Case report: Primary vulvar adenocarcinoma of mammary gland type-its genetic characteristics by focused next-generation sequencing.

Mammary-like vulvar adenocarcinoma (MLVA) is an exceedingly rare subtype of vulvar adenocarcinoma that shares features with mammary gland tissue. Due to its rarity and lack of consensus, MLVA presents diagnostic challenges to pathologists. We present the case of a 59-year-old female with an ulcerated mass on the right side of the external genitalia, diagnosed as MLVA. Comprehensive immunohistochemistry (IHC) and gene sequencing studies were performed to characterize the tumor. IHC analysis revealed triple expression of hormonal receptors (estrogen receptor, progesterone receptor, and HER2), supporting the mammary gland origin of the tumor. Gene sequencing identified unique genetic mutations associated with the expression of hormonal markers. One fusion gene (ERBB2-NAGLU) has not been reported in any tumors, and other mutations with unique mutation types have not been previously reported in MLVA. Our findings shed light on the molecular characteristics of MLV and may help improve the diagnosis and treatment of this rare type of vulvar adenocarcinoma.

Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation.

Lactation insufficiency affects many women worldwide. During lactation, a large portion of mammary gland alveolar cells become polyploid, but how these cells balance the hyperproliferation occurring during normal alveologenesis with terminal differentiation required for lactation is unknown. Here, we show that DNA damage accumulates due to replication stress during pregnancy, activating the DNA damage response. Modulation of DNA damage levels in vivo by intraductal injections of nucleosides or DNA damaging agents reveals that the degree of DNA damage accumulated during pregnancy governs endoreplication and milk production. We identify a mechanism involving early mitotic arrest through CDK1 inactivation, resulting in a heterogeneous alveolar population with regards to ploidy and nuclei number. The inactivation of CDK1 is mediated by the DNA damage response kinase WEE1 with homozygous loss of Wee1 resulting in decreased endoreplication, alveologenesis and milk production. Thus, we propose that the DNA damage response to replication stress couples proliferation and endoreplication during mammary gland alveologenesis. Our study sheds light on mechanisms governing lactogenesis and identifies non-hormonal means for increasing milk production.

Comorbid pathology of the mammary glands and endometriosis: risk factors and prognosis.

OBJECTIVE: Aim: based on a retrospective analysis, the relationship between external genital endometriosis and comorbid breast pathology was established and risk factors were identified, their comparison and the formation of a prognostic risk criterion were determined. PATIENTS AND METHODS: Materials and Methods: to address the objectives of the study, a retrospective analysis of 470 cases of patients treated for external genital endometriosis after surgical treatment and comorbid breast pathology was conducted. The control group included 30 healthy non-pregnant women. Statistical processing was performed on a personal computer using the statistical software package Statistica 10. RESULTS: Results: As a result of the analysis, the age of the patients ranged from 23 to 40 years. The average age of patients in the study group was (32.2}1.18) years, and in the control group (31.1}1.35) (p>0.05). The groups were homogeneous in terms of age (p>0.05), marital status (p>0.05) and level of education (p>0.05). Close relatives in 208 (44.25}2.18) % (OR=8.86; 95 % CI: (0.68-10.53); p<0.002) cases suffered from benign (hormone-dependent) tumours and tumour-like diseases of the uterus and appendages in isolation or in various combinations (fibroids, adenomyosis, endometrial hyperplasia). It was also found that 102 (21.70}1.67) % of patients had endometriosis, which may indicate a genetic predisposition to this disease. In the closest relatives of EM patients: in 118 (25.10}2.01) % of the examined parents, breast problems were noted, in 66 (14.04}1.12) % - diabetes mellitus, and in 98 (20.85}1.22) % thyroid diseases were detected, which in total amounted to (60.00}2.23) % (OR=9.12; 95 % CI: (0.58-11.54); p<0.002). Early menarche almost tripled the risk of EM (OR=2.72; 95% CI: (1.02-5.11); p<0.002), and menstrual irregularities doubled it (OR=2.04; 95% CI: (1.09-3.14); p<0.05), higher education, urban residents - 2.2 times higher (OR= 2.27; 95 % CI: (1.11-3.63); p<0.05), diseases of the gastrointestinal tract and hepatobiliary complex - 5.2 times higher (OR=5.27; 95 % CI: (1.89-12.03); p<0.05), frequently recurrent inflammatory diseases of the appendages - 3 times higher (OR=3.14; 95 % CI: (0.91-5.14); p<0.05), dysmetabolic manifestations (thyroid dysfunction) - 5 times higher (OR=5.11; 95 % CI: (1.61-9.503); p<0.002). CONCLUSION: Conclusions: Thus, in endometriosis and dyshormonal diseases of the mammary glands, menstrual and generative function disorders, along with clinical symptoms of pelvic pain, dysmenorrhoea, autonomic nervous system disorders and sexual dysfunction, are significant components of this problem, initiating comorbidity processes in target organs in the setting of hormonal maladaptation. Therefore, these comorbidities become a trigger for the activation of systemic hormonal imbalance and become an urgent interdisciplinary problem that requires further study.

