Mammary Gland Microenvironment Research Articles

An ovine model for investigation of the microenvironment of the male mammary gland.

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.

Microbiota Characterization of the Cow Mammary Gland Microenvironment and Its Association with Somatic Cell Count.

Subclinical mastitis is a common disease that threatens the welfare and health of dairy cows and causes huge economic losses. Somatic cell count (SCC) is the most suitable indirect index used to evaluate the degree of mastitis. To explore the relationship between SCC, diversity in the microbiome, and subclinical mastitis, we performed next-generation sequencing of the 16S rRNA gene of cow's milk with different SCC ranges. The data obtained showed that the microbiota was rich and coordinated with SCC below 2 × 105. SCC above 2 × 105 showed a decrease in the diversity of microbial genera. When SCC was below 2 × 105, the phylum Actinobacteriota accounted for the most. When SCC was between 2 × 105 and 5 × 105, Firmicutes accounted for the most, and when SCC exceeded 5 × 105, Firmicutes and Proteobacteria accounted for the most. Pathogenic genera such as Streptococcus spp. were absent, while SCC above 2 × 105 showed a decrease in the diversity of microbial genera. SCC was positively correlated with the percentage of Romboutsia, Turicibacter, and Paeniclostridium and negatively correlated with the percentage of Staphylococcus, Psychrobacter, Aerococcus, and Streptococcus. Romboutsia decreased 6.19 times after the SCC exceeded 2 × 105; the SCC increased exponentially from 2 × 105 to 5 × 105 and above 1 × 106 in Psychrobacter. Analysis of the microbiota of the different SCC ranges suggests that the development of mastitis may not only be a primary infection but may also be the result of dysbiosis in the mammary gland.

Alterations in the mammary gland and tumor microenvironment of formerly obese mice

BackgroundObesity is a risk factor for breast cancer, and women with obesity that develop breast cancer have a worsened prognosis. Within the mammary gland, obesity causes chronic, macrophage-driven inflammation and adipose tissue fibrosis. Weight loss is a recommended intervention to resolve obesity, but the impact of weight loss on the mammary gland microenvironment and in tumors has not been well identified.MethodsTo examine the effects of weight loss following obesity, mice were fed a high-fat diet for 16 weeks to induce obesity, then switched to a low-fat diet for 6 weeks. We examined changes in immune cells, including fibrocytes, which are myeloid lineage cells that have attributes of both macrophages and myofibroblasts, and collagen deposition within the mammary glands of non-tumor-bearing mice and within the tumors of mice that were transplanted with estrogen receptor alpha positive TC2 tumor cells. ResultsIn formerly obese mice, we observed reduced numbers of crown-like structures and fibrocytes in mammary glands, while collagen deposition was not resolved with weight loss. Following transplant of TC2 tumor cells into the mammary glands of lean, obese, and formerly obese mice, diminished collagen deposition and cancer-associated fibroblasts were observed in tumors from formerly obese mice compared to obese mice. Within tumors of obese mice, increased myeloid-derived suppressor cells and diminished CD8+ T cells were identified, while the microenvironment of tumors of formerly obese mice were more similar to tumors from lean mice. When TC2 tumor cells were mixed with CD11b+CD34+ myeloid progenitor cells, which are the cells of origin for fibrocytes, and transplanted into mammary glands of lean and obese mice, collagen deposition within the tumors of both lean and obese was significantly greater than when tumor cells were mixed with CD11b+CD34− monocytes or total CD45+ immune cells. ConclusionsOverall, these studies demonstrate that weight loss resolved some of the microenvironmental conditions within the mammary gland that may contribute to tumor progression. Additionally, fibrocytes may contribute to early collagen deposition in mammary tumors of obese mice leading to the growth of desmoplastic tumors.

High-fat diet, but not duration of lactation, increases mammary gland lymphatic vessel function and subsequent growth of inflammatory breast cancer cells

