A Unifying Theory of Branching Morphogenesis.

The morphogenesis of branched organs remains a subject of abiding interest. Although much is known about the underlying signaling pathways, it remains unclear how macroscopic features of branched organs, including their size, network topology, and spatial patterning, are encoded. Here, we show that, in mouse mammary gland, kidney, and human prostate, these features can be explained quantitatively within a single unifying framework of branching and annihilating random walks. Based on quantitative analyses of large-scale organ reconstructions and proliferation kinetics measurements, we propose that morphogenesis follows from the proliferative activity of equipotent tips that stochastically branch and randomly explore their environment but compete neutrally for space, becoming proliferatively inactive when in proximity with neighboring ducts. These results show that complex branched epithelial structures develop as a self-organized process, reliant upon a strikingly simple but generic rule, without recourse to a rigid and deterministic sequence of genetically programmed events.

Adjustment of dendritic cells to the breast-cancer microenvironment is subset specific.

The functions and transcriptional profiles of dendritic cells (DCs) result from the interplay between ontogeny and tissue imprinting. How tumors shape human DCs is unknown. Here we used RNA-based next-generation sequencing to systematically analyze the transcriptomes of plasmacytoid pre-DCs (pDCs), cell populations enriched for type 1 conventional DCs (cDC1s), type 2 conventional DCs (cDC2s), CD14+ DCs and monocytes-macrophages from human primary luminal breast cancer (LBC) and triple-negative breast cancer (TNBC). By comparing tumor tissue with non-invaded tissue from the same patient, we found that 85% of the genes upregulated in DCs in LBC were specific to each DC subset. However, all DC subsets in TNBC commonly showed enrichment for the interferon pathway, but those in LBC did not. Finally, we defined transcriptional signatures specific for tumor DC subsets with a prognostic effect on their respective breast-cancer subtype. We conclude that the adjustment of DCs to the tumor microenvironment is subset specific and can be used to predict disease outcome. Our work also provides a resource for the identification of potential targets and biomarkers that might improve antitumor therapies.

SnapShot: Branching Morphogenesis.

Ectodermal appendages such as feathers, hair, mammary glands, salivary glands, and sweat glands form branches, allowing much-increased surface for functional differentiation and secretion. Here, the principles of branching morphogenesis are exemplified by the mammary gland and feathers.

Epigenetic control of adult stem cell function.

Mammalian embryonic development is a tightly regulated process that, from a single zygote, produces a large number of cell types with hugely divergent functions. Distinct cellular differentiation programmes are facilitated by tight transcriptional and epigenetic regulation. However, the contribution of epigenetic regulation to tissue homeostasis after the completion of development is less well understood. In this Review, we explore the effects of epigenetic dysregulation on adult stem cell function. We conclude that, depending on the tissue type and the epigenetic regulator affected, the consequences range from negligible to stem cell malfunction and disruption of tissue homeostasis, which may predispose to diseases such as cancer.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Stem Cells and the Differentiation Hierarchy in Mammary Gland Development.

The mammary gland is a highly dynamic organ that undergoes profound changes within its epithelium during puberty and the reproductive cycle. These changes are fueled by dedicated stem and progenitor cells. Both short- and long-lived lineage-restricted progenitors have been identified in adult tissue as well as a small pool of multipotent mammary stem cells (MaSCs), reflecting intrinsic complexity within the epithelial hierarchy. While unipotent progenitor cells predominantly execute day-to-day homeostasis and postnatal morphogenesis during puberty and pregnancy, multipotent MaSCs have been implicated in coordinating alveologenesis and long-term ductal maintenance. Nonetheless, the multipotency of stem cells in the adult remains controversial. The advent of large-scale single-cell molecular profiling has revealed striking changes in the gene expression landscape through ontogeny and the presence of transient intermediate populations. An increasing number of lineage cell-fate determination factors and potential niche regulators have now been mapped along the hierarchy, with many implicated in breast carcinogenesis. The emerging diversity among stem and progenitor populations of the mammary epithelium is likely to underpin the heterogeneity that characterizes breast cancer.

Expanding the evo-devo toolkit: generation of 3D mammary tissue from diverse mammals.

