Normal Mammary Gland Research Articles

Progesterone and breast

Progesterone (Pg) is a pregnancy-related hormone that prepares the endometrium for the implantation of the fertilized zygote and suppresses myometrial contractility for the maintenance of pregnancy. At high concentrations, it acts as a natural immunosuppressant avoiding the rejection of a half allogeneic foetus. It is the precursor of many other related steroid hormones, but what is its role in the human breast? In this chapter, we will discuss some aspects related to Pg and its role in breast development and in the neoplastic disease. Understanding the mechanisms related to Pg-induced effects in the normal and neoplastic mammary gland will light the way to exploit this hormone signalling pathway therapeutically. We will introduce some aspects of the effects of progestins in normal breast development, breast cancer risk and in neoplastic growth, and we will describe ongoing clinical trials in breast cancer using progestins or antiprogestins.

Follicle Stimulating Hormone Receptor Expression In Histologically Normal And Neoplastic Canine Mammary Gland Tissues

The roles of Follicle Stimulating Hormone (FSH) in reproductive physiology have been recognized for a long time. The hormone achieves its functions by binding to the Follicle Stimulating Hormone Receptor (FSHRc) that is strongly expressed in both male and female gonads. Over the last 30 years FSH and its receptor have been discovered also in normal and neoplastic extragonadal tissues including: placenta, uterus, and most recently mammary glands. In neoplasias they contribute to cancer development, cancer cell migration, invasion and neovascularization. Canine mammary gland tumors (MGTs) are the most frequent tumors in intact bitches. Though frequently studied, their pathogenesis is still not completely understood and the presence of FSHRc has not been investigated. The purpose of this study was to evaluate histologically normal and neoplastic mammary glands for the presence of FSHRc using immunohistochemistry (IHC) and in situ hybridization (ISH). Positive immunohistochemical cytoplasmic staining is noticed in epithelial and the underlying myoepithelial cells of normal and neoplastic mammary glands and also in the cytoplasm of chondrocytes in mixed MGTs. In situ hybridization detected the expression of FSHRc in the nucleus and perinuclear cytoplasm of epithelial and myoepithelial cells of normal and neoplastic mammary glands. The signal was observed in the cytoplasm and, to a lesser degree, in the nucleus of chondrocytes in mixed MGTs. Further studies are necessary to elucidate the physiological and pathophysiological roles of FSH and FSHRc in histologically and neoplastic mammary glands and to investigate their potential use as targets for highly specific chemo‐ or immunotherapies of MGTs.

Monolayers of bovine milk‐derived stem cells present with distinctive multinucleated syncytial cells following self‐induced differentiation.

In an ongoing series of studies, our laboratory has been examining the biology of a putative stem cell population harvested from fresh bovine milk samples. Among the observations previously reported have been the long‐term survival of ductal‐like monolayer colonies which clearly undergo differentiation and generate several cell types consistent with the normal mammary gland. These include cells which produce large fat globules as well as supporting cells. In addition, after several month of maintenance, these colonies eventually generate localized 3D tissue features representative of mammary gland ducts and acinar structures. Now we are reporting on the development within these cultures of a subset of multinucleated syncytial cells. These cells appear to form in specific regional distributions and in smaller colonies are often spaced out relative to each other suggestive of a regulatory function in tissue feature development. In these studies, we have examined the relative development of milk ductal syncytial cells and sought evidence of a regulatory role using morphologic analysis and labeling studies. In addition, we are also examining the potential to artificially induce such syncytial cell formation using the cAMP activator forskolin. Preliminary experiments in this regard have focused on the effects of forskolin on the ductal cell line MCF10A. To date, results suggest that forskolin will in fact increase the incidence of syncytial cells in these monolayers. It remains to be examined whether this will also occur in our milk samples and then whether these syncytia are functionally equivalent to those already spontaneously occurring. While formation of syncytial cells is a common observation in breast ductal cancers and precancerous tissues, there is no indication in our bovine milk samples of any neoplastic transformation and the position, developmental time‐frame and relatively ubiquitous observation of these in every sample suggest a potentially functional regulatory role for these cells.