Inflammatory breast cancer (IBC) presents as rapid-onset swelling and breast skin changes caused by tumor emboli in the breast and breast skin lymphatics. IBC has been linked with obesity and duration of breastfeeding, but how these factors affect IBC tumor progression is not clear. We modeled the simultaneous effects of diet and weaning in mice on in vivo lymphatic function; on IBC tumor growth; and on aspects of the mammary gland microenvironment before and after IBC (SUM149) xenograft inoculation. We hypothesized that weaning status and diet would have synergistic effects on lymphatic function and the breast microenvironment to enhance IBC tumor growth. Changes in lymphatic structure and function were characterized with in vivo near-infrared fluorescence (NIRF) imaging. Mice were fed either a high-fat diet (HFD; 60 kcal%) or a normal/low-fat diet (LFD; 10 kcal%), bred twice, and subjected to either normal-duration nursing (NW) or forced weaning (FW). SUM149 IBC tumors were implanted at 14 months; images were obtained before and after implantation. Multiparous mice fed HFD showed increased pre-tumor lymphatic pulsing in both the FW and NW groups relative to mice fed LFD. HFD promoted tumor growth independent of weaning time (P = 0.04). Pre-tumor lymphatic pulsing was associated with tumor volume at 8 weeks (P = 0.02) and was significantly correlated with expression of the lymphatic tracking ligand CCL21 (P = 0.05, Table 1). HFD significantly increased the numbers of monocyte-derived IBA1+, CD163+, and CD11c+ cells (P < 0.0001, P < 0.0001, P = 0.0005) in the contralateral, non-tumor-bearing mammary gland. Numbers of lymphangiogenic podoplanin+/IBA1+ macrophages were increased in the ducts of HFD and FW mice (all P < 0.003). HFD in nulliparous mice had a similar increase in lymphatic pulsing at 14 weeks (P = 0.006), indicating that this functional change was independent of parity. We conclude that HFD induced increases in mammary gland lymphatic function, assessed as pulsing rate before tumor initiation, and correlated with inflammation in the mammary gland and increased SUM149 tumor growth. The relationship between diet, lymphatic pulsing, and tumor growth warrants further investigation.

Nonlinear microscopy and deep learning classification for mammary gland microenvironment studies.

Tumors, their microenvironment, and the mechanisms by which collagen morphology changes throughout cancer progression have recently been a topic of interest. Second harmonic generation (SHG) and polarization second harmonic (P-SHG) microscopy are label-free, hallmark methods that can highlight this alteration in the extracellular matrix (ECM). This article uses automated sample scanning SHG and P-SHG microscopy to investigate ECM deposition associated with tumors residing in the mammary gland. We show two different analysis approaches using the acquired images to distinguish collagen fibrillar orientation changes in the ECM. Lastly, we apply a supervised deep-learning model to classify naïve and tumor-bearing mammary gland SHG images. We benchmark the trained model using transfer learning with the well-known MobileNetV2 architecture. By fine-tuning the different parameters of these models, we show a trained deep-learning model that suits such a small dataset with 73% accuracy.

Nutritional Regulation of Mammary Tumor Microenvironment

The mammary gland is a heterogeneous organ comprising of immune cells, surrounding adipose stromal cells, vascular cells, mammary epithelial, and cancer stem cells. In response to nutritional stimuli, dynamic interactions amongst these cell populations can be modulated, consequently leading to an alteration of the glandular function, physiology, and ultimately disease pathogenesis. For example, obesity, a chronic over-nutritional condition, is known to disrupt homeostasis within the mammary gland and increase risk of breast cancer development. In contrast, emerging evidence has demonstrated that fasting or caloric restriction can negatively impact mammary tumorigenesis. However, how fasting induces phenotypic and functional population differences in the mammary microenvironment is not well understood. In this review, we will provide a detailed overview on the effect of nutritional conditions (i.e., overnutrition or fasting) on the mammary gland microenvironment and its impact on mammary tumor progression.

Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion, and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway.

Simple SummaryStudies from our group and others have established Kindlin-2 that is expressed in the cancer cells as a major driver of tumor progression and metastasis in breast cancer (BC). The role of Kindlin-2 that is expressed in the mammary glands (tumor microenvironment) in the pathogenesis of BC has, however, not been investigated. To this end, we generated a mouse strain that specifically lacks expression of Kindlin-2 in the basal cells within the mouse mammary glands. Loss of Kindlin-2 in this mammary gland compartment, while having no effect on mouse development, significantly inhibited BC tumors growth and metastasis when these Kindlin-2-deficient mice were challenged with mammary fat pad injection of cancer cells. At the molecular signaling level, we found that Kindlin-2 plays a significant role in regulating BC progression and metastasis in both the cancer cells and the tumor microenvironment (mammary epithelial cells) downstream of a TGF-β/EGF signaling axis.Breast cancer (BC) is one of the leading causes of cancer-related deaths due in part to its invasive and metastatic properties. Kindlin-2 (FERMT2) is associated with the pathogenesis of several cancers. Although the role of Kindlin-2 in regulating the invasion-metastasis cascade in BC is widely documented, its function in BC initiation and progression remains to be fully elucidated. Accordingly, we generated a floxed mouse strain by targeting the Fermt2 (K2lox/lox) locus, followed by tissue-specific deletion of Kindlin-2 in the myoepithelial compartment of the mammary glands by crossing the K2lox/lox mice with K14-Cre mice. Loss of Kindlin-2 in mammary epithelial cells (MECs) showed no deleterious effects on mammary gland development, fertility, and lactation in mice bearing Kindlin-2-deletion. However, in a syngeneic mouse model of BC, mammary gland, specific knockout of Kindlin-2 inhibited the growth and metastasis of murine E0771 BC cells inoculated into the mammary fat pads. However, injecting the E0771 cells into the lateral tail vein of Kindlin-2-deleted mice had no effect on tumor colonization in the lungs, thereby establishing a critical role of MEC Kindlin-2 in supporting BC tumor growth and metastasis. Mechanistically, we found the MEC Kindlin-2-mediated inhibition of tumor growth and metastasis is accomplished through its regulation of the TGF-β/ERK MAP kinase signaling axis. Thus, Kindlin-2 within the mammary gland microenvironment facilitates the progression and metastasis of BC.

