Multiparous Mice Research Articles

Alteration in Effects of Endometriosis on Fecundity According to Pregnancy Experience in Mouse Model.

This study is aimed at identifying variations in the effect of endometriosis on fecundity in a mouse model based on prior pregnancy experience. Endometriosis is one of the most prevalent gynecological diseases and is known to impact female fecundity adversely. In this study, an endometriosis mouse model was established by allografting uterine horn tissue using Pelch's method. The effect of endometriosis on fecundity was confirmed in primiparous and multiparous female mice. As fecundity indicators, the pregnancy rate, number of litters, pregnancy period, and survival rate of the pups were investigated. As a result of the experiment, the pregnancy rate decreased, and the pregnancy period tended to be shorter in primiparous female mice. However, there was no significant change in the multiparous mice. In addition, it has been established that correlations exist between the size of lesions and certain fecundity indicators of the lesion, even among primiparous and multiparous females with endometriosis. The study attempted to demonstrate a link between pregnancy experience and fecundity changes caused by endometriosis by experimentally reproducing clinical results using mouse models. These results suggest strategies for identifying several pathophysiological characteristics of endometriosis.

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.

SAT270 Parity Increases Corticosterone Response To Restraint-induced Stress After Mild Blast Traumatic Brain Injury In Mice

Abstract Disclosure: S. Anisowicz: None. M. Bodemeier Loayza Careaga: None. T. Wu: None. Background: Traumatic brain injury (TBI) is a common problem worldwide, with an estimated incidence of 69 million injuries per year. In the US military, one of the most common forms of TBI during operational deployments after Sep 11, 2001 was the blast TBI, thought to be responsible for over 80% of TBI sustained in theater. One consequence of TBI is dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis. HPA axis dysregulation is linked to increased risk for psychiatric disorders. Furthermore, prior research has shown that TBI may dysregulate the HPA axis in a sex-dependent manner. Throughout a female’s life span, there are several sentinel events of hormonal consequence, including pubarche, menopause, and parity, which are known to be times of changes in risks for psychiatric disorders. Objectives: The role of parity on TBI response has not yet been studied, though parity has been shown to decrease fear-related behaviors and decreased corticosterone load in multiparous mice when compared to nulliparous (NP) controls. Our objective is to determine if parity alters corticosterone (CORT) response to restraint, a mild psychological stressor, in mice exposed to mild blast TBI (mbTBI). Methods: One hundred thirteen NP or primiparous (PP) C57Bl6/J mice were randomly assigned to receive sham or mbTBI. Among those that received mbTBI (19.2 ± 0.12 psi), 32 were NP and 24 were PP. Among sham mice, 32 were NP and 26 were PP. All mice received isoflurane anesthesia prior to sham or mbTBI treatment. At 7-8 days after mbTBI, half the mice within each treatment group were subjected to a mild 20-min restraint before being anesthetized by CO2 followed by collection of trunk blood. Serum was harvested and analyzed for CORT concentrations by ELISA (Enzo Life Sciences). Results: All mice that received mbTBI had a longer (p&amp;lt;0.05) righting time compared to those that did not. There was no main effect of parity in the righting time (p&amp;gt;0.1). PP mice who experienced mbTBI demonstrated a greater (p&amp;lt;0.05) CORT response to restraint (157.3 ± 21 pg/mL) when compared to NP mice (95.9 ± 7.2 pg/mL). CORT response to restraint did not differ (p&amp;gt;0.1) between NP and PP mice who did not experience mbTBI (96.1 ± 7.6 vs 109.2 ± 16 pg/mL). Conclusions: Our data indicates that mbTBI alters HPA axis reactivity in PP mice compared to NP mice, increasing the CORT response to restraint induced stress. Parity alone did not alter CORT response to restraint stress or baseline CORT levels. The results of our finding highlight the importance of parity as a factor in an individual’s lifespan. Presentation: Saturday, June 17, 2023