Epidemiology and classification for canine and feline mammary gland tumors: a histopathological survey of 437 mammary gland tumor biopsies performed in a secondary care hospital in Chiang Mai, Thailand from 2012 to 2019.

Background: Metastatic disease resulting from mammary gland tumors (MGTs) is a known cause of death among dogs and cats. Keys to successful prevention and management strategies involve the accurate recording of diagnostic data. Methods: This retrospective study reviewed the epidemiology and classification of canine mammary gland tumors (CMTs) and feline mammary gland tumors (FMTs), as well as the factors including sex, age, and breed related to the occurrence of these tumors. Accordingly, 1,736 tumor biopsy cases were reported from 2012 to 2019 at Chiang Mai University Small Animal Hospital, Thailand, with 1,639 canine tumor biopsy cases and 97 feline tumor biopsy cases. Results: The proportion of CMTs was reported at 24.5% (401/1,639) for all canine tumor biopsy cases. Benign and malignant tumors were reported at 14.5% (58/401) and 85.5% (343/401) for all CMT cases, respectively. The mean age of dogs affected by benign CMTs was 9.0 ± 3.0 years, which was significantly lower than for malignant CMTs at 9.9 ± 2.8 years (P = 0.0239). According to histopathological classification, benign mixed tumors and simple carcinoma types were highest among benign and malignant CMT cases, respectively. Moreover, female dogs were at significantly higher risk of developing mammary gland tumors (OR = 45.8, 95% CI [3.9-86.0], P < 0.0001) than male dogs, as well as older dogs (>8 years) (OR = 1.7, 95% CI [1.2-2.2], P = 0.0001) compared to young ones (≤8 years). The proportion of FMTs was 37.1% (36/97) for all feline tumor biopsy cases. Benign and malignant tumors for all FMTs were reported at 16.7% (6/36) and 83.3% (30/36), respectively. According to histopathological classifications, adenoma and simple carcinoma were present in the highest proportion among benign and malignant FMTs, respectively. Female cats were at a significantly higher risk of developing mammary gland tumors than male cats (OR = 25.7, 95% CI [3.9-272.8], P < 0.0001). Conclusions and clinical importance: There was a high proportion of MGT cases compared with other tumor cases reported in a secondary care hospital in Chiang Mai, Thailand from 2012 to 2019, and malignant tumor biopsies have been more frequently observed than benign tumor biopsies in both CMT and FMT cases. The resulting data originating from this study can be an aid for veterinary oncologists in better educating clients and planning treatment and prevention strategies and it can be used as a basis for further experimental studies in the oncology section.

Mechanisms underlying the therapeutic effects of Xiaoyaosan in treating hyperplasia of mammary glands based on network pharmacology.

This study utilized network pharmacology to investigate the effects of Xiaoyaosan (XYS) on the intervention of hyperplasia of mammary glands (HMG) by targeting specific genes and signaling pathways. The active ingredients and targets of XYS, which consisted of 8 traditional Chinese medicines (TCM), were identified using TCMSP. The gene targets associated with HMG were obtained from the GeneCards Database, and the intersection data between the 2 was integrated. Cytoscape 3.8.1 software was used to construct a network diagram illustrating the relationship between compounds, drug active ingredients, target proteins, and the disease. The protein-protein interaction network diagram was generated using STRING, and the core targets were analyzed. A total of 133 active ingredients in XYS and 7662 active ingredient targets were identified. Among them, 6088 targets were related to HMG, and 542 were common targets between the active ingredients and the disease. The protein-protein interaction (PPI) core network contained 15 targets, with 5 key targets playing a crucial role. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses have indicated that XYS has the potential to treat HMG by interfering with the AGE-RAGE signaling pathway in diabetic complications, the MAPK signaling pathway, and the PI3K-Akt signaling pathway. Additionally, molecular docking studies have shown excellent binding properties between the drug components and key targets. Thus, this study provides a theoretical foundation for a better understanding of the pharmacological mechanism and clinical application of XYS in the comprehensive treatment of HMG.

Using Organoids to Tap Mammary Gland Diversity for Novel Insight.

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

Defining and targeting macrophage heterogeneity in the mammary gland and breast cancer.