Chronic Isolation Stress Affects Central Neuroendocrine Signaling Leading to a Metabolically Active Microenvironment in a Mouse Model of Breast Cancer

Social isolation is a powerful stressor capable of affecting brain plasticity and function. In the case of breast cancer, previous data indicate that stressful experiences may contribute to a worse prognosis, activating neuroendocrine and metabolism pathways, although the mechanisms underlying these effects are still poorly understood. In this study, we tested the hypothesis that chronic isolation stress (IS) may boost hypothalamic–pituitary–adrenal (HPA) axis activity, leading to changes in the hypothalamic expression of genes modulating both mood and metabolism in an animal model of breast cancer. This centrally activated signaling cascade would, in turn, affect the mammary gland microenvironment specifically targeting fat metabolism, leading to accelerated tumor onset. MMTVNeuTg female mice (a model of breast cancer developing mammary hyperplasia at 5 months of age) were either group-housed (GH) or subjected to IS from weaning until 5 months of age. At this time, half of these subjects underwent acute restraint stress to assess corticosterone (CORT) levels, while the remaining subjects were characterized for their emotional profile in the forced swimming and saccharin preference tests. At the end of the procedures, all the mice were sacrificed to assess hypothalamic expression levels of Brain-derived neurotrophic factor (Bdnf), Neuropeptide Y (NpY), Agouti-Related Peptide (AgRP), and Serum/Glucocorticoid-Regulated Protein Kinase 1 (SgK1). Leptin and adiponectin expression levels, as well as the presence of brown adipose tissue (BAT), were assessed in mammary fat pads. The IS mice showed higher CORT levels following acute stress and decreased expression of NpY, AgRP, and SgK1, associated with greater behavioral despair in the forced swimming test. Furthermore, they were characterized by increased consumption of saccharin in a preference test, suggesting an enhanced hedonic profile. The IS mice also showed an earlier onset of breast lumps (assessed by palpation) accompanied by elevated levels of adipokines (leptin and adiponectin) and BAT in the mammary fat pads. Overall, these data point to IS as a pervasive stressor that is able to specifically target neuronal circuits, mastered by the hypothalamus, modulating mood, stress reactivity and energy homeostasis. The activation of such IS-driven machinery may hold main implications for the onset and maintenance of pro-tumorigenic environments.

Mammary Organoids and 3D Cell Cultures: Old Dogs with New Tricks.

3D cell culture methods have been an integral part of and an essential tool for mammary gland and breast cancer research for half a century. In fact, mammary gland researchers, who discovered and deciphered the instructive role of extracellular matrix (ECM) in mammary epithelial cell functional differentiation and morphogenesis, were the pioneers of the 3D cell culture techniques, including organoid cultures. The last decade has brought a tremendous increase in the 3D cell culture techniques, including modifications and innovations of the existing techniques, novel biomaterials and matrices, new technological approaches, and increase in 3D culture complexity, accompanied by several redefinitions of the terms "3D cell culture" and "organoid". In this review, we provide an overview of the 3D cell culture and organoid techniques used in mammary gland biology and breast cancer research. We discuss their advantages, shortcomings and current challenges, highlight the recent progress in reconstructing the complex mammary gland microenvironment in vitro and ex vivo, and identify the missing 3D cell cultures, urgently needed to aid our understanding of mammary gland development, function, physiology, and disease, including breast cancer.

Single-Cell RNA-seq Reveals Obesity-Induced Alterations in the Brca1-Mutated Mammary Gland Microenvironment.

