Aquaporins in Glandular Secretion.

Exocrine and endocrine glands deliver their secretory product, respectively, at the surface of the target organs or within the bloodstream. The release of their products has been shown to rely on secretory mechanisms often involving aquaporins (AQPs). This chapter will provide insight into the role of AQPs in secretory glands located within the gastrointestinal tract, including salivary glands, gastric glands, duodenal Brunner's glands, liver, gallbladder, intestinal goblets cells, and pancreas, as well and in other parts of the body, including airway submucosal glands, lacrimal glands, mammary glands, and eccrine sweat glands. The involvement of AQPs in both physiological and pathophysiological conditions will also be highlighted.

High-throughput formation and image-based analysis of basal-in mammary organoids in 384-well plates.

This manuscript describes a new method for forming basal-in MCF10A organoids using commercial 384-well ultra-low attachment (ULA) microplates and the development of associated live-cell imaging and automated analysis protocols. The use of a commercial 384-well ULA platform makes this method more broadly accessible than previously reported hanging drop systems and enables in-incubator automated imaging. Therefore, time points can be captured on a more frequent basis to improve tracking of early organoid formation and growth. However, one major challenge of live-cell imaging in multi-well plates is the rapid accumulation of large numbers of images. In this paper, an automated MATLAB script to handle the increased image load is developed. This analysis protocol utilizes morphological image processing to identify cellular structures within each image and quantify their circularity and size. Using this script, time-lapse images of aggregating and non-aggregating culture conditions are analyzed to profile early changes in size and circularity. Moreover, this high-throughput platform is applied to widely screen concentration combinations of Matrigel and epidermal growth factor (EGF) or heparin-binding EGF-like growth factor (HB-EGF) for their impact on organoid formation. These results can serve as a practical resource, guiding future research with basal-in MCF10A organoids.

Mammary basal cells: Stars of the show.

Nearly all mammals rely on lactation to support their young and to ensure the continued survival of their species. Despite its importance, relatively little is known about how milk is produced and how it is ejected from the lumen of mammary alveoli and ducts. This review focuses on the latter. We discuss how a relatively small number of basal cells, wrapping around each alveolar unit, contract to forcibly expel milk from the alveolar lumen. We consider how individual basal cells coordinate their activity, the fate of these cells at the end of lactation and avenues for future deliberation and exploration.

In Vitro Flow Visualization in a Lactating Human Breast Model.

Human milk extraction from the breast is affected by the infant's oral activities. Natural suckling by the infant includes both intraoral vacuum and peripheral oral compression during breastfeeding. However, the contribution of each of these motions to milk extraction at the outlet and at the duct bifurcations is unclear. In this work, we investigated the flow field in a lactating breast model considering bifurcated milk ducts and multiphase breast-infant interactions. A bio-inspired breastfeeding simulator device was utilized to mimic an infant's oral feeding mechanism during breastfeeding and extract the human milk-mimicking Fluid from the transparent and elastic lactating breast phantom during experiments. Using a particle image velocimetry system, we found that the oscillatory flow under vacuum pressure provides a higher velocity field at the outlet compared to that when an infant applies both vacuum and oral compression pressures. Additionally, the intraoral vacuum coordinated with the oral peripheral compression causes stronger vorticities and secondary flows at the adjunction of the bifurcated ducts than the vacuum-only case. Vacuum-only extraction yields an increase in flow velocity at the outlet and could be one of the reasons for nipple pain, whereas infant's oral activities on the breast generated more vortices in the milk duct adjunctions and might cause milk duct clogs. This phenomenon is rationalized based on the validation of a previous in vivo clinical study of milk production compared between commercial pumps and infant suckling. The fact that milk consumption of vacuum-only extraction is less than that of vacuum plus oral compression further explains the effectiveness of applying a natural suckling pattern in human lactation.

Resolving Human Lactation Heterogeneity Using Single Milk-Derived Cells, a Resource at the Ready.

Single cell RNA sequencing (scRNAseq) of human milk-derived cells (HMDCs) makes highly detailed analyses of the biology of human lactation possible. We explore this powerful application as an exciting tool to inspect the cellular composition of human milk. We point out some important challenges unique to this approach and highlight the importance of collaborations between biologists and well-trained bioinformaticians to utilize these data to their maximum potential. We extend this focus by discussing the first two such studies that describe HMDCs via scRNAseq and a variety of important questions in the field that warrant attention through further research. The stage is set to apply scRNAseq in human lactation biology, potentially leading to new insights regarding the molecular and cellular diversity of human secretory mammary epithelial cells.