Multiparity increases the risk of diabetes by impairing the proliferative capacity of pancreatic β cells

Pregnancy imposes a substantial metabolic burden on women, but little is known about whether or how multiple pregnancies increase the risk of maternal postpartum diabetes. In this study, we assessed the metabolic impact of multiple pregnancies in humans and in a rodent model. Mice that underwent multiple pregnancies had increased adiposity, but their glucose tolerance was initially improved compared to those of age-matched virgin mice. Later, however, insulin resistance developed over time, but insulin secretory function and compensatory pancreatic β cell proliferation were impaired in multiparous mice. The β cells of multiparous mice exhibited aging features, including telomere shortening and increased expression of Cdkn2a. Single-cell RNA-seq analysis revealed that the β cells of multiparous mice exhibited upregulation of stress-related pathways and downregulation of cellular respiration- and oxidative phosphorylation-related pathways. In humans, women who delivered more than three times were more obese, and their plasma glucose concentrations were elevated compared to women who had delivered three or fewer times, as assessed at 2 months postpartum. The disposition index, which is a measure of the insulin secretory function of β cells, decreased when women with higher parity gained body weight after delivery. Taken together, our findings indicate that multiple pregnancies induce cellular stress and aging features in β cells, which impair their proliferative capacity to compensate for insulin resistance.

Role of Secreted Frizzled-Related Protein 1 in Early Breast Carcinogenesis and Breast Cancer Aggressiveness

A human transcriptome array on ERα-positive breast cancer continuum of risk identified Secreted Frizzled-Related Protein 1 (SFRP1) as decreased during breast cancer progression. In addition, SFRP1 was inversely associated with breast tissue age-related lobular involution, and differentially regulated in women with regard to their parity status and the presence of microcalcifications. The causal role of SFRP1 in breast carcinogenesis remains, nevertheless, not well understood. In this study, we characterized mammary epithelial cells from both nulliparous and multiparous mice in organoid culture ex vivo, in the presence of estradiol (E2) and/or hydroxyapatite microcalcifications (HA). Furthermore, we have modulated SFRP1 expression in breast cancer cell lines, including the MCF10A series, and investigated their tumoral properties. We observed that organoids obtained from multiparous mice were resistant to E2 treatment, while organoids obtained from nulliparous mice developed the luminal phenotype associated with a lower ratio between Sfrp1 and Esr1 expression. The decrease in SFRP1 expression in MCF10A and MCF10AT1 cell lines increased their tumorigenic properties in vitro. On the other hand, the overexpression of SFRP1 in MCF10DCIS, MCF10CA1a, and MCF7 reduced their aggressiveness. Our results support the hypothesis that a lack of SFRP1 could have a causal role in early breast carcinogenesis.

Multiparity and Aging Impact Chondrogenic and Osteogenic Potential at Symphyseal Enthesis: New Insights into Interpubic Joint Remodeling

Pregnancy and childbirth cause adaptations to the birth canal to allow for delivery and fast recovery. To accommodate delivery through the birth canal, the pubic symphysis undergoes changes that lead to the interpubic ligament (IpL) and enthesis formation in primiparous mice. However, successive deliveries influence joint recovery. We aimed to understand tissue morphology and chondrogenic and osteogenic potential at symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. Morphological and molecular differences were found at the symphyseal enthesis among the study groups. Despite the apparent incapacity to restore cartilage in multiparous senescent animals, the symphyseal enthesis cells are active. However, these cells have reduced expression of chondrogenic and osteogenic markers and are immersed in densely packed collagen fibers contiguous to the persistent IpL. These findings may indicate alterations of key molecules in the progenitor cell population maintenance of the chondrocytic and osteogenic lineages at the symphyseal enthesis in multiparous senescent animals, possibly compromising the mouse joint histoarchitecture recovery. This sheds light on the distention of the birth canal and the pelvic floor that may play a role in pubic symphysis diastasis (PSD) and pelvic organ prolapse (POP), both in orthopedic and urogynecological practice in women.