The varying pathways of mammary gland development across species and evolutionary history are underexplored, largely due to a lack of model systems. Recent progress in organoid technology holds the promise of enabling in-depth studies of the developmental adaptations that have occurred throughout the evolution of different species, fostering beneficial phenotypes. The practical application of this technology for mammary glands has been mostly confined to rodents and humans. In the current study, we have successfully created next-generation 3D mammary gland organoids from eight eutherian mammals and the first branched organoid of a marsupial mammary gland. Using mammary organoids, we identified a role for ROCK protein in regulating branching morphogenesis, a role that manifests differently in organoids from different mammals. This finding demonstrates the utility of the 3D organoid model for understanding the evolution and adaptations of signaling pathways. These achievements highlight the potential for organoid models to expand our understanding of mammary gland biology and evolution, and their potential utility in studies of lactation or breast cancer.

Slug and Sox9 cooperatively determine the mammary stem cell state.

Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.

The epithelial polarity axis controls the resting membrane potential and Cl- co-transport in breast glandular structures.

The membrane potential (MP) controls cell homeostasis by directing molecule transport and gene expression. How the MP is set upon epithelial differentiation is unknown. Given that tissue architecture also controls homeostasis, we investigated the relationship between basoapical polarity and resting MP in three-dimensional culture of the HMT-3522 breast cancer progression. A microelectrode technique to measure MP and input resistance reveals that the MP is raised by gap junction intercellular communication (GJIC), which directs tight-junction mediated apical polarity, and is decreased by the Na+/K+/2Cl- (NKCC, encoded by SLC12A1 and SLC12A2) co-transporter, active in multicellular structures displaying basal polarity. In the tumor counterpart, the MP is reduced. Cancer cells display diminished GJIC and do not respond to furosemide, implying loss of NKCC activity. Induced differentiation of cancer cells into basally polarized multicellular structures restores widespread GJIC and NKCC responses, but these structures display the lowest MP. The absence of apical polarity, necessary for cancer onset, in the non-neoplastic epithelium is also associated with the lowest MP under active Cl- transport. We propose that the loss of apical polarity in the breast epithelium destabilizes cellular homeostasis in part by lowering the MP.

Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1+/- tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER)+ , HER2+ , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1+/- tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8+ T cells characterized triple-negative and HER2+ cancers but not ER+ tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER+ tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.

The immune environment of the mammary gland fluctuates during post-lactational regression and correlates with tumour growth rate.

Post-lactational mammary gland regression encompasses extensive programmed cell death and removal of milk-producing epithelial cells, breakdown of extracellular matrix components and redifferentiation of stromal adipocytes. This highly regulated involution process is associated with a transient increased risk of breast cancer in women. Using a syngeneic tumour model, we show that tumour growth is significantly altered depending on the stage of involution at which tumour cells are implanted. Tumour cells injected at day 3 involution grew faster than those in nulliparous mice, whereas tumours initiated at day 6 involution grew significantly slower. These differences in tumour progression correlate with distinct changes in innate immune cells, in particular among F4/80-expressing macrophages and among TCRδ+ unconventional T cells. Breast cancer post-pregnancy risk is exacerbated in older first-time mothers and, in our model, initial tumour growth is moderately faster in aged mice compared with young mice. Our results have implications for breast cancer risk and the use of anti-inflammatory therapeutics for postpartum breast cancers.

Bisphenol A replacement chemicals, BPF and BPS, induce protumorigenic changes in human mammary gland organoid morphology and proteome.

SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.

Using Organoids to Tap Mammary Gland Diversity for Novel Insight.

This article offers a comprehensive perspective on the transformative role of organoid technology on mammary gland biology research across a diverse array of mammalian species.The mammary gland's unique development and regenerative capabilities render this organ an ideal model for studying developmental evolution, stem cell behavior, and regenerative processes. The discussion extends to the use of cross-species mammary organoids to address key biological inquiries in evolution, tissue regeneration, cancer research, and lactation, highlighting the limitations of traditional mouse models and the benefits of incorporating a more diverse range of animal models.Advances in organoid biology have been critical in overcoming ethical and practical constraints of in-vivo studies, especially in human research. The generation of human and mouse mammary organoids that faithfully recapitulate in-vivo tissues marks a significant stride in this field. Parallel capabilities are now emerging for other mammals, as well.Utilizing mammary organoids from various species has the potential to make invaluable contributions to our understanding of mammary gland biology, with implications for regenerative medicine, cancer research, and lactation studies, thereby contributing to advancements in human health, agriculture, and nutrition science.