Clinical and experimental studies have shown that obesity increases the development and progression of breast cancer. The impact of obesity on the tumor microenvironment plays an important role in tumorigenesis, yet the precise mechanisms underlying obesity-mediated effects on cell-to-cell communication within the tumor microenvironment have been difficult to define. In this study, we conducted single-cell RNA sequencing (scRNA-seq) studies to investigate the impact of high-fat diet (HFD)-induced obesity on transcriptomic landscapes of stromal and immune cells in mammary glands of Brca1−/−; p53+/− mice, an animal breast cancer model. Hierarchical clustering and gene pathway enrichment analyses of scRNA-seq data showed that five different subtypes of stromal fibroblasts existed in mouse Brca1-mutated mammary glands. HFD-induced obesity led to upregulated expression of extracellular matrix (ECM) genes (Col3a1, Col6a3, Eln, and Sparc) and downregulated expression of immunoregulatory genes (Iigp1 and Cxcl10) in these stromal subtype cells. These findings, taken together, suggest that obesity alters the ECM composition and immune ecosystem through modulating the functionality of mammary stromal fibroblasts. Moreover, scRNA-seq analysis of mammary immune cells indicated that HFD-induced obesity promoted the generation and/or recruiting of pro-tumorigenic M2 macrophages in mammary glands. Our studies provide new insight into a mechanistic paradigm wherein obesity modulates the functions of stromal and immune cells to create the tumorigenic microenvironment for promoting breast tumorigenesis.

Abstract P6-15-04: Modeling limited breastfeeding and diet on IBC like tumor progression

Abstract Introduction/Motivation: We previously reported IBC patients with limited breast-feeding history have a worse prognosis than those with greater breast feeding history. Further, we reported pregnancy related factors including multiple early pregnancies and not breast feeding as well as obesity are risk factors for triple negative and ER+ subtypes. Significant evidence suggests the microenvironment drives IBC symptoms and growth pattern. We sought to characterize the impact of purported risk factors on tumor growth, IBC-like skin symptoms and mammary gland microenvironment in animal models. Methods: We commissioned breeding of nulliparous, multiparous (x 2) force weaned, and multiparous naturally weaned (labeled nursing) mice. Mice in each of the three groups were fed either a low fat (10 Kcal %) or high fat (60 Kcal %) diet for three weeks before tumor cell injections. SUM 149 GFP Luc were injected into the left ventral #4 mammary fat pad. Mice were sacrificed, scored for the IBC-like symptom of gross skin invasion or evident skin symptoms, and tissue from the contralateral mammary gland were sectioned and stained with H and E. Analysis was performed on the 31 animals that developed primary tumors and were scored for skin invasion at the time of resection. Of these 26 mice had contralateral gland tissue assessed by H and E. Phenotypes including duct dilation, degree of adipose tissue, duct density, inflammation and necrosis were scored manually, 1-3 scale. Quadratic mixed models with random intercept were fitted to compare tumor growth. Fisher’s exact test and T-tests were performed to compare variables. Results: Tumor incidence and latency were not different by risk factor group. Tumor growth was faster in multiparous force weaned versus others, P &amp;lt; 0.0001, but unchanged by diet, P = 0.187. Force weaned animals on a high fat diet had a trend for increased skin invasion compared to others (force weaned high fat diet vs. other 85% vs. 42%; P = 0.08) suggesting a synergy for this IBC-like symptom. Contralateral gland ductal density (less dense) and ductal dilation (not dilated) were normal histologic variables which correlated with skin invasion on the tumor side (P =0.006 and P = 0.011, respectively). Gland density but not dilated ducts trended to correlation with force weaning, P = 0.07, but not diet. Discussion: In this effect size finding pilot study, combined risk factors were associated with a higher incidence of skin invasion of SUM149 xenografts. Changes in the microenvironment in the contralateral gland typical of involution were significantly associated with skin invasion, and gland density trends towards association with force weaning. These data provide provocative pilot results linking histologic effects in the microenvironment to tumor growth characteristics and support the hypothesis that involution induced by forced weaning may facilitate IBC-like tumor growth. These studies facilitate well-powered additional work to establish mechanism and automate normal tissue evaluation. Citation Format: Wintana Balema, Richard Larson, Renae Van Whye, Rong Ye, Jing Ning, Omar Rahal, Shane Stecklein, Savitri Krishnamurthy, Randa El-Zein, Wendy Woodward. Modeling limited breastfeeding and diet on IBC like tumor progression [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 P6-15-04.

Quantitative Phosphoproteomics Reveals System-Wide Phosphorylation Network Altered by Spry in Mouse Mammary Stromal Fibroblasts.