Retinal Venule Coverage by Pericytes Decreases in Multiparous Mice in a Time-Dependent Manner Post-Delivery.

Structural changes in the retinal vasculature have been linked to increased cardiovascular risks and also change as a function of age. Because multiparity has been associated with poorer cardiovascular health scores, we hypothesized that changes in retinal vascular caliber would be observed in multiparous, compared to nulliparous, females and retired breeder males. Age-matched nulliparous (n = 6) and multiparous (n = 11, retired breeder females with 4 ± 1 litters), and male breeder (n = 7) SMA-GFP reporter mice were included for assessment of retinal vascular structure. Multiparous females had higher body mass, heart weight, and kidney weight compared to nulliparous mice, with lower kidney and higher brain weight compared to male breeders. There was no difference in number of retinal arterioles or venules, or arteriole or venule diameter among groups; however, venous pericyte density (number per venule area) decreased in multiparous vs. nulliparous mice and was negatively associated with the time since last litter and with age. Our results suggest that the time elapsed since delivery is an important factor to be considered in multiparity studies. Taken together, changes in vascular structure and potentially function, are time- and age-dependent. Ongoing and future work will determine whether structural changes are associated with functional consequences at the blood-retinal barrier.

Do Aging and Parity Affect VEGF-A/VEGFR Content and Signaling in the Ovary?-A Mouse Model Study.

In this study, the effects of aging and parity on VEGF-A/VEGFR protein content and signaling in the mice ovaries were determined. The research group consisted of nulliparous (virgins, V) and multiparous (M) mice during late-reproductive (L, 9-12 months) and post-reproductive (P, 15-18 months) stages. Whilst ovarian VEGFR1 and VEGFR2 remained unchanged in all the experimental groups (LM, LV, PM, PV), protein content of VEGF-A and phosphorylated VEGFR2 significantly decreased only in PM ovaries. VEGF-A/VEGFR2-dependent activation of ERK1/2, p38, as well as protein content of cyclin D1, cyclin E1, and Cdc25A were then assessed. In ovaries of LV and LM, all of these downstream effectors were maintained at a comparable low/undetectable level. Conversely, the decrease recorded in PM ovaries did not occur in the PV group, in which the significant increase of kinases and cyclins, as well phosphorylation levels mirrored the trend of the pro-angiogenic markers. Altogether, the present results demonstrated that, in mice, ovarian VEGF-A/VEGFR2 protein content and downstream signaling can be modulated in an age- and parity-dependent manner. Moreover, the lowest levels of pro-angiogenic and cell cycle progression markers detected in PM mouse ovaries sustains the hypothesis that parity could exert a protective role by downregulating the protein content of key mediators of pathological angiogenesis.

Multiparity Differentially Affects Specific Aspects of the Acute Neuroinflammatory Response to Traumatic Brain Injury in Female Mice

Pregnancy is associated with profound acute and long-term physiological changes, but the effects of such changes on brain injury outcomes are unclear. Here, we examined the effects of previous pregnancy and maternal experience (parity) on acute neuroinflammatory responses to lateral fluid percussion injury (FPI), a well-defined experimental traumatic brain injury (TBI) paradigm. Multiparous (2–3 pregnancies and motherhood experiences) and age-matched nulliparous (no previous pregnancy or motherhood experience) female mice received either FPI or sham injury and were euthanized 3 days post-injury (DPI). Increased cortical Iba1, GFAP, and CD68 immunolabeling was observed following TBI independent of parity and microglia morphology did not differ between TBI groups. However, multiparous females had fewer CD45+ cells near the site of injury compared to nulliparous females, which was associated with preserved aquaporin-4 polarization, suggesting that parity may influence leukocyte recruitment to the site of injury and maintenance of blood brain barrier permeability following TBI. Additionally, relative cortical Il6 gene expression following TBI was dependent on parity such that TBI increased Il6 expression in nulliparous, but not multiparous, mice. Together, this work suggests that reproductive history may influence acute neuroinflammatory outcomes following TBI in females.