Characterization of Gene Expression Signatures for the Identification of Cellular Heterogeneity in the Developing Mammary Gland.

The developing mammary gland depends on several transcription-dependent networks to define cellular identities and differentiation trajectories. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the epithelial cellular heterogeneity across the diverse stages of gland maturation. Still, a deeper dive into expanded molecular signatures would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation approaches and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage-defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including adipocytes, fibroblasts and rare immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cells, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development.

WNT4 Balances Development vs Disease in Gynecologic Tissues and Women's Health.

The WNT family of proteins is crucial in numerous developmental pathways and tissue homeostasis. WNT4, in particular, is uniquely implicated in the development of the female phenotype in the fetus, and in the maintenance of müllerian and reproductive tissues. WNT4 dysfunction or dysregulation can drive sex-reversal syndromes, highlighting the key role of WNT4 in sex determination. WNT4 is also critical in gynecologic pathologies later in life, including several cancers, uterine fibroids, endometriosis, and infertility. The role of WNT4 in normal decidualization, implantation, and gestation is being increasingly appreciated, while aberrant activation of WNT4 signaling is being linked both to gynecologic and breast cancers. Notably, single-nucleotide polymorphisms (SNPs) at the WNT4 gene locus are strongly associated with these pathologies and may functionally link estrogen and estrogen receptor signaling to upregulation and activation of WNT4 signaling. Importantly, in each of these developmental and disease states, WNT4 gene expression and downstream WNT4 signaling are regulated and executed by myriad tissue-specific pathways. Here, we review the roles of WNT4 in women's health with a focus on sex development, and gynecologic and breast pathologies, and our understanding of how WNT4 signaling is controlled in these contexts. Defining WNT4 functions provides a unique opportunity to link sex-specific signaling pathways to women's health and disease.

Human mammary epithelial cells in a mature, stratified epithelial layer flatten and stiffen compared to single and confluent cells.

BACKGROUND: The epithelium forms a protective barrier against external biological, chemical and physical insults. So far, AFM-based, micro-mechanical measurements have only been performed on single cells and confluent cells, but not yet on cells in mature layers. METHODS: Using a combination of atomic force, fluorescence and confocal microscopy, we determined the changes in stiffness, morphology and actin distribution of human mammary epithelial cells (HMECs) as they transition from single cells to confluency to a mature layer. RESULTS: Single HMECs have a tall, round (planoconvex) morphology, have actin stress fibers at the base, have diffuse cortical actin, and have a stiffness of 1 kPa. Confluent HMECs start to become flatter, basal actin stress fibers start to disappear, and actin accumulates laterally where cells abut. Overall stiffness is still 1 kPa with two-fold higher stiffness in the abutting regions. As HMECs mature and form multilayered structures, cells on apical surfaces become flatter (apically more level), wider, and seven times stiffer (mean, 7 kPa) than single and confluent cells. The main drivers of these changes are actin filaments, as cells show strong actin accumulation in the regions where cells adjoin, and in the apical regions. CONCLUSIONS: HMECs stiffen, flatten and redistribute actin upon transiting from single cells to mature, confluent layers. GENERAL SIGNIFICANCE: Our findings advance the understanding of breast ductal morphogenesis and mechanical homeostasis.

Integrin-mediated adhesion and mechanosensing in the mammary gland.

The mammary gland is dynamically remodelled during its postnatal development and the reproductive cycles. This inherent plasticity has been suggested to increase the susceptibility of the organ to carcinogenesis. Morphological changes in the mammary epithelium involve cell proliferation, differentiation, apoptosis, and migration which, in turn, are affected by cell adhesion to the extracellular matrix (ECM). Integrin adhesion receptors function in the sensing of the biochemical composition, patterning and mechanical properties of the ECM surrounding the cells, and strongly influence cell fate. This review aims to summarize the existing literature on how different aspects of integrin-mediated adhesion and mechanosensing, including ECM composition; stiffness and topography; integrin expression patterns; focal adhesion assembly; dynamic regulation of the actin cytoskeleton; and nuclear mechanotransduction affect mammary gland development, function and homeostasis. As the mechanical properties of a complex tissue environment are challenging to replicate in vitro, emphasis has been placed on studies conducted in vivo or using organoid models. Outright, these studies indicate that mechanosensing also contributes to the regulation of mammary gland morphogenesis in multiple ways.

