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.

Alveolar cells in the mammary gland: lineage commitment and cell death.

The mammary gland provides a spectacular example of physiological cell death whereby the cells that produce milk during lactation are removed swiftly, efficiently, and without inducing inflammation upon the cessation of lactation. The milk-producing cells arise primarily during pregnancy and comprise the alveolar lineage that is specified by signalling pathways and factors that are activated in response to pregnancy hormones. There are at least two alveolar sub-lineages, one of which is marked by the presence of binucleate cells that are especially susceptible to programmed cell death during involution. This process of post-lactational regression, or involution, is carefully orchestrated and occurs in two phases, the first results in a rapid switch in cell fate with the secretory epithelial cells becoming phagocytes whereupon they destroy dead and dying cells from milk. This reversible phase is followed by the second phase that is marked by an influx of immune cells and a remodelling of the gland to replace the alveolar cells with re-differentiated adipocytes, resulting in a return to the pre-pregnant state in preparation for any subsequent pregnancies. The mouse mammary gland provides an excellent experimental tool with which to investigate lineage commitment and the mechanisms of programmed cell death that occur in a normal physiological process. Importantly, involution has highlighted a role for lysoptosis, a mechanism of cell death that is mediated by lysosomal cathepsins and their endogenous inhibitors, serpins. In this review, I discuss alveolar lineage commitment during pregnancy and the programmed cell death pathways that destroy these cells during involution.

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.

Gpr125 is a unifying hallmark of multiple mammary progenitors coupled to tumor latency.

Gpr125 is an orphan G-protein coupled receptor, with homology to cell adhesion and axonal guidance factors, that is implicated in planar polarity and control of cell movements. By lineage tracing we demonstrate that Gpr125 is a highly specific marker of bipotent mammary stem cells in the embryo and of multiple long-lived unipotent basal mammary progenitors in perinatal and postnatal glands. Nipple-proximal Gpr125+ cells express a transcriptomic profile indicative of chemo-repulsion and cell movement, whereas Gpr125+ cells concentrated at invasive ductal tips display a hybrid epithelial-mesenchymal phenotype and are equipped to bind chemokine and growth factors and secrete a promigratory matrix. Gpr125 progenitors acquire bipotency in the context of transplantation and cancer and are greatly expanded and massed at the pushing margins of short latency MMTV-Wnt1 tumors. High Gpr125 expression identifies patients with particularly poor outcome within the basal breast cancer subtype highlighting its potential utility as a factor to stratify risk.

In conversation with Christine Watson.

Christine J. Watson is Professor of Cell and Cancer Biology at the University of Cambridge. Christine obtained her Bachelor's (honors) degree in Biochemistry at the University of Glasgow in 1975 and, after a soujourn in Glauco Tocchini-Valentini's lab at the Institute of Cell Biology, Consiglio Nazionale delle Ricerche in Rome, she undertook a PhD in Molecular Genetics at Imperial College London. During her PhD, she looked at differences in gene expression between differentiated and undifferentiated embryonal carcinoma stem cells, inspiring an early interest in gene expression and cell fate determination. Between 1986 and 1992, Christine undertook three postdoctoral research positions that took her from London back to Scotland, where she was first introduced to mammary gland biology through her work with John Clark at the Roslin Institute in Edinburgh. During her time in the Clark lab, Christine identified a factor - later shown to be STAT5 - that binds to the promoter of the milk protein gene ß-lactoglobulin. This prompted further work identifying the key role played by the STAT family of transcription factors in mammary gland development. Shortly afterwards, Christine became a group leader at the Roslin Institute and later relocated to the University of Edinburgh to collaborate with Andrew Wyllie. This led to her recruitment to the University of Cambridge in 1998, where she has remained to date. Over the last two decades, the Watson lab has focused on elucidating the mechanisms underlying lineage commitment of mammary stem and progenitor cells and the regulation of cell death in involuting mammary gland. In this interview, Christine discusses her research highlights and provides a glimpse into her personal interests, as she moves towards retirement.

The ever-expanding landscape of cancer therapeutic approaches.

Cancer is a leading cause of death and a major health problem worldwide, particularly in more developed countries. There is, therefore, an urgent clinical need to develop more effective therapies to treat cancer and metastatic disease. In this Editorial, the content of The FEBS Journal's Special Issue on Cancer Therapeutics is outlined. The interesting collection of recent articles in this issue covers a wide repertoire of cancer therapeutic approaches. While some of the articles discuss broad-spectrum applications such as immunotherapy and oncolytic virus therapy, others focus on a particular type of cancer or a signalling pathway that has gone awry such as aberrant Ca2+ signalling, glycosylation or pre-mRNA processing. Finally, an article featured in this issue reviews our current understanding of how cancer cells can become dormant, often for decades, and which pathways reactivate these cells to cause relapse. I am sure there is something for everyone in this issue.