Exposure to environmentally relevant doses of plasticizers alters maternal behavior and related neuroendocrine processes in primiparous and multiparous female mice

Phthalates are organic pollutants frequently detected in the environment. The effects of these substances on male reproduction have been extensively studied but their potential impact on female reproductive behaviors in particular at environmental doses still remains to be documented. In the present study, we examined the effects of chronic exposure to di (2-ethylhexyl) phthalate (DEHP) alone at 5 or 50 μg/kg/d, or in an environmental phthalate mixture on maternal behavior of lactating female mice after a first (primiparous) and a second gestation (multiparous). Exposure of DEHP alone or in a phthalate mixture reduced pup-directed behaviors, increased self-care and forced nursing behaviors and altered nest quality for both primiparous and multiparous dams. In pup-retrieval test, primiparous and multiparous dams exposed to DEHP alone or in a phthalate mixture retrieved their pups more rapidly, probably due to a higher emission of ultrasonic vocalizations by the pups. At lactational day 2 following the third and last gestational period, the neural circuitry of maternal behavior was analyzed. A lower number of oxytocin-immunoreactive neurons in the paraventricular and anterior commissural nuclei was found in dams exposed to DEHP alone or in a phthalate mixture, while no changes were observed in the number of arginine-vasopressin immunoreactive cells. In the medial preoptic area, exposure to DEHP alone or in a phthalate mixture reduced ERα-immunoreactive cell number. Dendritic spine density assessed for DEHP at 5 μg/kg/d was also reduced. Thus, exposure to DEHP alone or in a phthalate mixture altered maternal behavior probably through a neuroendocrine mode of action involving oxytocin and estrogen through ERα, key pathways necessary for neuroplasticity and behavioral processing.

Abstract 1351: Lauryn Werner

Abstract Clinical studies have linked usage of progestins (synthetic progesterone) to breast cancer risk. However, little is understood regarding the role of native progesterone (P4), signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation. To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared to placebo-treated mice. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR is overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands by flow cytometry, which revealed decreased numbers of various immune cell populations. Transgenic mice were also monitored for mammary gland tumor development over a 2-year timespan. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry. Upon long-term monitoring, we determined that multi-parous PR overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR overexpressing mice contained fewer infiltrating immune cells, and RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR overexpressing mice as compared to control mice. Finally, we utilized syngeneic mammary gland tumor models to evaluate the effect of P4 on the growth of PR+ mammary gland tumors in vivo, which revealed that P4 promoted tumor growth and decreased immune cell infiltration of PR+ mammary gland tumors. Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of anti-progestin therapies to promote immune-mediated elimination of mammary gland tumors. Citation Format: Lauryn Rose Werner, Katelin A. Gibson, Merit L. Goodman, Dominika E. Helm, Katherine R. Walter, Sean M. Holloran, Gloria M. Trinca, Richard C. Hastings, Howard H. Yang, Ying Hu, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Rashna Madan, Alfredo A. Molinolo, Mary A. Markiewicz, Prabhakar Chalise, Margaret L. Axelrod, Justin M. Balko, Kent W. Hunter, Zachary C. Hartman, Carol A. Lange, Christy R. Hagan. Lauryn Werner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1351.