Toward Characterizing Lymphatic Vasculature in the Mammary Gland During Normal Development and Tumor-Associated Remodeling.

Lymphatic vasculature has been shown to promote metastatic spread of breast cancer. Lymphatic vasculature, which is made up of larger collecting vessels and smaller capillaries, has specialized cell junctions that facilitate cell intravasation. Normally, these junctions are designed to collect immune cells and other cellular components for immune surveillance by lymph nodes, but they are also utilized by cancer cells to facilitate metastasis. Although lymphatic development overall in the body has been well-characterized, there has been little focus on how the lymphatic network changes in the mammary gland during stages of remodeling such as pregnancy, lactation, and postpartum involution. In this review, we aim to define the currently known lymphangiogenic factors and lymphatic remodeling events during mammary gland morphogenesis. Furthermore, we juxtapose mammary gland pubertal development and postpartum involution to show similarities of pro-lymphangiogenic signaling as well as other molecular signals for epithelial cell survival that are critical in these morphogenic stages. The similar mechanisms include involvement of M2-polarized macrophages that contribute to matrix remodeling and vasculogenesis; signal transducer and activator of transcription (STAT) survival and proliferation signaling; and cyclooxygenase 2 (COX2)/Prostaglandin E2 (PGE2) signaling to promote ductal and lymphatic expansion. Investigation and characterization of lymphangiogenesis in the normal mammary gland can provide insight to targetable mechanisms for lymphangiogenesis and lymphatic spread of tumor cells in breast cancer.

Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells.

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed  cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

A novel preclinical model of the normal human breast.

Improved screening and treatment have decreased breast cancer mortality, although incidence continues to rise. Women at increased risk of breast cancer can be offered risk reducing treatments, such as tamoxifen, but this has not been shown to reduce breast cancer mortality. New, more efficacious, risk-reducing agents are needed. The identification of novel candidates for prevention is hampered by a lack of good preclinical models. Current patient derived in vitro and in vivo models cannot fully recapitulate the complexities of the human tissue, lacking human extracellular matrix, stroma, and immune cells, all of which are known to influence therapy response. Here we describe a normal breast explant model utilising a tuneable hydrogel which maintains epithelial proliferation, hormone receptor expression, and residency of T cells and macrophages over 7 days. Unlike other organotypic tissue cultures which are often limited by hyper-proliferation, loss of hormone signalling, and short treatment windows (< 48h), our model shows that tissue remains viable over 7 days with none of these early changes. This offers a powerful and unique opportunity to model the normal breast and study changes in response to various risk factors, such as breast density and hormone exposure. Further validation of the model, using samples from patients undergoing preventive therapies, will hopefully confirm this to be a valuable tool, allowing us to test novel agents for breast cancer risk reduction preclinically.

A role for fibroblast-derived SASP factors in the activation of pyroptotic cell death in mammary epithelial cells.

In normal tissue homeostasis, bidirectional communication between different cell types can shape numerous biological outcomes. Many studies have documented instances of reciprocal communication between fibroblasts and cancer cells that functionally change cancer cell behavior. However, less is known about how these heterotypic interactions shape epithelial cell function in the absence of oncogenic transformation. Furthermore, fibroblasts are prone to undergo senescence, which is typified by an irreversible cell cycle arrest. Senescent fibroblasts are also known to secrete various cytokines into the extracellular space; a phenomenon that is termed the senescence-associated secretory phenotype (SASP). While the role of fibroblast-derived SASP factors on cancer cells has been well studied, the impact of these factors on normal epithelial cells remains poorly understood. We discovered that treatment of normal mammary epithelial cells with conditioned media from senescent fibroblasts (SASP CM) results in a caspase-dependent cell death. This capacity of SASP CM to cause cell death is maintained across multiple senescence-inducing stimuli. However, the activation of oncogenic signaling in mammary epithelial cells mitigates the ability of SASP CM to induce cell death. Despite the reliance of this cell death on caspase activation, we discovered that SASP CM does not cause cell death by the extrinsic or intrinsic apoptotic pathway. Instead, these cells die by an NLRP3, caspase-1, and gasdermin D-dependent induction of pyroptosis. Taken together, our findings reveal that senescent fibroblasts can cause pyroptosis in neighboring mammary epithelial cells, which has implications for therapeutic strategies that perturb the behavior of senescent cells.