Characterization of Mammary Cells Co‐expressing Separate Lineage Markers

Epithelial cell hierarchy is a highly disputed topic in mammary gland biology. Studying the differentiation potential among cell types within the mammary gland is crucial for understanding normal development and would inform studies in tumorigenesis on a molecular and cellular level. Our recent study uncovers alterations occurring in the aging mouse mammary gland by utilizing single‐cell RNA‐sequencing to study the transcriptomic changes that occur between young and aged mice. From this data, we identified a cluster of luminal cells that expressed canonical lineage markers for two cell types, hormone‐sensing (HS) and alveolar (AV) cells, and that these HS‐AV cells decrease in abundance with age. The co‐expression of these markers contests the presently accepted epithelial hierarchy, as the HS and AV cells are currently thought to be maintained by separate pools of unipotent progenitor cells. Here, we characterized these murine HS‐AV cells by their gene expression pattern consisting of canonical HS and AV markers and confirmed their presence using immunofluorescence staining. In these stainings, we used two sets of canonical HS and AV markers to show co‐localization of both types in single cells, thus uncovering their histological localization within the luminal layer. We further quantified the abundance of these cells and confirmed the population’s decrease with age. Additionally, we have examined human tissues for the presence of HS‐AV cells and have confirmed their existence in the human mammary gland. Future multi‐variate analysis on a larger set of human samples is needed to determine the correlation of HS‐AV cell abundance with age in human mammary glands, as confounding factors such as history of pregnancy, family history, or environmental factors makes it challenging to exclusively analyze the age‐dependent effect on the cell type’s population size. Finally, the lineage relationship of HS‐AV cells to HS and AV cells remains unclear. We are performing functional experiments on isolated HS‐AV cells in order to study their differentiation lineages in vitro as organoid cultures. Unmasking the function of these cells within the normal mammary gland could provide significant insight into the study of the mammary epithelial differentiation hierarchy. A better understanding of this previously less well understood cell type may improve our fundamental knowledge of mammary cell states during normal physiology. Given that the mammary gland is a major organ for cancer development, a more detailed understanding of its cell states may also inform mechanisms of disease development.Support or Funding InformationSusan G. Komen Postdoctoral Fellowship, Croucher Postdoctoral Fellowship (CML), NIH F31 Fellowship (GKG), Hope Funds Postdoctoral Fellowship, Marsha Rivkin Center for Ovarian Cancer Research (YO), Chan Zuckerberg Initiative Donor‐Advised Fund, Silicon Valley Community Foundation, NHGRI Career Development Award (LP), Susan G. Komen Breast Cancer Foundation, and Breast Cancer Research Foundation (JSB).

R-spondin-mediated WNT signaling potentiation in mammary and breast cancer development.

The mammary gland is a secretory organ, which develops as a network of growing epithelial ducts composed of luminal and basal cells that invade the surrounding adipose tissue through a series of developmental cycles. Mammary stem cells (MaSCs) maintain an accurate tissue homeostasis, and their proliferation and cell fate determination are regulated by multiple hormones and local factors. The WNT pathway plays a critical role in controlling the enormous tissue expansion and remodeling during mammary gland development through the maintenance and differentiation of MaSCs, and its deregulation has been implicated in breast cancer (BC) initiation and progression. The R-spondins (RSPOs) are four secreted proteins that strongly enhance target cell sensitivity to WNT ligands. Moreover, leucine-rich repeat-containing G-protein-coupled receptors (LGRs) 4-6 are considered obligate high-affinity receptors for RSPOs and have been described as stem cell markers. Importantly, elevated RSPO expression has been recently identified in several tumor types from patients, including BC, and it has been reported that they play a significant role in mammary tumor progression in experimental models. In this review, exploring our present knowledge, we summarize the role of the RSPO-LGR axis as a WNT-enhancing signaling cascade in the MaSC compartment and during the normal and neoplastic mammary gland development. In addition, we include an updated expression profile of the RSPOs and their action mediators at the cell membrane, the LGRs, and the ubiquitin-ligases ZNRF3/RNF43, in different BC subtypes. Finally and based on these data, we discuss the significance of tumor-associated alterations of these proteins and their potential use as molecular targets for detection and treatment of BC.

The Presence and Distribution of TRPM7 in the Canine Mammary Glands.