Aging accelerates while multiparity delays tumorigenesis in mouse models of high-grade serous carcinoma

ObjectivesThe “incessant ovulation” hypothesis links increased risk for tubo-ovarian high-grade serous carcinoma (HGSC) due to more ovulations and reduced risk conferred by pre-menopausal exposures like oral contraceptive use, multiparity, and breastfeeding. However, most women diagnosed with HGSC are postmenopausal, implying age is a major risk factor for HGSC. Our mouse model for HGSC, based on tamoxifen (TAM)-induced somatic inactivation of the Brca1, Trp53, Rb1, and Nf1 (BPRN) tumor suppressor genes in oviductal epithelium, recapitulates key genetic, histopathologic, and biological features of human HGSCs. We aimed to credential the model for future efforts to define biological and risk modification factors in HGSC pathogenesis. MethodsBPRN mice were treated with TAM to induce tumors at defined ages and parity status. ResultsBPRN mice aged 9-months prior to tumor induction had markedly shorter survival than 6–8 week old mice induced to form tumors (median 46.5 weeks versus 61.5 weeks, log-rank test P = 0.0006). No significant differences in cancer phenotypes were observed between multiparous versus nulliparous BPRN mice. However, using a modified tumor model with one wild-type Nf1 allele (BPRNfl/+), nulliparous mice had more advanced tumors than multiparous mice (Mantel-Haenszel Chi-square test of association, P = 0.01). ConclusionsOur findings show aging is associated with significantly shortened survival post tumor induction in the BRPN model and multiparity delays development and/or progression of HGSC in certain genetic contexts. The findings support relevance of our mouse model to gain mechanistic insights into how known factors exert their protective effects and to test novel approaches for HGSC prevention.

Abstract P5-02-01: A FACS-free purification method to study estrogen signaling, organoid formation, and metabolic reprogramming in mammary epithelial cells

Abstract Mammary epithelial cells (MECs) are known to have their metabolism reprogrammed following pregnancy to allow the increased energy requirements for lactation. The estrogen receptor α (ERα) is mainly associated with the regulation of biological pathways linked to mammary gland development but its influence on MECs metabolism is still unknown. Our hypothesis is that ERα reprograms cell metabolism in normal MECs, a phenomenon that would be reprogrammed during breast carcinogenesis. Few in vitro models are used to study MECs, and most of them do not express ERα. Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs to study ERα metabolic functions using mass spectrometry (MS). Purified MECs from nulliparous mice remain differentiated for up to six days with &amp;gt;85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favouring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. To further understand this reprogramming, stable isotope tracer analysis in primary culture organoids are currently performed. In addition, pregnancy and breast-feeding are known to be protective against breast carcinogenesis. Consequently, we also performed MEC purification and organoid culture using mammary glands from multiparous mice. Intriguingly, organoid phenotypic characterization indicated a difference in organoid structures between MECs from nulliparous and multiparous mice. Furthermore, we observe significant differences in estrogenic response between both conditions, suggesting that pregnancy and/or lactation promotes the establishment of specific epigenetic marks that are preserved even ex vivo. Chromatin immunoprecipitation of specific histone marks and MS-based metabolic studies are ongoing to better understand the different responses of mammary organoids to estrogens between nulliparous and multiparous mice. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures and for MS-based metabolomics. We demonstrated that these organoids retain a functional ERα pathway over time and that ERα significantly reprograms multiple metabolic pathways. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis. Citation Format: Aurélie Lacouture, Cynthia Jobin, Alisson Clemenceau, Cindy Weidmann, Line Berthiaume, Dominic Bastien, Isabelle Laverdière, Martin Pelletier, Caroline Diorio, Francine Durocher, Étienne Audet-Walsh. A FACS-free purification method to study estrogen signaling, organoid formation, and metabolic reprogramming in mammary epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-01.

Abstract P5-06-11: Secreted frizzled related protein 1: From lobular involution to breast osteoimmunological disorder