In vitro and in vivo evaluation of anti-tumoral effect of M1 phenotype induction in macrophages by miR-130 and miR-33 containing exosomes.

In the tumor microenvironment, macrophages polarize into the M2 phenotype to facilitate tumorigenesis. Tumor-derived exosomes can act as mediators between the tumor microenvironment and stromal cells by transporting proteins, mRNAs, and miRNAs. Exosomal miRNAs play a pivotal role in modulating tumor microenvironment and macrophage polarization. Here, we overexpressed miR-130 and miR-33 in exosomes of MDA-MB-231 cells and investigated their effect on macrophage polarization and tumor progression. For this purpose, exosomes were extracted from MDA-MB-231 cells and characterized using dynamic light scattering, electron microscopy, and western blotting of exosomal markers. Then, miR-130 or miR-33 containing exosomes were used to treat IL4-induced M2 or tumor-associated macrophages (TAMs). After treatment, the polarization status of macrophages, including the expression of M1 specific genes, and the secretion of cytokines were evaluated. Finally, the conditioned medium from exosome-treated macrophages was incubated with cancer cells to evaluate its effect on the migration and invasion ability of cancer cells and, in vivo experiments investigated the effect of exosome-treated macrophages on breast cancer progression. Exosomes characterization results approved the range of size and homogeneity of extracted exosomes. Overexpression of miR-130 and miR-33 in exosomes increased the expression of M1 signature genes (IRF5, MCP1, CD80) and secretion of cytokines (IL-1ß and TNF-α) as well as yeast phagocytic activity of macrophages. Besides, the conditioned medium of macrophages treated with miRNA containing exosomes declined the migration and invasion ability of cancer cells. The in vivo results indicated the inhibitory effect of exosome-treated macrophages on tumor growth. Furthermore, the results showed that in response to exosome-treated macrophages, the production of TNF-α by spleen cells increased, while the production of IL-10 and TGF-ß by these cells decreased. These findings suggest that overexpression of miR-130 and miR-33 in exosomes can decrease tumor progression by shifting macrophage polarization from M2 to M1 phenotype and can be a potential therapeutic strategy for tumor interventions.

The effects of papaya leaf juice for breastfeeding and working mothers on increasing prolactin hormone levels and infant's weight in Tangerang

Based on preliminary studies, it is known that 80% of working mothers fail to make exclusive breastfeeding, on average babies are given formula milk 2-4 months old and stop breastfeeding at 6-18 months of age. Papaya leaves are one of the galactagogues that contain quercetin which can activate the hormone prolactin and help increase breastmilk. The purpose of this study was to determine the effect of giving papaya leaf juice for nursing mothers who work toward increasing levels of the hormone prolactin and infant weight in Tangerang. The research design used was a pre-experimental model with one group pretest-posttest design. Samples taken as many as 10 mothers who have babies aged 0-6 months who have difficulty in breastfeeding because the amount of breastmilk was a little. The study was conducted in several clinics in Tangerang. The intervention was giving papaya leaf juice for 7 consecutive days. The results obtained from this study are as follows: 90% of mothers aged between 20 and 35 years, 70% of mothers have a history of spontaneous labor and have no complications at the time of delivery, 40% of mother's education is junior high school (SMP), and 70% of mothers are primiparas. The average increase in the amount of prolactin after the intervention was 19.59ng/ml, while the average weight gain of the newborn after the intervention was 165g. Wilcoxon test results for increased levels of the hormone prolactin p-value of 0.047<0.05 and for an increase in body weight of newborns p-value of 0.009<0.05. CONCLUSION: There is an effect of giving papaya leaf juice for nursing mothers who work on increasing levels of the hormone prolactin and infant weight in Tangerang. Papaya leaf juice can be a galactagogue for mothers who experience problems with a small amount of breastmilk

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Amino acid transportation, sensing and signal transduction in the mammary gland: key molecular signalling pathways in the regulation of milk synthesis.

The mammary gland, a unique exocrine organ, is responsible for milk synthesis in mammals. Neonatal growth and health are predominantly determined by quality and quantity of milk production. Amino acids are crucial maternal nutrients that are the building blocks for milk protein and are potential energy sources for neonates. Recent advances made regarding the mammary gland further demonstrate that some functional amino acids also regulate milk protein and fat synthesis through distinct intracellular and extracellular pathways. In the present study, we discuss recent advances in the role of amino acids (especially branched-chain amino acids, methionine, arginine and lysine) in the regulation of milk synthesis. The present review also addresses the crucial questions of how amino acids are transported, sensed and transduced in the mammary gland.