Simple SummaryMammary gland tumors are one of the major causes of mortality in dogs. It is, therefore, imperative to clarify the nature of this disease. Transient receptor potential melastatin-subfamily member 7 (TRPM7), a bifunctional ion channel found in human cells, plays a role in normal physiological processes such as cell development, survival, proliferation, differentiation, and migration. However, TRPM7 is also active in several types of cancers in humans, and blocking the expression of this pathway leads to a decrease in the proliferation, migration, and invasion of cancer cells. We have proved the presence of TRPM7 in healthy canine mammary tissues, which could help further studies that aim to establish the relationship between TRPM7 and its physiological and pathophysiological effects in the canine mammary glands and, possibly, tumors.The transient receptor potential melastatin-subfamily member 7 (TRPM7) cation channel is a bifunctional ion channel with intrinsic kinase activity and is ubiquitously expressed in the animal/human body. Accumulated knowledge of TRPM7 suggests that it plays an essential role in normal physiological processes, including the development, survival, proliferation, differentiation, and migration of cells. The aim of this study was to demonstrate the presence and expression patterns of TRPM7 in normal canine mammary glands using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Normal mammary gland tissue samples were obtained from five female beagle dogs. RT-PCR and sequencing of the amplified PCR products demonstrated the presence of TRPM7 mRNA in normal mammary glands, and the presence of TRPM7 protein was confirmed by Western blotting. Immunohistochemical investigations demonstrated the expression of TRPM7 in the apical membrane of acinar and ductal epithelial cells in the canine mammary glands. These results provide the first evidence of the presence and distribution of TRPM7 in the canine mammary gland and could help explain the physiological and pathological roles of TRPM7 in the canine mammary gland; however, additional studies are required to elucidate these roles.

GPR30 expression and function in breast cancer cells are induced through a cis‑acting element targeted by ETS factors.

The capacity that G protein‑coupled receptor30 (GPR30) has demonstrated for triggering estrogen‑dependent signaling pathways has attracted the interest of breast cancer researchers; however, the reported expression profiles and functions of GPR30 in breast cancer are inconsistent. The main purpose of the present investigation was to identify transcriptional mechanisms underlying the expression of GPR30 that allow a better understanding of its role in breast cancer progression. In the cell lines used as different polarity models in the present study, it was determined immunologically that GPR30 is expressed in normal mammary gland cells and that this expression decreased considerably during breast cancer development, where cell identity is lost. However, it was also determined that, in spite of low GPR30 expression levels in breast cancer cells with little differentiation, this membrane estrogen receptor (ER) is able to increase cell viability and suppress migration in cells that have acquired metastatic capacity. In addition, through transient expression assays in breast cancer cells, it was revealed that a transcriptional mechanism dependent on protein kinase A and susceptible to retinoic acid in ER‑positive cells induces GPR30 expression through a cis‑regulatory element for E26 transformation‑specific transcription factors, located between ‑631 and ‑625bp from the GPR30 translation start codon. Overall, these results suggested that invitro transcriptional regulation of GPR30 expression in breast cancer cells may serve a relevant role in the conservation of an epithelial phenotype, and also may be important to avoid the transition to metastasis.

Abstract B102: Epithelium-derived cytokines in breast carcinogenesis

Abstract Novel approaches in cancer immunotherapy have led to effective therapeutics against late-stage cancers. However, the role of the immune system in protection against early-stage cancers remains uncertain. We have previously discovered that thymic stromal lymphopoietin (TSLP) blocks early stages of breast cancer development through activation of CD4+ T cells. TSLP is an epithelium-derived danger signal that plays a key role in initiating an immune response upon cell damage and stress. The aim of this study is to investigate the potential involvement of other epithelial-derived cytokines in the activation of an immune response against premalignant breast lesions. To identify these signals, we analyzed the expression of a wide range of epithelium-derived cytokines in normal mammary glands, premalignant lesions, and late-stage tumors of MMTV-PyMT (PyMTtg) and K14-Cre, Brca1f/f, p53f/f mouse models. These mice spontaneously develop breast tumors that resemble human luminal and basal-like breast cancers, respectively. Of the cytokines examined, IL-1a was highly expressed in premalignant lesions compared to mammary glands and tumors. Additionally, IL-12p40 was upregulated in both early and late stages of breast cancer compared to normal mammary gland. Other cytokines, like IL-7, IL-11, and IL-24, were highly expressed in the mammary gland, but their expression was lost during breast tumorigenesis. In conclusion, we identified upregulation of IL-1a and IL-12p40 in early-stage breast cancer, suggesting a role for these cytokines in inducing antitumor immunity in the breast. Furthermore, we demonstrate selection against expression of IL-7, IL-11, and IL-24 during the process of cancer initiation and promotion, suggesting a role in normal mammary gland homeostasis and possibly immunity against breast carcinogenesis. Taken together, we have identified potential novel therapeutic targets that can be leveraged for breast cancer immunoprevention. Citation Format: Sanne Kroon, Kaitlin Dempsey, Margherita Boieri, Shawn Demehri. Epithelium-derived cytokines in breast carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B102.