Understanding the fundamental role of the stroma in normal development and cancer progression has been an emerging focus in recent years. The receptor tyrosine kinase (RTK) signaling pathway has been reported playing critical roles in regulating the normal and cancer microenvironment, but the underlying mechanism is still not very clear. By applying the quantitative phosphoproteomic analysis of Sprouty proteins (SPRYs), generic modulators of RTK signaling and deleted mouse mammary fibroblasts, we quantified a total of 11,215 unique phosphorylation sites. By contrast, 554 phosphorylation sites on 425 proteins had SPRY-responsive perturbations. Of these, 554 phosphosites, 362 sites on 277 proteins, were significantly increased, whereas 192 sites on 167 proteins were decreased. Among the regulated proteins, we identified 31 kinases, 7 phosphatases, and one phosphatase inhibitor that were not systematically characterized before. Furthermore, we reconstructed a phosphorylation network centered on RTK signaling regulated by SPRY. Collectively, this study uncovered a system-wide phosphorylation network regulated by SPRY, providing an additional insight into the complicated RTK signaling pathways involved in the mammary gland microenvironment.

Abstract LB-183: Anti-PD1 blunts obesity associated triple negative breast cancer progression

Abstract Few targeted therapies exist for triple negative breast cancer (TNBC), resulting in elevated recurrence and metastasis and a great unmet clinical need. Immune checkpoint therapy is promising; a recent clinical trial reported improved responses with anti-PD-L1 in advanced TNBC. Obesity increases poor outcomes in TNBC due to elevated invasion, metastasis, and mortality. Despite being an inflammatory state, obesity also can increase immune checkpoint ligands PD-1 and PD-L1 in some tumors and expression on PD-L1 on T cells and adipocytes, which drive immunosuppression. Greater PD-1 and PD-L1 may be due to hypoxia and inflammatory cytokines through increased expression and stability. Critically, immune checkpoint inhibitors appear to be more effective in obese patients, but underlying mechanisms remain unclear. Thus, we hypothesize that hypoxia and inflammation may synergistically increase immune checkpoint ligands which would alter immune checkpoint inhibitor efficacy in TNBC. To address this problem, syngeneic tumors were generated via orthotopic engraftment of E0771 basal-like breast cancer cell line into age-matched immune-competent C57BL/6J female littermates made obese on high fat diet (HFD, 60% kcal from fat) or lean on low fat diet (LFD, 10% fat). Mice were started on diets at weaning. By sacrifice at 27 weeks of age, lean and obese mice weighed 24.39g or 45.54g, respectively (p value &amp;lt;0.0001, N=15-16). Body composition showed no change in lean mass; obese mice had 48.64% adiposity (g fat/g body weight) compared to 15.99% in lean mice as 27 weeks (P=&amp;lt;0.0001). E0771 cells (0.25 x106) were injected after 24 weeks on diet and mice were sacrificed 24 days later. Mice were treated with anti-mouse anti-PD-1 or isotype control (IgG2a) (200 ug/kg i.p.) every 3rd day until sacrifice. Eleven out of 16 obese mice developed tumors (68.8% incidence). Surprisingly, only 2 tumors arose in N=16 lean mice (12.5% incidence) despite randomized same-day cell injections of lean and obese mice. Importantly, anti-PD-1 significantly reduced tumor volume in obese mice as measured by IVIS and caliper (P=0.0147, N=4-5, 22 days after tumor implantation). Ongoing studies include comprehensive evaluation of the immune microenvironment of both the tumors and normal mammary gland by flow and RNAseq. Immunohistology quantification of tumor, immune cell, and microenvironment characteristics will determine to what extent anti-PD-1 altered modifiers of tumor progression such as proliferation, apoptosis/necrosis, angiogenesis, stem cell content, epithelial to mesenchymal transition parameters. In conclusion, these results suggest that obesity-induced mammary microenvironmental effects augmented tumor incidence in immune intact C57BL/6J mice. Furthermore, immune checkpoint inhibitor successfully reduced tumor progression in obese mice. Potential inflammatory changes unique to the mammary gland or tumor microenvironment could be targeted in concert with checkpoint inhibitors in future interventions. Citation Format: Ajeeth Pingili, Emily Miller, Bin Teng, Liza Makowski. Anti-PD1 blunts obesity associated triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-183.

Studies of postpartum mammary gland involution reveal novel pro-metastatic mechanisms.