How should we define mammary stem cells?

Mammary stem cells (MaSCs) have been defined by cell surface marker expression and their ability to repopulate a cleared fat pad, a capacity now known to result from reprogramming upon transplantation. Furthermore, lineage-tracing studies have provoked controversy as to whether MaSCs are unipotent or bi/multipotent. Various innovative experimental approaches, including single-cell RNA sequencing (scRNA-Seq), epigenetic analyses, deep tissue and live imaging, and advanced mouse models, have provided new and unexpected insights into stem and progenitor cells; thus, it is now timely to reappraise our concept of the MaSC hierarchy. Here, I highlight misconceptions, suggest definitions of stem and progenitor cells, and propose a way forward in our search for an understanding of MaSCs.

Mammary development in the embryo and adult: new insights into the journey of morphogenesis and commitment.

The mammary gland is a unique tissue and the defining feature of the class Mammalia. It is a late-evolving epidermal appendage that has the primary function of providing nutrition for the young, although recent studies have highlighted additional benefits of milk including the provision of passive immunity and a microbiome and, in humans, the psychosocial benefits of breastfeeding. In this Review, we outline the various stages of mammary gland development in the mouse, with a particular focus on lineage specification and the new insights that have been gained by the application of recent technological advances in imaging in both real-time and three-dimensions, and in single cell RNA sequencing. These studies have revealed the complexity of subpopulations of cells that contribute to the mammary stem and progenitor cell hierarchy and we suggest a new terminology to distinguish these cells.

Dynamic architectural interplay between leucocytes and mammary epithelial cells.

The adult mammary gland undergoes dynamic changes during puberty and the postnatal developmental cycle. The mammary epithelium is composed of a bilayer of outer basal, or myoepithelial, cells and inner luminal cells, the latter lineage giving rise to the milk-producing alveolar cells during pregnancy. These luminal alveolar cells undergo Stat3-mediated programmed cell death following the cessation of lactation. It is established that immune cells in the microenvironment of the gland have a role to play both in the ductal outgrowth during puberty and in the removal of dead cells and remodelling of the stroma during the process of postlactational regression. However, most studies have focussed on the role of the stromal immune cell compartment or have quantified immune cell populations in tissue extracts. Our recent development of protocols for deep imaging of the mammary gland in three dimensions (3D) has enabled the architectural relationship between immune cells and the epithelium to be examined in detail, and we have discovered a surprisingly dynamic relationship between the basal epithelium and leucocytes. Furthermore, we have observed morphological changes in the myoepithelial cells, as involution progresses, which were not revealed by previous work in 2D tissue sections and whole tissue. This dynamic architecture suggests a role for myoepithelial cells in the orderly progression of involution. We conclude that deep imaging of mammary gland and other tissues is essential for analysing complex interactions between cellular compartments.

Tumour cell invasiveness and response to chemotherapeutics in adipocyte invested 3D engineered anisotropic collagen scaffolds.

Breast cancers are highly heterogeneous and their metastatic potential and response to therapeutic drugs is difficult to predict. A tool that could accurately gauge tumour invasiveness and drug response would provide a valuable addition to the oncologist's arsenal. We have developed a 3-dimensional (3D) culture model that recapitulates the stromal environment of breast cancers by generating anisotropic (directional) collagen scaffolds seeded with adipocytes and culturing tumour fragments therein. Analysis of tumour cell invasion in the presence of various therapeutic drugs, by immunofluorescence microscopy coupled with an optical clearing technique, demonstrated the utility of this approach in determining both the rate and capacity of tumour cells to migrate through the stroma while shedding light also on the mode of migration. Furthermore, the response of different murine mammary tumour types to chemotherapeutic drugs could be readily quantified.

Neutral lineage tracing of proliferative embryonic and adult mammary stem/progenitor cells.

Mammary gland development occurs over multiple phases, beginning in the mammalian embryo and continuing throughout reproductive life. The remarkable morphogenetic capacity of the mammary gland at each stage of development is attributed to the activities of distinct populations of mammary stem cells (MaSCs) and progenitor cells. However, the relationship between embryonic and adult MaSCs, and their fate during different waves of mammary gland morphogenesis, remains unclear. By employing a neutral, low-density genetic labelling strategy, we characterised the contribution of proliferative stem/progenitor cells to embryonic, pubertal and reproductive mammary gland development. Our findings further support a model of lineage restriction of MaSCs in the postnatal mammary gland, and highlight extensive redundancy and heterogeneity within the adult stem/progenitor cell pool. Furthermore, our data suggest extensive multiplicity in their foetal precursors that give rise to the primordial mammary epithelium before birth. In addition, using a single-cell labelling approach, we revealed the extraordinary capacity of a single embryonic MaSC to contribute to postnatal ductal development. Together, these findings provide tantalising new insights into the disparate and stage-specific contribution of distinct stem/progenitor cells to mammary gland development.