FGF13 promotes metastasis of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes, due to the high propensity to develop distant metastases. Hence, new molecular targets for therapeutic intervention are needed for TNBC. We recently conducted a rigorous phenotypic and genomic characterization of four isogenic populations of MDA-MB-231 human triple-negative breast cancer cells that possess a range of intrinsic spontaneous metastatic capacities in vivo, ranging from nonmetastatic (MDA-MB-231_ATCC) to highly metastatic to lung, liver, spleen and spine (MDA-MB-231_HM). Gene expression profiling of primary tumours by RNA-Seq identified the fibroblast growth factor homologous factor, FGF13, as highly upregulated in aggressively metastatic MDA-MB-231_HM tumours. Clinically, higher FGF13 mRNA expression was associated with significantly worse relapse free survival in both luminal A and basal-like human breast cancers but was not associated with other clinical variables and was not upregulated in primary tumours relative to normal mammary gland. Stable FGF13 depletion restricted in vitro colony forming ability in MDA-MB-231_HM TNBC cells but not in oestrogen receptor (ER)-positive MCF-7 or MDA-MB-361 cells. However, despite augmenting MDA-MB-231_HM cell migration and invasion in vitro, FGF13 suppression almost completely blocked the spontaneous metastasis of MDA-MB-231_HM orthotopic xenografts to both lung and liver while having negligible impact on primary tumour growth. Together, these data indicate that FGF13 may represent a therapeutic target for blocking metastatic outgrowth of certain TNBCs. Further evaluation of the roles of individual FGF13 protein isoforms in progression of the different subtypes of breast cancer is warranted.

Abstract P1-02-09: Diagnostic system of breast ultrasound images using Convolutional Neural Network

Abstract Mammography and ultrasound are widely used for breast cancer diagnosis. In 2003, the American Society of Radiological Surgery created “Breast Imaging Reporting and Data System (BI-RADS)” to standardize the terms in breast ultrasonic diagnosis. Although BI-RADS categorization of ultrasound is globally used, quality consistency remains an urgent task in breast ultrasound diagnosis. Although there are such quality control problems, the importance of ultrasonic examination in breast cancer diagnosis is increasing. "Comparative Exam for Verifying the Effectiveness of Ultrasound in Breast Cancer Screening (Japan Strategic Anti-cancer Randomized Trial: J-START )”, a randomized controlled trial, showed that breast screening by combined application of mammography and ultrasound was significantly better in detecting early breast cancer compared to mammography alone among women in their 40s. Despite some argument about the increase of false positive cases, breast screening is becoming more important and popular in the countermeasure-type screening settings in Japan. Moreover, the education of doctors and technicians responsible for ultrasonographic screening and improvement of examination accuracy is an urgent matter. Since the accuracy of the breast ultrasound examination depends on the equipment, environment, technicians and doctors, it is necessary to solve the problem in quality control. Therefore, we are collaborating with a company which specializes in Artificial Intelligence (AI) to develop a “quality-controlled” breast ultrasonographic diagnosis system by using AI technology. The system can detect the presence of a lesion and make diagnosis real-time during the ultrasonic examination. This system uses a technology called Convolutional Neural Network, which is frequently used in image analysis by AI. We selected ultrasonographic images for machine learning from patients who underwent ultrasonographic screening between January 2016 and December 2018 in Keio University Hospital, Japan. All patients who were diagnosed as malignant underwent biopsy and were diagnosed histologically. The patients who were diagnosed as benign either underwent biopsy or a half-year or more observation. Trained physicians added annotations to each selected image. Annotation was added on 901 images, of which 630 images (138 images of normal mammary gland, 242 images of benign tumor, 250 images of malignant tumor) were used as training data. We tested the system by using 271 images(70 images of normal mammary gland, 97 images of benign tumors, 104 images of malignant tumors) as test data. As a result, sensitivity of mass (benign or malignant) detection among normal breast was 91.8%, and specificity was 91.4%. The sensitivity in the detection of malignant lesion among masses was 86.2%, and the specificity was 88.4%. In conclusion, we succeeded to develop a real time working, quality-controlled, breast ultrasonographic diagnosis system using artificial intelligence. We plan to improve both sensitivity and specificity by adding more learning data for clinical application in near future. Citation Format: Masayuki Kikuchi, Tetsu Hayashida, Rurina Watanuki, Ayako Nakashoji, Yuko Kawai, Aiko Nagayama, Tomoko Seki, Maiko Takahashi, Yuko Kitagawa. Diagnostic system of breast ultrasound images using Convolutional Neural Network [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-02-09.