INTRODUCTION: Macrophages are innate immune cells that are associated with extensive phenotypic and functional plasticity and contribute to normal development, tissue homeostasis, and diseases such as cancer. In this review, we discuss the heterogeneity of tissue resident macrophages in the normal mammary gland and tumor-associated macrophages in breast cancer. Tissue resident macrophages are required for mammary gland development, where they have been implicated in promoting extracellular matrix remodeling, apoptotic clearance, and cellular crosstalk. In the context of cancer, tumor-associated macrophages are key drivers of growth and metastasis via their ability to promote matrix remodeling, angiogenesis, lymphangiogenesis, and immunosuppression. METHOD: We identified and summarized studies in Pubmed that describe the phenotypic and functional heterogeneity of macrophages and the implications of targeting individual subsets, specifically in the context of mammary gland development and breast cancer. We also identified and summarized recent studies using single-cell RNA sequencing to identify and describe macrophage subsets in human breast cancer samples. RESULTS: Advances in single-cell RNA sequencing technologies have yielded nuances in macrophage heterogeneity, with numerous macrophage subsets identified in both the normal mammary gland and breast cancer tissue. Macrophage subsets contribute to mammary gland development and breast cancer progression in differing ways, and emerging studies highlight a role for spatial localization in modulating their phenotype and function. CONCLUSION: Understanding macrophage heterogeneity and the unique functions of each subset in both normal mammary gland development and breast cancer progression may lead to more promising targets for the treatment of breast cancer.

Method for evaluating milk production in mouse mammary gland.

Lactation is a characteristic physiological function of mammals and is important for nourishing infants and the dairy industry; however, the molecular mechanisms underlying the function remain to be elucidated. A technique to directly evaluate the quantity and quality of milk in mice is necessary for the study of the lactation mechanism in vivo. By measuring the changes in milk amount after different durations of milk accumulation (0-24 h) using a ductal cannulation technique and oxytocin supplementation, we estimated the milk production rate at a single mammary gland level. In addition, collected milk was available to assess milk quality, including creamatocrit, osmolarity, and concentrations of ions, lactose, and total protein. Moreover, as a proof of principle, the effects of intraductal administration of a hypertonic solution to the abdominal mammary gland were examined. This stimulation increased milk amount, possibly by osmosis, compared with the contralateral control gland. These results demonstrated that this method is useful for examining the lactation ability and mechanisms in vivo. Studies using this method will contribute to the further understanding of lactation mechanisms in mammals.

Synergistic Anti-Cancer Effects of ERB-041 and Genistein through Estrogen Receptor Suppression-Mediated PI3K/AKT Pathway Downregulation in Canine Mammary Gland Tumor Cells.

Canine-mammary-gland tumors (CMTs) are prevalent in female dogs, with approximately 50% of them being malignant and often presenting as inoperable owing to their size or metastasis. Owing to poor outcomes, effective alternatives to conventional chemotherapy for humans are necessary. Two estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), which act in opposition to each other, are involved, and CMT growth involves ERα through the phosphoinositide 3-kinases (PI3K)/AKT pathway. In this study, we aimed to identify the synergistic anti-cancer effects of ERB-041, an ERß agonist, and genistein, an isoflavonoid from soybeans known to have ERß-specific pseudo-estrogenic actions, on CMT-U27 and CF41.Mg CMT cell lines. ERB-041 and genistein synergistically inhibited cell proliferation and increased the number of annexin V-positive cells in both cell lines. Furthermore, we observed a synergistic increase in the Bax/Bcl-2 ratio and cleaved caspase-3 expression. Additionally, cell-cycle arrest occurred through the synergistic regulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). We also found a synergistic decrease in the expression of ERα, and the expression of proteins involved in the PI3K/AKT pathway, including p-PI3K, phosphatase and tensin homolog (PTEN), AKT, and mechanistic target of rapamycin (mTOR). In conclusion, ERB-041 and genistein exhibited a synergistic anticancer effect on CMTs, suggesting that cotreatment with ERB-041 and genistein is a promising treatment for CMTs.

A Decision Tree to Guide Human and Mouse Mammary Organoid Model Selection.