Postpartum involution is the process by which the lactating mammary gland returns to the pre-pregnant state after weaning. Expression of tumor-promotional collagen, upregulation of matrix metalloproteinases, infiltration of M2 macrophages, and remodeling of blood and lymphatic vasculature are all characteristics shared by the involuting mammary gland and breast tumor microenvironment. The tumor promotional nature of the involuting mammary gland is perhaps best evidenced by cases of postpartum breast cancer (PPBC), or those cases diagnosed within 10 years of most recent childbirth. Women with PPBC experience more aggressive disease and higher risk of metastasis than nulliparous patients and those diagnosed outside the postpartum window. Semaphorin 7a (SEMA7A), cyclooxygenase-2 (COX-2), and collagen are all expressed in the involuting mammary gland and, together, predict for decreased metastasis free survival in breast cancer. Studies investigating the role of these proteins in involution have been important for understanding their contributions to PPBC. Postpartum involution thus represents a valuable model for the identification of novel molecular drivers of PPBC and classical cancer hallmarks. In this review, we will highlight the similarities between involution and cancer in the mammary gland, and further define the contribution of SEMA7A/COX-2/collagen interplay to postpartum involution and breast tumor progression and metastasis.

Abstract 2234: Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking diet, development and breast cancer risk

Abstract Evidence supports the notion that critical events during mammary development permanently alter developmentally regulated programs which influence the breast microenvironment to increase breast cancer risk. This is analogous to metabolic memory in diabetic patients where early metabolic events have been found to be remembered and affect disease severity later in life. Advanced glycation end products (AGEs) are highly reactive metabolites that irreversibly accumulate in tissues as we age. AGE accumulation can contribute to pro-inflammatory and -oxidant phenotypes when signaling through the receptor for advanced glycation end products (RAGE). The pathogenic effects of AGE-RAGE signaling include tissue degeneration, protein dysfunction, aberrant cell signaling, and reduced genetic fidelity. AGEs form during normal metabolism but critically, lifestyle factors such as poor diet, a sedentary lifestyle and being obese also contribute to the AGE accumulation pool. The permanence of AGE adducts and their ability to mediate chronic and persistent inflammatory and oxidative stresses is particularly compatible to the concept of metabolic memory. Our dietary studies in pubertal FVB/n mice fed a high AGE diet show a significant dysregulation of mammary gland development and hyperplastic lesion formation. We observe delayed mammary ductal extension, increased ductal branching and aberrant terminal end-bud (TEB) morphology. The basal myoepithelial cell layer surrounding mammary ducts and TEBs was irregular and epithelial cell proliferation was increased. Molecular characterization of these hyperplastic lesions were defined using DCIS progression markers by histopathological staining and qRT-PCR. Elevated AGE levels accompanied increased RAGE expression and increased macrophage and fibroblast infiltration around the TEBs. In attempt to reverse the effects caused by a high AGE diet, mice were fed a control diet after a pubertal high AGE diet. Hyperplastic lesions persisted despite diet intervention. Importantly, hyperplastic lesions were not observed in mice fed a control diet during puberty, then switched to a high AGE diet. These data indicate that exposure to AGE induced changes during puberty may leave a long-lasting imprint analogous to metabolic memory. In conclusion, increased AGE consumption during pubertal growth results in significant disruption of normal mammary development and the appearance of hyperplastic lesions by adulthood. Consumption of a high AGE diet despite a control diet intervention, reveals hyperplastic lesions indicative of metabolic memory. We hypothesize that the high AGE diet may leave a metabolic imprint on the mammary gland microenvironment, increasing the risk of future breast cancer development. Citation Format: Jaime F. Randise, Bradley A. Krisanits, Lourdes M. Nogueira, Kristi L. Helke, Taaliah Campbell, Victoria J. Findlay, David P. Turner. Dietary-AGE ingestion during puberty modifies the breast microenvironment to alter mammary gland development: Linking diet, development and breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2234.

Abstract PR07: Elucidating mechanisms of p53-deficient breast cancer development via lineage tracing and clonal analysis