Abstract P3-02-10: The possible association among breast cancer, diabetes mellitus and GLP-1 receptor

Abstract Introduction: Diabetes mellitus (DM) has been generally considered one of the risk factors of breast cancer (BC) development. Glucagon-like-peptide-1 (GLP-1) has been reported to be associated with increased risks of cancer development in some human malignancies. Koehler et.al.,reported that GLP-1 analogues could be associated with intestinal tumor growth through increased Fibroblast Growth Factor 7 (FGF7). However, the involvement of GLP-1 has not been studied in BC. Therefore, in this study, we explored the possible association of GLP-1 with BC risk in DM patients, by comparing GLP-1 receptor (GLP-1R), FGF7 and Fibroblast Growth Factor Receptor2 (FGFR2) status of BC between DM and non-DM BC patients. Method: We immunolocalized GLP-1R, FGFR2 and FGF7, the ligand of FGFR2 in 125 BC cases with DM and 57 without DM (NDM), operated from 2005 to 2013 and 2017 at Nahanishi Clinic and Tohoku University Hospital, Japan, excluding those who received neoadjuvant chemotherapy. Result: The status of GLP-1R was significantly more frequent in BC tissue (Ca) than in normal mammary gland tissue (NCa) regardless of DM (DM Ca 67.2% vs NCa 19.5% P<0.001, NDM Ca 51.7% vs NCa 16.4% P<0.001). That of GLP-1R in Ca of the DM group was significantly more frequent than in the NDM group (DM 67.2% vs NDM 51.7% P=0.044). That of FGFR2 was significantly more frequent in Ca than NCa of DM group (Ca 92% vs NCa 59.3% P<0.01), although no significant difference detected in NDM group (Ca 82.8% vs NCa 74.5% P=0.29). There were no significant differences of FGFR2 status in BC between the DM and NDM groups (DM 92% vs NDM 82.8% P=0.062). H score of FGF7 tended to be higher in Ca than NCa in the DM group (Ca 176.5 vs NCa 158.5 P=0.058) but this was not detected in the NDM group (Ca 166.3 vs NCa 161.1 P=0.7). The status of FGF7 in Ca was not different between the DM and NDM groups (DM 176.5 vs NDM 166.3 P=0.37). That of FGF7 in Ca harboring GLP-1R was significantly higher than Ca without GLP-1R regardless of DM (DM GLP-1R+ 189.4 vs GLP-1R- 150 P<0.01, NDM GLP-1R+ 185.5 vs GLP-1R- 146.4 P<0.01) with the same findings obtained in NCa tissue (DM GLP-1R+ 237.8 vs GLP-1R- 137.8 P<0.001, NDM GLP-1R+ 228.8 vs GLP-1R- 149.3 P=0.032). Conclusion: Results of our present study did demonstrate that GLP-1R was more frequently detected in BC tissue than in normal mammary gland tissue with more pronounced in DM patients. These results also indicated an association between GLP1 and FGF7 in the development of BC in DM patients. Citation Format: Naoko Takigami, Shimpei Kuniyoshi, Yasuhiko Miki, Kentaro Tamaki, Yoshihiko Kamada, Kano Uehara, Seiko Tsuchiya, Shigeharu Terukina, Takanori Ishida, Minoru Miyashita, Ayako Kanai, Keely May McNamara, Nobumitsu Tamaki, Hironobu Sasano. The possible association among breast cancer, diabetes mellitus and GLP-1 receptor [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-10.