Abstract Breast cancer is the leading cause of cancer-related death in women worldwide. Our group previously performed a human transcriptome array on normal tissue, atypical ductal hyperplasia, ductal carcinoma in situ and estrogen receptor-α positive invasive ductal carcinoma. Among the transcripts down-regulated during carcinogenesis: Secreted Frizzled-Related Protein 1 (SFRP1), a Wnt signaling pathway antagonist inversely associated with age-related lobular involution has been identified.Interestingly, SFRP1 expression is higher in presence of hydroxyapatite microcalcifications in non-tumoral breast tissue. SFRP1 is also known to be involved in the increase of bone resorption, notably by downregulating osteoblastic differentiation. This suggests that the lack of SFRP1 could be involved in both osteoblast-like phenotype acquisition and early breast carcinogenesis. Unfortunately, data on the mechanism of microcalcifications formation and their impacts on breast carcinogenesis are still lacking.We hypothesize that loss of SFRP1 is responsible for microcalcifications accumulation and early breast carcinogenesis. Our objectives are:1. To define the impact of microcalcifications on SFRP1 expression in non-tumoral mammary gland and 2. to decipher the causal role of SFRP1 in microcalcifications formation. Organoid culture using freshly purified epithelial cells from nulliparous and multiparous mice mammary glands, in presence or in absence of hydroxyapatite crystals has been performed. Organoids number and phenotype have been studied (aim 1). We also have modulated SFRP1 expression in mammary cell lines by lentiviral transduction and performed proliferation, migration and alizarin assays to assess the tumoral aggressivity and cells abilities to produce calcium deposits in vitro (aim 2). SFRP1 and markers of osteoblastic differentiation (Runt-related transcription factor 2, RUNX2) and bone formation (Alkaline Phosphatase, ALPL; Osteopontin, SPP1; Periostin, POSTN) will also be quantified by quantitative PCR in both mice organoids and human mammary cell lines (aims 1 and 2). 1. The ratio between the mammary gland and body weight obtained from nulliparous mice (n = 12) is significantly lower compared with multiparous mice (n = 12; fold-change = 1.84; p-value &amp;lt; 0.001). Hydroxyapatite crystals induced an increase in the proportion of organoids with lumen and branching compared with the control without hydroxyapatite specifically in organoids obtained from nulliparous mice. Organoid’s size and gene expression profiles are still to be assessed. 2. SFRP1 downregulation in a non-tumoral atypical ductal hyperplasia cell line (MCF10AT1) induced an increase in proliferative and migratory cells abilities. It was also responsible for an increase in estrogen receptor 1 (ESR1) expression, suggesting that SFRP1 is related to luminal carcinogenesis. Results also demonstrate that decreasing SFRP1 in MCF10AT1 induced a profile similar to growth stage in bone tissue, which notably means an increase of expression of the osteoblastic transcription factor RUNX2 and the marker of mineralization SPP1, also involved in lactation-related pink adipose tissue formation. We also observed an increase of POSTN expression, known to be involved in decreased bone mineral density during lactation.SFRP1 upregulation in the invasive luminal A cell line MCF7 reduced cell migration and ESR1 expression. Likewise, it increased MCF7 proliferation compared to control. In addition, it induced an increase of RUNX2 and SPP1 expression, similar to what is reported in matrix mineralization profile in bone. These results support our hypothesis that SFRP1 has a causal role in the acquisition of a malignant phenotype but also in osteoblast-like phenotype in mammary epithelial cells. The cause of SFRP1 downregulation in women remains to be explored. Citation Format: Alisson Clemenceau, Aurélie Lacouture, Juliette Bherer, Audet-Walsh Étienne, Caroline Diorio, Francine Durocher. Secreted frizzled related protein 1: From lobular involution to breast osteoimmunological disorder [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-11.

Abstract P4-04-07: Progesterone promotes immunomodulation and tumor development in the murine mammary gland