Over the past 50 years, researchers from the mammary gland field have launched a collection of distinctive 3D cell culture systems to study multiple aspects of mammary gland physiology and disease. As our knowledge about the mammary gland evolves, more sophisticated 3D cell culture systems are required to answer more and more complex questions. Nowadays, morphologically complex mammary organoids can be generated in distinct 3D settings, along with reproduction of multiple aspects of the gland microenvironment. Yet, each 3D culture protocol comes with its advantages and limitations, where some culture systems are best suited to study stemness potential, whereas others are tailored towards the study of mammary gland morphogenesis. Therefore, prior to starting a 3D mammary culture experiment, it is important to consider and select the ideal culture model to address the biological question of interest. The number and technical requirements of novel 3D cell culture methods vastly increased over the past decades, making it currently challenging and time consuming to identify the best experimental testing. In this chapter, we provide a summary of the most promising murine and human 3D organoid models that are currently used in mammary gland biology research. For each model, we will provide a brief description of the protocol and an overview of the expected morphological outcome, the advantages of the model, and the potential pitfalls, to guide the reader to the best model of choice for specific applications.

Calcitriol promotes M2 polarization of tumor-associated macrophages in 4T1 mouse mammary gland cancer via the induction of proinflammatory cytokines.

Our research found that vitamin D3 (VD3) treatment increased lung metastasis in mice with 4T1 murine breast cancer (BC). This study aims to investigate the impact of VD3 on the activation of tumor-associated macrophages (TAMs) in BC. Mice bearing 4T1, E0771, 67NR BC cells, and healthy mice, were fed diets with varying VD3 contents (100-deficient, 1000-normal, and 5000 IU/kg-elevated). Some mice in the 1000 and 100 IU/kg groups received calcitriol. We studied bone metastasis and characterized TAMs and bone marrow-derived macrophages (BMDMs). 4T1 cells had higher bone metastasis potential in the 5000 IU/kg and calcitriol groups. In the same mice, an elevated tumor osteopontin level and M2 polarization of TAMs (MHCIIlow CD44high phenotype) were observed. Gene expression analysis confirmed M2 polarization of 4T1 (but not 67NR) TAMs and BMDMs, particularly in the 100 IU + cal group (increased Mrc1, Il23, and Il6). This polarization was likely due to COX-2/PGE2 induction in 4T1 calcitriol-treated cells, leading to increased proinflammatory cytokines like IL-6 and IL-23. Future studies will explore COX-2/PGE2 as a primary mediator of calcitriol-stimulated inflammation in the BC microenvironment, especially relevant for BC patients with VD3 deficiency and supplementation.

In vitro evaluation of the antitumor activity of axitinib in canine mammary gland tumor cell lines.

BACKGROUND: Axitinib, a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor (VEGFR) tyrosine kinase 1,2 and 3, is used in chemotherapy because it inhibits tumor angiogenesis by blocking the VEGF/VEGFR pathway. In veterinary medicine, attempts have been made to apply tyrosine kinase inhibitors with anti-angiogenic effects to tumor patients, but there are no studies on axitinib in canine mammary gland tumors (MGTs). OBJECTIVES: This study aimed to confirm the antitumor activity of axitinib in canine mammary gland cell lines. METHODS: We treated canine MGT cell lines (CIPp and CIPm) with axitinib and conducted CCK, wound healing, apoptosis, and cell cycle assays. Additionally, we evaluated the expression levels of angiogenesis-associated factors, including VEGFs, PDGF-A, FGF-2, and TGF-ß1, using quantitative real-time polymerase chain reaction. Furthermore, we collected canine peripheral blood mononuclear cells (PBMCs), activated them with concanavalin A (ConA) and lipopolysaccharide (LPS), and then treated them with axitinib to investigate changes in viability. RESULTS: When axitinib was administered to CIPp and CIPm, cell viability significantly decreased at 24, 48, and 72 h (p < 0.001), and migration was markedly reduced (6 h, p < 0.05; 12 h, p < 0.005). The apoptosis rate significantly increased (p < 0.01), and the G2/M phase ratio showed a significant increase (p < 0.001). Additionally, there was no significant change in the viability of canine PBMCs treated with LPS and ConA. CONCLUSION: In this study, we confirmed the antitumor activity of axitinib against canine MGT cell lines. Accordingly, we suggest that axitinib can be applied as a new treatment for patients with canine MGTs.

A xenotransplantation mouse model to study physiology of the mammary gland from large mammals.

