sequencing and functional assays to decipher mammary cell states and lineage hierarchies.

The identification and molecular characterization of cellular hierarchies in complex tissues is key to understanding both normal cellular homeostasis and tumorigenesis. The mammary epithelium is a heterogeneous tissue consisting of two main cellular compartments, an outer basal layer containing myoepithelial cells and an inner luminal layer consisting of estrogen receptor-negative (ER-) ductal cells and secretory alveolar cells (in the fully functional differentiated tissue) and hormone-responsive estrogen receptor-positive (ER+) cells. Recent publications have used single-cell RNA-sequencing (scRNA-seq) analysis to decipher epithelial cell differentiation hierarchies in human and murine mammary glands, and reported the identification of new cell types and states based on the expression of the luminal progenitor cell marker KIT (c-Kit). These studies allow for comprehensive and unbiased analysis of the different cell types that constitute a heterogeneous tissue. Here we discuss scRNA-seq studies in the context of previous research in which mammary epithelial cell populations were molecularly and functionally characterized, and identified c-Kit+ progenitors and cell states analogous to those reported in the recent scRNA-seq studies.

CXCL12γ induces human prostate and mammary gland development.

BACKGROUND: Epithelial stem cells (ESCs) demonstrate a capacity to maintain normal tissues homeostasis and ESCs with a deregulated behavior can contribute to cancer development. The ability to reprogram normal tissue epithelial cells into prostate or mammary stem-like cells holds great promise to help understand cell of origin and lineage plasticity in prostate and breast cancers in addition to understanding normal gland development. We previously showed that an intracellular chemokine, CXCL12γ induced cancer stem cells and neuroendocrine characteristics in both prostate and breast adenocarcinoma cell lines. However, its role in normal prostate or mammary epithelial cell fate and development remains unknown. Therefore, we sought to elucidate the functional role of CXCL12γ in the regulation of ESCs and tissue development. METHODS: Prostate epithelial cells (PNT2) or mammary epithelial cells (MCF10A) with overexpressed CXCL12γ was characterized by quantitative real-time polymerase chain reaction, Western blots, and immunofluorescence for lineage marker expression, and fluorescence activated cell sorting analyses and sphere formation assays to examine stem cell surface phenotype and function. Xenotransplantation animal models were used to evaluate gland or acini formation in vivo. RESULTS: Overexpression of CXCL12γ promotes the reprogramming of cells with a differentiated luminal phenotype to a nonluminal phenotype in both prostate (PNT2) and mammary (MCF10A) epithelial cells. The CXCL12γ-mediated nonluminal type cells results in an increase of epithelial stem-like phenotype including the subpopulation of EPCAMLo /CD49fHi /CD24Lo /CD44Hi cells capable of sphere formation. Critically, overexpression of CXCL12γ promotes the generation of robust gland-like structures from both prostate and mammary epithelial cells in in vivo xenograft animal models. CONCLUSIONS: CXCL12γ supports the reprogramming of epithelial cells into nonluminal cell-derived stem cells, which facilitates gland development.

Multidimensional Imaging of Mammary Gland Development: A Window Into Breast Form and Function.

An in-depth appreciation of organ form and function relies on the ability to image intact tissues across multiple scales. Difficulties associated with imaging deep within organs, however, can preclude high-resolution multidimensional imaging of live and fixed tissues. This is particularly challenging in the mammary gland, where the epithelium lies deeply encased within a stromal matrix. Recent advances in deep-tissue and live imaging methodologies are increasingly facilitating the visualization of complex cellular structures within their native environment. Alongside, refinements in optical tissue clearing and immunostaining methods are enabling 3D fluorescence imaging of whole organs at unprecedented resolutions. Collectively, these methods are illuminating the dynamic biological processes underlying tissue morphogenesis, homeostasis, and disease. This review provides a snapshot of the current and state-of-the-art multidimensional imaging techniques applied to the postnatal mammary gland, illustrating how these approaches have revealed important new insights into mammary gland ductal development and lactation. Continual evolution of multidimensional image acquisition and analysis methods will undoubtedly offer further insights into mammary gland biology that promises to shed new light on the perturbations leading to breast cancer.

In vivo Mouse Mammary Gland Formation.

For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several in vitro assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for in vivo mammary gland formation assay, including preparation of "cleared" recipients and mammary cells for implantation, the surgery process and how to assess the experimental results. Combined with manipulation of mammary cells via gene editing and /or drug treatment, this protocol could be very useful in the researches of mammary stem cells and mammary development.