The Multifaceted Role of STAT3 in Mammary Gland Involution and Breast Cancer.

Since seminal descriptions of signal transducer and activator of transcription 3 (STAT3) as a signal transducer and transcriptional regulator, which is most usually activated by phosphorylation of a specific tyrosine residue, a staggering wealth of research has delineated the key role of this transcription factor as a mediator of mammary gland postlactational regression (involution), and paradoxically, a pro-survival factor in breast cancer and some breast cancer cell lines. STAT3 is a critical regulator of lysosomal-mediated programmed cell death (LM-PCD) during mammary gland involution, where uptake of milk fat globules, and consequent high levels of free fatty acids, cause permeabilisation of lysosomal vesicle membranes, in turn leading to cathepsin protease leakage and cell death. A recent proteomic screen of STAT3-induced changes in lysosomal membrane protein components has highlighted wide-ranging effects of STAT3, which may coordinate LM-PCD via the stimulation of endocytosis, intracellular trafficking, and lysosome biogenesis. In parallel, STAT3 regulates the acute phase response during the first phase of involution, and it contributes to shaping the pro-tumourigenic 'wound healing' signature of the gland during the second phase of this process. STAT3 activation during involution is important across species, although some differences exist in the progression of involution in dairy cows. In breast cancer, a number of upstream regulators can lead to STAT3 activation and the effects of phosphorylation of STAT3 are equally wide-ranging. Recent studies have implicated microRNAs in some regulatory pathways. In this review, we will examine the multifaceted role of STAT3 in mammary gland involution and tumourigenesis, incorporating a review of these fundamental processes in tandem with a discussion of recent developments in this field.

Sinus-like dilatations of the mammary milk ducts, Ki67 expression, and CD3-positive T lymphocyte infiltration, in the mammary gland of wild European rabbits during pregnancy and lactation.

Sinus-like dilatations of the mammary duct are recognisable in the mammary gland of pregnant and lactating wild European rabbits. These dilatations exhibit a bilaminar epithelial lining, with luminal epithelial cells expressing basal and lateral E-cadherin. Occasional binucleated mammary epithelial cells are present in the luminal layer. Underlying the luminal epithelial cells is a basal layer of cytokeratin 14-positive cells, supported by a thin layer of fibrous tissue. Multi-segmental epithelial proliferation, as indicated by Ki67 expression, is apparent in the luminal epithelial cells, suggesting a capacity for division during pregnancy and lactation. CD3-positive T lymphocytes are present both intraepithelially, suggesting exocytosis, and in foci subjacent to the ductular epithelium. We consider that sinus-like dilatations of the mammary duct may have the potential to give rise to a subset of the mammary gland neoplasms classified as ductal in origin. Milk accumulation in these sinus-like dilatations is likely to provide a niche for bacterial replication in cases of mastitis in rabbits. These structures are an important component of the innate immune system of the mammary gland, both as a physical barrier and as an interface between the milk and mammary immune cells.

The Mammary Microenvironment in Mastitis in Humans, Dairy Ruminants, Rabbits and Rodents: A One Health Focus.

The One Health concept promotes integrated evaluation of human, animal, and environmental health questions to expedite advances benefiting all species. A recognition of the multi-species impact of mastitis as a painful condition with welfare implications leads us to suggest that mastitis is an ideal target for a One Health approach. In this review, we will evaluate the role of the mammary microenvironment in mastitis in humans, ruminants and rabbits, where appropriate also drawing on studies utilising laboratory animal models. We will examine subclinical mastitis, clinical lactational mastitis, and involution-associated, or dry period, mastitis, highlighting important anatomical and immunological species differences. We will synthesise knowledge gained across different species, comparing and contrasting disease presentation. Subclinical mastitis (SCM) is characterised by elevated Na/K ratio, and increased milk IL-8 concentrations. SCM affecting the breastfeeding mother may result in modulation of infant mucosal immune system development, whilst in ruminants notable milk production losses may ensue. In the case of clinical lactational mastitis, we will focus on mastitis caused by Staphylococcus aureus and Escherichia coli. Understanding of the pathogenesis of involution-associated mastitis requires characterization of the structural and molecular changes occurring during involution and we will review these changes across species. We speculate that milk accumulation may act as a nidus for infection, and that the involution 'wound healing phenotype' may render the tissue susceptible to bacterial infection. We will discuss the impact of concurrent pregnancy and a 'parallel pregnancy and involution signature' during bovine mammary involution.