Although highly conserved in structure and function, many (patho)physiological processes of the mammary gland vary drastically between mammals, with mechanisms regulating these differences not well understood. Large mammals display variable lactation strategies and mammary cancer incidence, however, research into these variations is often limited to in vitro analysis due to logistical limitations. Validating a model with functional mammary xenografts from cryopreserved tissue fragments would allow for in vivo comparative analysis of mammary glands from large and/or rare mammals and would improve our understanding of postnatal development, lactation, and premalignancy across mammals. To this end, we generated functional mammary xenografts using mammary tissue fragments containing mammary stroma and parenchyma isolated via an antibody-independent approach from healthy, nulliparous equine and canine donor tissues to study these species in vivo. Cryopreserved mammary tissue fragments were xenotransplanted into de-epithelialized fat pads of immunodeficient mice and resulting xenografts were structurally and functionally assessed. Preimplantation of mammary stromal fibroblasts was performed to promote ductal morphogenesis. Xenografts recapitulated mammary lobule architecture and contained donor-derived stromal components. Mammatropic hormone stimulation resulted in (i) upregulation of lactation-associated genes, (ii) altered proliferation index, and (iii) morphological changes, indicating functionality. Preimplantation of mammary stromal fibroblasts did not promote ductal morphogenesis. This model presents the opportunity to study novel mechanisms regulating unique lactation strategies and mammary cancer induction in vivo. Due to the universal applicability of this approach, this model serves as proof-of-concept for developing mammary xenografts for in vivo analysis of virtually any mammals, including large and rare mammals.

Kv 11.1 Expression Is Associated With Malignancy of Canine Mammary Gland Tumors.

BACKGROUND/AIM: The expression level of the voltage-dependent potassium channel Kv 11.1 was shown to be associated with the clinicopathological features, aggressiveness, and prognosis of human breast cancer. Canine mammary gland tumor (cMGT) is the most common tumor type in intact female dogs; however, the significance of Kv 11.1 in cMGT is unknown. The aim of this study was to identify Kv 11.1 expression in 57 benign and malignant cMGT tissues from dogs and to investigate the correlation of Kv 11.1 expression with the clinicopathological parameters and prognosis of cMGT. MATERIALS AND METHODS: A total of 57 samples were collected from cMGTs surgically resected at the Veterinary Medical Teaching Hospital, Seoul National University and subjected to immunohistochemistry assay using rabbit anti-Kv 11.1 polyclonal antibody. Immunohistochemical staining results were evaluated as the sum of intensity and percentage scores. The correlation between immunohistochemistry scores and clinicopathological parameters was investigated. RESULTS: Immunohistochemical analysis revealed that Kv 11.1 immunoreactivity was higher in benign cMGTs than in malignant cMGTs. Kv 11.1 expression was significantly associated with tumor malignancy (p<0.001), tumor size (p<0.001), histological grade (p<0.05), and age at the time of mastectomy (p<0.05). CONCLUSION: This study presents the first evidence of Kv 11.1 expression in cMGTs and indicates an inverse correlation between Kv 11.1 expression and tumor malignancy. Kv 11.1 expression can be used as a prognostic biomarker and a tool for the management of cMGTs.

Borrelia burgdorferi colonizes the mammary glands of lactating C3H mice: does not cause congenital Lyme disease.

Transplacental transmission of syphilis causing spirochete, Treponema pallidum subspecies pallidum, from mother to child results in congenital syphilis, an ever-expanding devastating disease worldwide. Although adverse effects of untreated gestational Lyme disease, caused by a related spirochete, Borrelia burgdorferi on fetus viability and development have been observed, cases of congenital Lyme disease are not reported. In this study, we show that B. burgdorferi colonizes mammary glands of C3H mice only postpartum; however, neither transmission of these spirochetes from dams-to-pups occurs nor congenital Lyme disease is observed in pups.

The role of lymph nodes and their drainage in canine mammary gland tumours: Systematic review.

Mammary gland tumours are the most common neoplasms in intact bitches. Over the last decades, veterinary oncology has evolved in detecting and determining the lymph nodes to be removed in these patients for an accurate staging and prognosis, as well as to achieve better disease control and higher overall survival time. Our objective was to describe recent advances related to lymphatic drainage in bitches with mammary gland tumours, focusing on surgery, diagnosis, and prognosis. Through a systematic review using PubMed as the database, a thorough multi-step search reduced 316 studies to 30 for analysis. Vital dyes appear to be crucial in reducing the overall surgery time through transoperative staining of the lymph nodes. Imaging contrasts provide information regarding specific tumour drainage; however, there is still little evidence for their use. The axillary and superficial inguinal lymph nodes are well-established as regional lymph nodes of the cranial and caudal mammary glands. In sequence, accessory axillary, medial iliac, popliteal, and sternal lymph nodes should receive attention if they demonstrate contrast drainage, even considering that the literature has not shown a relationship between drainage and metastasis in these cases. In conclusion, recent studies have provided us with more support in regional lymph node excision regarding the TNM staging system. Studies are highly heterogeneous and method comparisons do not fit due to the non-uniformity of samples, materials, and procedures. We suggest further studies with a larger sample size, complete follow-up of patients, contrast use, and lymph node morphological and immunohistochemical analysis.

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

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