Abstract BL1: Deciphering stem and progenitor cells to understand breast cancer

Abstract Breast cancer is a highly heterogeneous disease at the molecular and pathological levels. To understand heterogeneity and potential ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial differentiation hierarchy as well as the cellular composition of breast tumors. Discrete populations of mouse and human mammary epithelial cells have been isolated on the basis of cell surface marker expression, yielding three primary populations including a stem cell-enriched population and two luminal populations. Their responsiveness to female steroid hormones was investigated, revealing the importance of paracrine regulation of ER/PR– stem/progenitor cells by ER/PR+ luminal ‘hormone sensor’ cells. RANKL was found to be a key paracrine effector of progesterone signaling, underscoring the central role that female hormones play in normal mammary gland homeostasis and cancer. Consistent with this observation, a RANK+ luminal progenitor cell was found to be the likely target cell that gives rise to breast cancer in BRCA1 mutation carriers. Pre-clinical studies further indicate that it may be possible to target this ‘culprit’ cell with the RANKL inhibitor denosumab. This finding is now being tested in a randomized phase III prevention study, ‘BRCA-P’. In further studies, a quiescent stem cell that remains hormone responsive has been identified. Lineage tracing, combined with high resolution 3D imaging (visualizing large regions of intact tissue), was carried out to track stem and progenitor cells in situ. These studies have revealed the presence of long-lived stem cells that replenish mammary epithelium, while unipotent progenitors appear to play an essential role in sustaining the independent lineages on a day-to-day basis. In order to generate a comprehensive single cell expression resource and explore molecular heterogeneity in the mouse mammary gland, single cell profiling at key developmental stages was performed. This has revealed novel intermediates and unexpected changes in the transcriptional landscape that could inform a role for distinct cell types during mammary oncogenesis. To gain insight into cancer heterogeneity and clonal dynamics at cellular resolution, 3D imaging was combined with lineage tracing to track specific cells during cancer development. Through the application of a novel pipeline, the epithelial-to-mesenchymal transition (EMT) was found to be a frequent event, thus highlighting the inherent plasticity of mammary tumors. These findings are currently being extended to human tumors to further understand inter- and intra-tumoral heterogeneity. In parallel, our group has established a bank of patient-derived xenograft (PDX) and patient derived organoid (PDO) models to facilitate the transfer of laboratory findings to the clinic. These serve as useful pre-clinical models to test promising agents. Initial efforts have focused on BH3 mimetics, a new class of drug that targets the BCL-2 family of pro-survival proteins. This work is leading to early phase clinical trials, currently focused on patients with metastatic ER+ breast cancer. Citation Format: JE Visvader, GJ Lindeman. Deciphering stem and progenitor cells to understand breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr BL1.

Abstract P6-03-04: Microenvironment-induced epigenetic reprogramming imposes drug-tolerant states in breast cancer cells

Abstract Development of resistance to breast cancer (BC) therapy is frustratingly common. Multiple mechanisms of resistance have been identified including selection of genetic resistance, redundant signaling, and others. More recently, rare cancer cells were shown to be sporadically reprogrammed by the presence of anti-cancer drugs into transiently resistant states, then to expand later after cessation of therapy. We have demonstrated that the physical character and composition of tumor microenvironment (ME) components are determinants of BC cell drug resistance, however, the mechanism by which this phenomenon occurs is unclear. We hypothesize that epigenetic reprogramming is not rare or sporadic, indeed, that specific tumor ME components impose epigenetic states that beget the expression of gene clusters that put cancer cells into drug-tolerant states. Our current pharmacopeia targets cancer cells, not their ME. Some tumor ME components, e.g. extracellular matrices (ECM), have half-lives that span years, and growth factors with typically short half-lives can be stabilized by direct association with ECM. Thus, a ME-imposed drug tolerance mechanism would explain how resistance can arise rapidly, in multiple locations, without the need for genetic selection. Because ME-cell interactions lead to activation of specific pathways, based on canonical receptor-ligand models, we should be able to circumvent ME-imposed drug tolerance by addition of molecules that alter ME-induced signaling. Clusters of genes that are activated in multidrug resistant breast cancer cells identified potential markers of drug tolerance. ME-drivers of the drug tolerance markers were identified in high-throughput cell-based screens with the MicroEnvironment MicroArray (MEMA) platform. BC cells were exposed in parallel to over 1000 unique combinatorial MEs, which were chosen to mimic normal mammary gland, and luminal or basal BC tumor MEs. Single-cell resolution analysis revealed MEs that induce and maintain drug-tolerance markers. Longer-term functional experiments validated that the identified MEs also increased BC cells drug tolerance. Chromatin accessibility profiling with ATAC-sequencing demonstrated that BC cells cultured in those drug-tolerance inducing MEs exhibit chromatin opening in promoter regions of genes linked to epithelial plasticity (e.g. Axl, SNAI2, Vimentin, TWIST1) within 24h of exposure. These results support the concept that specific MEs drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance. Citation Format: Tiina A Jokela, Dustin Schones, Mark A LaBarge. Microenvironment-induced epigenetic reprogramming imposes drug-tolerant states in breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-04.