Abstract TP53 is the most frequently mutated gene in human breast cancers and its mutations have been found in all breast cancer subtypes (e.g., luminal, basal-like, claudin-low). Genome-sequencing studies predict that TP53 mutation is an early event in breast cancer development. However, how p53-deficiency contributes to breast cancer initiation, leading to development of different subtypes of breast cancer, remains largely elusive. To address this, we developed a novel approach to trace development of breast cancer from luminal mammary epithelial cells (MECs), which may represent cells of origin for most breast cancers. Our approach is based on induced loss of p53 in luminal MECs at the level of single cells, via intraductal injection of Cre-expressing adenovirus under the control of the pan-luminal Keratin 8 promoter (Ad-K8-Cre). This genetic approach permits evolution of single mutant luminal MECs in their native habitat through interaction with the microenvironment (e.g., neighboring p53-wild type MECs, stromal cells, immune cells), and allows us to dissect contributions from genetic, epigenetic, and environmental factors to breast tumorigenesis. Lineage and clonal analyses revealed that induced loss of p53 alone in luminal MECs triggered their clonal expansion without affecting their original luminal identity, leading to formation of a p53-deficient luminal premalignant field. Molecular analysis of luminal MECs in this premalignant field revealed potential involvement of environmental factors (e.g., cyclic ovarian hormones, immune cells in the mammary gland microenvironment) in its formation. Claudin-low mammary tumors eventually emerged from this p53-deficient luminal premalignant field with 100% penetrance, apparently due to acquisition of spontaneous, recurrent mutations in p53-deficient MECs. These data support that p53-deficiency alone does not affect the fate of luminal MECs directly, but it facilitates development of mammary tumors with loss of the luminal identity after a long latency. As deficiencies of both p53 and BRCA1 in luminal MECs predispose them to development of basal-like breast cancer (rather than claudin-low cancer), we studied whether induced loss of BRCA1 in p53-deficient luminal MECs would lead to their cell fate alteration. In fact, when we induced loss of both p53 and BRCA1 in luminal MECs, we observed luminal-to-basal cell fate alteration only after a short latency. Together, our data suggest that loss of p53 in single luminal MECs leads to their clonal dominance over wild-type neighbors without directly impairing their luminal fate; it is the cooperating oncogenic events (e.g., loss of BRCA1, or acquisition of recurrent secondary mutations) that drive development of breast cancer from p53-deficient luminal cells toward different subtypes. Our results also predict that loss of p53 in luminal MECs of BRCA1 mutation carriers would not only lead to their clonal dominance, but also cause luminal-to-basal change within the mutant clones (as an early event), and ultimately development of basal-like breast cancer from these aberrant clones. This abstract is also being presented as Poster A35. Citation Format: Dongxi Xiang, Luwei Tao, Zhe Li. Elucidating mechanisms of p53-deficient breast cancer development via lineage tracing and clonal analysis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR07.

Abstract 2974: Obesity-induced modifications in the mammary gland microenvironment promote TNBC progression even after weight loss

Abstract Obesity contributes to 20% of US female cancer deaths and is linked to increased risk for triple negative breast cancer (TNBC), as well as increased risk of vascular invasion, metastasis, and mortality in breast cancer patients. We previously showed that obesity-induced elevation of hepatocyte growth factor (HGF) signaling through its receptor, cMET, accelerated development of tumors in the C3(1)-TAg GEMM of TNBC compared to low fat diet (LFD)-fed lean controls. Weight loss significantly reduced HGF/cMET signaling in normal mammary glands and cMET in tumors, and reversed high fat diet (HFD)-driven tumor progression, while crizotinib-mediated cMET inhibition limited tumor development and microvascular density in both lean and obese C3(1)-TAg mice. To differentiate tumor-intrinsic versus microenvironment-associated mechanisms, we employed an orthotopic transplant model of a C3(1)-TAg TNBC cell line to investigate tumor growth dynamics in lean, obese, or formerly obese (FOb) microenvironments. We hypothesized that TNBC tumors derived from lean C3(1)-TAg mice and transplanted into obese mice would grow more rapidly, contain more macrophages (MFs) and exhibit greater blood vessel density than tumors in lean or FOb mice due to obesity-induced MF and/or endothelial secretion of HGF. At 8 weeks of age, female FVB/NJ mice were randomized to diet groups (N=20/diet): 1) lean (LFD, 10% kcal from fat), 2) obese (HFD, 60% kcal from fat) 3) or FOb (HFD, 5 weeks; LFD, 5 weeks). Following 10 weeks on diets mice were injected with 2x105 luciferase-tagged C3(1)-TAg cells. Tumor volume was measured by calipers. At 21 days post-injection mice were sacrificed for tissue collection. Normal mammary gland, tumors, and tumor-adjacent adipose + tumor-draining lymph node were collected for histological examination and gene expression analysis. Mammary fat pad mass in obese FVB/NJ mice (0.27 g) was significantly greater than lean (0.14 g) or FOb animals (0.11 g) (P&amp;lt;0.0001). Tumor volume at sacrifice was significantly greater in obese vs lean animals (190 mm3 vs 89.96 mm3, respectively; P&amp;lt;0.05). However, mean final tumor volume in FOb animals (148.0 mm3) did not differ significantly from either lean or obese mice. Our findings show that obesity accelerated tumor progression relative to lean mice. C3(1)-TAg cells transplanted into FOb mice resulted in an intermediate rate of tumor growth, suggesting that obesity-mediated changes to the microenvironment were not fully corrected by weight loss despite normalization of body weight and adiposity. To elucidate how the FOb mammary microenvironment persists in driving TNBC tumor promotion, planned analyses include histological examination of candidate mediators, including MF infiltration and regional lymph node metastasis by diet group, as well as quantification of pro-growth and pro-angiogenic signaling molecules (e.g., HGF, VEGF, Ang2) in lysates of tumors and tumor-adjacent adipose. Citation Format: Alyssa J. Cozzo, Ottavia Zattra, Alex J. Freemerman, Liza Makowski. Obesity-induced modifications in the mammary gland microenvironment promote TNBC progression even after weight loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2974. doi:10.1158/1538-7445.AM2017-2974