Abstract Clinical studies have linked usage of progestins (synthetic progesterone) to breast cancer risk. However, little is understood regarding the role of native progesterone (P4), signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation. To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared to placebo-treated mice. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR is overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands by flow cytometry, which revealed decreased numbers of various immune cell populations. Transgenic mice were also monitored for mammary gland tumor development over a 2-year timespan. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry. Upon long-term monitoring, we determined that multi-parous PR overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR overexpressing mice contained fewer infiltrating immune cells, and RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR overexpressing mice as compared to control mice. Finally, we utilized syngeneic mammary gland tumor models to evaluate the effect of P4 on the growth of PR+ mammary gland tumors in vivo, which revealed that P4 promoted tumor growth and decreased immune cell infiltration of PR+ mammary gland tumors. Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of anti-progestin therapies to promote immune-mediated elimination of mammary gland tumors. Citation Format: Lauryn R Werner, Katelin A Gibson, Merit L Goodman, Dominika E Helm, Katherine R Walter, Sean M Holloran, Gloria M Trinca, Richard C Hastings, Howard H Yang, Ying Hu, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Rashna Madan, Alfred A Molinolo, Mary A Markiewicz, Prabhakar Chalise, Margaret L Axelrod, Justin M Balko, Kent W Hunter, Zachary C Hartman, Carol A Lange, Christy R Hagan. Progesterone promotes immunomodulation and tumor development in the murine mammary gland [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-07.

Abstract P1-24-06: Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression

Abstract Background. Lymphovascular invasion (LVI) and breast tumor emboli within dermal and breast lymphatic vessels are prognostic for metastatic spread and poor outcomes, and are abundant in Inflammatory breast cancer (IBC). IBC is an aggressive breast cancer that presents suddenly with breast swelling and redness due to tumor emboli in lymphatics. Lack of breast-feeding and obesity are IBC risk factors. We sought to demonstrate the combinatorial effects of a high-fat diet and nursing on lymphatic function and compare these to IBC tumor induced changes in lymphatic function. We hypothesize that risk factors for aggressive breast cancer may alter lymphatic function in the normal gland prior to tumor initiation. Methods. Following two rounds of pregnancy in 20 multiparous SCID Beige immunocompromised mice, half of the mice were force weaned while half nursed pups. Prior to forced weaning, half of each of these groups were fed a high fat diet (HFD: 60 Kcal %, N = 10) while the other half received a low-fat diet (LFD: 10 Kcal %, N = 10). Consecutive dynamic near-infrared fluorescence (NIRF) lymphatic imaging was performed at 6-7 months (covid interruption) and 14 months after initiating the diet by injecting a near-IR fluorophore into the mammary fatpad and recording lymphatic pulsing over 8 minutes using V++. Matlab and ImageJ were used to quantify pulsing rates on the ventral lymphatics in each animal. Fatpads were subsequently inoculated with SUM149 IBC cells and imaging was repeated 16 months post diet initiation. Lymphatic imaging over time by HFD vs LFD was further studied in nulliparous animals. Tissues were collected for further analyses. ResultsData analysis prior to tumor injection, demonstrated lymphatic pulsing (pulses/4 minutes) increased over time in HFD force weaned (HFFW) and HFD nursing (HFN) animals only (65.5 vs 72.6, P=0.059; 60.1 vs 76.6, P=0.0099, respectively). Comparing HFFW and HFN to matched LFD groups (LFFW and LFN), at 14 weeks HFD was associated with increased pumping after forced weaning (62.3 vs. 72.6, P=0.074), and nursing (62.5 vs 76.6, P=0.0023). There was an increase in pulsing after tumor initiation (16 months after initiation of diet) in all groups (80.1, 84.1, 83.2, 82.4, P &amp;gt; 0.05 all comparisons to initial timepoint). In a separate experiment examining HFD (N=5) vs LFD (N=5) in nulliparous mice, lymphatic contractile activity increased in all animals over. time, average ventral lymphatic contractile frequency for LFD and HFD at week 8,11 and 14 weeks after diet initiation were 5, 8.64, 15.9 pumps/4 mins vs 11.8, 18.5, 28.2 pumps/4 mins, (P = 0.01, 0.05, and 0.0005 respectively). ConclusionsHFD increased lymphatic pulsing rate over time to a significantly greater extent than LFD continuing over 14 months independent of reproductive and nursing status. Tumor initiation prompted further increased pulsing rates beyond that observed after HFD across all groups. The magnitude of the effect of HFD on lymphatic pulsing approached the rate after tumor initiation, while reproductive variables did not impact lymphatic pulsing. Further studies are warranted to demonstrate the relationship if any between lymphatic pumping pre-initiation and LVI after tumor initiation and examine the role of intervention on reducing LVI. Citation Format: Wintana Balema, Janelle Morton, Richard Larson, Fred C Velasquez, Eva Sevick-Muraca, Wendy A Woodward. Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-06.