Function of ion transporters in maintaining acid-base homeostasis of the mammary gland and the pathophysiological role in breast cancer.

The incidence of breast cancer is increasing year by year, and the pathogenesis is still unclear. Studies have shown that the high metabolism of solid tumors leads to an increase in hypoxia, glycolysis, production of lactic acid and carbonic acid, and extracellular acidification; a harsh microenvironment; and ultimately to tumor cell death. Approximately 50% of locally advanced breast cancers exhibit hypoxia and/or local hypoxia, and acid-base regulatory proteins play an important role in regulating milk secretion and maintaining mammary gland physiological function. Therefore, ion transporters have gradually become a hot topic in mammary gland and breast cancer research. This review focuses on the research progress of ion transporters in mammary glands and breast cancer. We hope to provide new targets for the treatment and prognosis of breast cancer.

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.

Unraveling the Breast: Advances in Mammary Biology and Cancer Methods.

The field of mammary gland biology and breast cancer research encompasses a wide range of topics and scientific questions, which span domains of molecular, cell and developmental biology, cancer research, and veterinary and human medicine, with interdisciplinary overlaps to non-biological domains. Accordingly, mammary gland and breast cancer researchers employ a wide range of molecular biology methods, in vitro techniques, in vivo approaches as well as in silico analyses. The list of techniques is ever-expanding; together with the refinement of established, staple techniques in the field, new technologies keep emerging thanks to technological advances and scientific creativity. This issue of the Journal of Mammary Gland Biology and Neoplasia represents a compilation of original articles and reviews focused on methods used in mammary gland biology and breast cancer research.

Morphological, hormonal, and molecular changes in different maternal tissues during lactation and post-lactation.

Milk supply and quality during lactation are critical for progeny survival. Maternal tissues and metabolism, influenced by hormonal changes, undergo modification during lactation to sustain breastfeeding. Two organs that suffer essential adjustment are the mammary glands and the bone; however, renal calcium conservation and calcium absorption from the intestine are also modified. Lactation leads to a transient loss of bone minerals to provide adequate amounts of minerals, including calcium for milk production. Physiological, metabolic, and molecular changes in different tissues participate in providing nutrients for milk production. After weaning, the histological, metabolic, and hormonal modifications that take place in lactation are reverted, and bone remineralization is a central function at this time. This study focuses on the hormonal, metabolic, molecular, and tissue modifications that occur in mammary glands, bone, intestine, and kidneys in the mother during lactation and post-weaning periods.

Mammary stem cells, where art thou?

Tremendous progress has been made in the field of stem cell biology. This is in part due to the emergence of various vertebrate organs, including the mammary gland, as an amenable model system for adult stem cell studies and remarkable technical advances in single cell technology and modern genetic lineage tracing. In the current review, we summarize the recent progress in mammary gland stem cell biology at both the adult and embryonic stages. We discuss current challenges and controversies, and potentially new and exciting directions for future research. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

Distinct oncogenes drive different genome and epigenome alterations in human mammary epithelial cells.

Molecular subtypes of breast cancer are defined on the basis of gene expression and genomic/epigenetic pattern differences. Different subtypes are thought to originate from distinct cell lineages, but the early activation of an oncogene could also play a role. It is difficult to discriminate the respective inputs of oncogene activation or cell type of origin. In this work, we wished to determine whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. To this aim, we transduced shp53 immortalized HMECs in parallel with the CCNE1, WNT1 and RASv12 oncogenes which activate distinct oncogenic pathways and characterized them at sequential stages of transformation for changes in their genetic and epigenetic profiles. We show that initial activation of CCNE1, WNT1 and RASv12, in shp53 HMECs results in different and reproducible changes in mRNA and micro-RNA expression, copy number alterations (CNA) and DNA methylation profiles. Noticeably, HMECs transformed by RAS bore very specific profiles of CNAs and DNA methylation, clearly distinct from those shown by CCNE1 and WNT1 transformed HMECs. Genes impacted by CNAs and CpG methylation in the RAS and the CCNE1/WNT1 clusters showed clear differences, illustrating the activation of distinct pathways. Our data show that early activation of distinct oncogenic pathways leads to active adaptive events resulting in specific sets of CNAs and DNA methylation changes. We, thus, propose that activation of different oncogenes could have a role in reshaping the genetic landscape of breast cancer subtypes.