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

SummaryThe link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.

Postmenopausal Dense Breasts Maintain Premenopausal Levels of GH and Insulin-like Growth Factor Binding Proteins in Vivo.

Dense breast tissue is associated with 4 to 6 times higher risk of breast cancer by poorly understood mechanisms. No preventive therapy for this high-risk group is available. After menopause, breast density decreases due to involution of the mammary gland. In dense breast tissue, this process is haltered by undetermined biological actions. Growth hormone (GH) and insulin-like binding proteins (IGFBPs) play major roles in normal mammary gland development, but their roles in maintaining breast density are unknown. To reveal in vivo levels of GH, IGFBPs, and other pro-tumorigenic proteins in the extracellular microenvironment in breast cancer, in normal breast tissue with various breast density in postmenopausal women, and premenopausal breasts. We also sought to determine possible correlations between these determinants. Microdialysis was used to collect extracellular in vivo proteins intratumorally from breast cancers before surgery and from normal human breast tissue from premenopausal women and postmenopausal women with mammographic dense or nondense breasts. Estrogen receptor positive breast cancers exhibited increased extracellular GH (P < .01). Dense breasts of postmenopausal women exhibited similar levels of GH as premenopausal breasts and significantly higher levels than in nondense breasts (P < .001). Similar results were found for IGFBP-1, -2, -3, and -7 (P < .01) and for IGFBP-6 (P <.05). Strong positive correlations were revealed between GH and IGFBPs and pro-tumorigenic matrix metalloproteinases, urokinase-type plasminogen activator, Interleukin 6, Interleukin 8, and vascular endothelial growth factor in normal breast tissue. GH pathways may be targetable for cancer prevention therapeutics in postmenopausal women with dense breast tissue.

The sensory innervation of the human nipple

Nipples represent a highly specialized skin with capital importance in mammals for breastfeeding and additionally in humans due to sexuality. The histological studies regarding this region are scarce, so 42 human nipples were studied to describe the morphology of the nipple innervation. Our results exclude the presence of a rich innervation on nipple’s skin or superficial dermis, thus definitely excluding nipple skin from the concept glabrous skin. The presence of mechanoreceptors is limited to scarce Merkel cells on the epidermis and some corpuscular capsulated and non-capsulated structures in the dermis; Merkel cells progressively decrease with ageing. No Meissner corpuscles were found and the rare Pacinian corpuscles identified were close to vascular structures and embroidered in the mammary fatty tissue. The great sensitivity observed functionally on the breast and especially in the nipple can be morphologically explained by two elements; on the one hand there is a rich smooth muscle innervation present in the deep dermis; on the other hand the mammary gland demonstrate Piezo2 expression in many glandular cells, with two differentiated patterns in the ductal and in the acinar tissue of the breast. The role of Piezo2 in the normal mammary gland is discussed.

A new pipeline for pathophysiological analysis of the mammary gland based on organoid transplantation and organ clearing.