An Engineered Human Adipose/Collagen Model for In Vitro Breast Cancer Cell Migration Studies.

Adipocytes are one of the major stromal cell components of the human breast. These cells play a key role in the development of the gland and are implicated in breast tumorigenesis. Frequently, directional stromal collagen I fibers are found surrounding aggressive breast tumors. These fibers enhance breast cancer cell migration and are associated with poor patient prognosis. We sought to recapitulate these stromal components in vitro to provide a three-dimensional (3D) model comprising human adipose tissue and anisotropic collagen fibers. We developed a human mesenchymal stem cell (hMSC) cell line capable of undergoing differentiation into mature adipocytes by immortalizing hMSCs, isolated from breast reduction mammoplasties, through retroviral transduction. These immortalized hMSCs were seeded in engineered collagen I scaffolds with directional internal architecture, and adipogenesis was chemically induced, resulting in human adipose tissue being synthesized in vitro in an architectural structure associated with breast tumorigenesis. Subsequently, fluorescently labeled cells from an established breast cancer cell line were seeded into this model, cocultured for 7 days and imaged using multiphoton microscopy. Enhanced breast cancer cell migration was observed in the adipose-containing model over empty scaffold controls, demonstrating an adipocyte-mediated influence on breast cancer cell migration. Thus, this 3D in vitro model recapitulates the migratory effects of adipocytes observed on breast cancer cells and suggests that it could have utility with fresh breast tumor biopsies as an assay for cancer therapeutic efficacy in personalized medicine strategies.

Stat3-mediated alterations in lysosomal membrane protein composition.

Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome.

Mammary Stem Cells: Premise, Properties, and Perspectives.

Adult mammary stem cells (MaSCs) drive postnatal organogenesis and remodeling in the mammary gland, and their longevity and potential have important implications for breast cancer. However, despite intense investigation the identity, location, and differentiation potential of MaSCs remain subject to deliberation. The application of genetic lineage-tracing models, combined with quantitative 3D imaging and biophysical methods, has provided new insights into the mammary epithelial hierarchy that challenge classical definitions of MaSC potency and behaviors. We review here recent advances - discussing fundamental unresolved properties of MaSC potency, dynamics, and plasticity - and point to evolving technologies that promise to shed new light on this intractable debate. Elucidation of the physiological mammary differentiation hierarchy is paramount to understanding the complex heterogeneous breast cancer landscape.

Development of three-dimensional collagen scaffolds with controlled architecture for cell migration studies using breast cancer cell lines.

Cancer is characterized by cell heterogeneity and the development of 3D in vitro assays that can distinguish more invasive or migratory phenotypes could enhance diagnosis or drug discovery. 3D collagen scaffolds have been used to develop analogues of complex tissues in vitro and are suited to routine biochemical and immunological assays. We sought to increase 3D model tractability and modulate the migration rate of seeded cells using an ice-templating technique to create either directional/anisotropic or non-directional/isotropic porous architectures within cross-linked collagen scaffolds. Anisotropic scaffolds supported the enhanced migration of an invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. In addition, MDA-MB-468 showed increased migration upon epithelial-to-mesenchymal transition (EMT) in anisotropic scaffolds. The provision of controlled architecture in this system may act both to increase assay robustness and as a tuneable parameter to capture detection of a migrated population within a set time, with consequences for primary tumour migration analysis. The separation of invasive clones from a cancer biomass with in vitro platforms could enhance drug development and diagnosis testing by contributing assay metrics including migration rate, as well as modelling cell-cell and cell-matrix interaction in a system compatible with routine histopathological testing.

Analysis of the Involuting Mouse Mammary Gland: An In Vivo Model for Cell Death.

Involution of the mammary gland occurs at the end of every period of lactation and is an essential process to return the gland to a pre-pregnant state in readiness for the next pregnancy. Involution is a complex process of regulated alveolar cell death coupled with tissue remodeling and requires exquisite control of transcription and signaling. These processes can be investigated using a variety of molecular and morphological approaches.In this chapter we describe how to initiate involution and collect mammary glands, measure involution morphologically, and quantify lysosomal leakiness in mammary tissue and in cultured mammary epithelial cells. These procedures encompass a range of microscopy and molecular biology techniques.

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.

BACKGROUND: High-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated. METHODS: Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed. RESULTS: Varying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue. CONCLUSIONS: The utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development.