Loss of monocyte chemoattractant protein-1 expression delays mammary tumorigenesis and reduces localized inflammation in the C3(1)/SV40Tag triple negative breast cancer model

ABSTRACTMonocyte chemoattractant protein 1 (MCP-1) has been implicated as a major modulator in the progression of mammary tumorigenesis, largely due to its ability to recruit macrophages to the tumor microenvironment. Macrophages are key mediators in the connection between inflammation and cancer progression and have been shown to play an important role in tumorigenesis. Thus, MCP-1 may be a potential therapeutic target in inflammatory and difficult-to-treat cancers such as triple negative breast cancer (TNBC). We examined the effect of MCP-1 depletion on mammary tumorigenesis in a model of TNBC. Tumor measurements were conducted weekly (until 22 weeks of age) and at sacrifice (23 weeks of age) in female C3(1)/SV40Tag and C3(1)/SV40Tag MCP-1 deficient mice to determine tumor numbers and tumorvolumes. Histopathological scoring was performed at 12 weeks of age and 23 weeks of age. Gene expression of macrophage markers and inflammatory mediators were measured in the mammary gland and tumor microenvironment at sacrifice. As expected, MCP-1 depletion resulted in decreased tumorigenesis, indicated by reduced primary tumor volume and multiplicity, and a delay in tumor progression represented by histopathological scoring (12 weeks of age). Deficiency in MCP-1 significantly downregulated expression of macrophage markers in the mammary gland (Mertk and CD64) and the tumor microenvironment (CD64), and also reduced expression of inflammatory cytokines in the mammary gland (TNFα and IL-1β) and the tumor microenvironment (IL-6). These data support the hypothesis that MCP-1 expression contributes to increased tumorigenesis in a model of TNBC via recruitment of macrophages and subsequent increase in inflammatory mediators.

Mammary gland stem cells and their application in breast cancer.

The mammary gland is an organ comprising two primary lineages, specifically the inner luminal and the outer myoepithelial cell layers. Mammary gland stem cells (MaSCs) are highly dynamic and self-renewing, and can give rise to these mammary gland lineages. The lineages are responsible for gland generation during puberty as well as expansion during pregnancy. In recent years, researchers have focused on understanding how MaSCs are regulated during mammary gland development and transformation of breast cancer. Here, we summarize the identification of MaSCs, and how they are regulated by the signaling transduction pathways, mammary gland microenvironment, and non-coding RNAs (ncRNAs). Moreover, we debate the evidence for their serving as the origin of breast cancer, and discuss the therapeutic perspectives of targeting breast cancer stem cells (BCSCs). In conclusion, a better understanding of the key regulators of MaSCs is crucial for the clinical treatment of breast cancer.

Marginal zinc intake reduces the protective effect of lactation on mammary gland carcinogenesis in a DMBA-induced tumor model in mice

Breastfeeding can reduce breast cancer risk; however, unknown factors modify this protective effect. Zinc (Zn) modulates an array of cellular functions including oxidative stress, cell proliferation, motility and apoptosis. Marginal Zn intake is common in women and is associated with breast cancer. We reported that marginal Zn intake in mice leads to mammary gland hypoplasia and hallmarks of pre-neoplastic lesions. In the present study, we tested the hypothesis that marginal Zn intake confounds the protective effect of lactation on breast cancer. Nulliparous mice fed control (ZA, 30 mg Zn/kg) or a marginal Zn diet (ZD, 15 mg Zn/kg), were bred and offspring were weaned naturally. Post-involution, mice were gavaged with corn oil or 7,12-dimethylbenz(a)anthracene (DMBA, 1 mg/wk for 4 weeks) and tumor development was monitored. A ZD diet led to insufficient involution, increased fibrosis and oxidative stress. Following DMBA treatment, mice fed ZD had higher oxidative stress in mammary tissue that correlated with reduced levels of peroxiredoxin-1 and p53 and tended to have shorter tumor latency and greater incidence of non-palpable tumors. In summary, marginal Zn intake creates a toxic mammary gland microenvironment and abrogates the protective effect of lactation on carcinogenesis.