ABSTRACTRecent developments in techniques for tissue clearing and size reduction have enabled optical imaging of whole organs and the study of rare tumorigenic events in vivo. The adult mammary gland provides a unique model for investigating physiological or pathological processes such as morphogenesis or epithelial cell dissemination. Here, we establish a new pipeline to study rare cellular events occurring in the mammary gland, by combining orthotopic transplantation of mammary organoids with the uDISCO organ size reduction and clearing method. This strategy allows us to analyze the behavior of individually labeled cells in regenerated mammary gland. As a proof of concept, we analyzed the localization of rare epithelial cells overexpressing atypical protein kinase C iota (also known as PRKCI, referred to here as aPKCι) with an N-terminal eGFP fusion (GFP-aPKCι+) in the normal mammary gland. Using this analytical pipeline, we were able to visualize epithelial aPKCι+ cells escaping from the normal mammary epithelium and disseminating into the surrounding stroma. This technical resource should benefit mammary development and tumor progression studies.

Neoadjuvant Chemotherapy Shifts Breast Tumor Microbiota Populations to Regulate Drug Responsiveness and the Development of Metastasis.

Breast tumors have their own specific microbiota, distinct from normal mammary gland tissue. Patients with breast cancer that present with locally advanced disease often undergo neoadjuvant chemotherapy to reduce tumor size prior to surgery to allow breast conservation or limit axillary lymph node dissection. The purpose of our study was to evaluate whether neoadjuvant chemotherapy modulates the tumor microbiome and the potential impact of microbes on breast cancer signaling. Using snap-frozen aseptically collected breast tumor tissue from women who underwent neoadjuvant chemotherapy (n = 15) or women with no prior therapy at time of surgery (n = 18), we performed 16S rRNA-sequencing to identify tumoral bacterial populations. We also stained breast tumor microarrays to confirm presence of identified microbiota. Using bacteria-conditioned media, we determined the effect of bacterial metabolites on breast cancer cell proliferation and doxorubicin therapy responsiveness. We show chemotherapy administration significantly increased breast tumor Pseudomonas spp. Primary breast tumors from patients who developed distant metastases displayed increased tumoral abundance of Brevundimonas and Staphylococcus. We confirmed presence of Pseudomonas in breast tumor tissue by IHC staining. Treatment of breast cancer cells with Pseudomonas aeruginosa conditioned media differentially effected proliferation in a dose-dependent manner and modulated doxorubicin-mediated cell death. Our results indicate chemotherapy shifts the breast tumor microbiome and specific microbes correlate with tumor recurrence. Further studies with a larger patient cohort may provide greater insights into the role of microbiota in therapeutic outcome and develop novel bacterial biomarkers that could predict distant metastases. IMPLICATIONS: Breast tumor microbiota are modified by therapy and affects molecular signaling.

Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle in mice.

Female mice heterozygous for a genetic mutation in transcription factor forkhead box p3 (Foxp3) spontaneously develop mammary cancers; however, the underlying mechanism is not well understood. We hypothesised that increased cancer susceptibility is associated with an underlying perturbation in mammary gland development. The role of Foxp3 in mammary ductal morphogenesis was investigated in heterozygous Foxp3Sf/+ and wildtype Foxp3+/+ mice during puberty and at specific stages of the oestrous cycle. No differences in mammary ductal branching morphogenesis, terminal end bud formation or ductal elongation were observed in pubertal Foxp3Sf/+ mice compared with Foxp3+/+ mice. During adulthood, all mice underwent normal regular oestrous cycles. No differences in epithelial branching morphology were detected in mammary glands from mice at the oestrus, metoestrus, dioestrus and pro-oestrus stages of the cycle. Furthermore, abundance of Foxp3 mRNA and protein in the mammary gland and lymph nodes was not altered in Foxp3Sf/+ mice compared with Foxp3+/+ mice. These studies suggest that Foxp3 heterozygosity does not overtly affect mammary gland development during puberty or the oestrous cycle. Further studies are required to dissect the underlying mechanisms of increased mammary cancer susceptibility in Foxp3Sf/+ heterozygous mice and the function of this transcription factor in normal mammary gland development.

Deep imaging reveals new insights into mammary gland architecture and breast cancer susceptibility.

The abundance of adipose tissue in the mammary gland obscures vision of the 3-dimensional architecture. Hitchcock et al. employed a new technique of deep tissue imaging that has enabled visualisation of dynamic interactions between mammary gland epithelial and immune cells with unprecedented 3-dimensional clarity. Deep imaging will help further our understanding of the complex biological interactions that underpin both normal mammary gland development and the susceptibility of this tissue to cancer. This knowledge will assist the development of much-needed prevention strategies to reduce the incidence of breast cancer. Comment on: https://doi.org/10.1111/febs.15126.