Progesterone promotes immunomodulation and tumor development in the murine mammary gland

BackgroundClinical studies have linked usage of progestins (synthetic progesterone [P4]) to breast cancer risk. However, little is understood regarding the role of native P4, signaling through the progesterone receptor (PR),...

H2AX Promoter Demethylation at Specific Sites Plays a Role in STAT5-Induced Tumorigenesis.

Deregulated STAT5 activity in the mammary gland of transgenic mice results in parity-dependent latent tumorigenesis. The trigger for cell transformation was previously associated with hyperactivation of the H2AX proximal promoter in a small basal cell population during pregnancy. The current study focuses on the latent activation of tumor development. H2AX was highly expressed in carcinoma and adenocarcinoma as compared to the multiparous mammary gland, whereas pSTAT5 expression decreased in a tumor type-dependent manner. In contrast to the pregnant gland, no positive correlation between H2AX and pSTAT5 expression could be defined in carcinoma and adenocarcinoma. Using targeted methylation analysis, the methylation profile of the H2AX promoter was characterized in the intact gland and tumors. Average H2AX promoter methylation in the tumors was relatively high (~90%), but did not exceed that of the multiparous gland; 5mC methylation was higher in the differentiated tumors and negatively correlated with its oxidative product 5hmC and H2AX expression. Individual analysis of 25 H2AX promoter-methylation sites revealed two consecutive CpGs at positions -77 and - 54 that were actively demethylated in the multiparous gland, but not in their age-matched virgin counterpart. The different methylation profiles at these sites distinguished tumor types and may assume a prognostic role. In-silico and ChIP analyses revealed overlapping methylation-independent SP1-binding and methylation-dependent p53-binding to these sites. We propose that interference with SP1-assisted p53-binding to these sites abrogates H2AX's ability to arrest the cell cycle upon DNA damage, and contributes to triggering latent development of STAT5-induced tumors in estrapausal multiparous mice.

Discordance between GLP-1R gene and protein expression in mouse pancreatic islet cells

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in β-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces β-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized that Glp1r expression is heterogeneous among β-cells and that metabolic stress decreases the number of GLP-1R-positive β-cells. Here, analyses of publicly available single-cell RNA-Seq sequencing (scRNASeq) data from mouse and human β-cells indicated that significant populations of β-cells do not express the Glp1r gene, supporting heterogeneous GLP-1R expression. To check these results, we used complementary approaches employing FACS coupled with quantitative RT-PCR, a validated GLP-1R antibody, and flow cytometry to quantify GLP-1R promoter activity, gene expression, and protein expression in mouse α-, β-, and δ-cells. Experiments with Glp1r reporter mice and a validated GLP-1R antibody indicated that >90% of the β-cells are GLP-1R positive, contradicting the findings with the scRNASeq data. α-cells did not express Glp1r mRNA and δ-cells expressed Glp1r mRNA but not protein. We also examined the expression patterns of GLP-1R in mouse models of metabolic stress. Multiparous female mice had significantly decreased β-cell Glp1r expression, but no reduction in GLP-1R protein levels or GLP-1R-mediated insulin secretion. These findings suggest caution in interpreting the results of scRNASeq for low-abundance transcripts such as the incretin receptors and indicate that GLP-1R is widely expressed in β-cells, absent in α-cells, and expressed at the mRNA, but not protein, level in δ-cells.

Parity Attenuates Intraepithelial Corneal Sensory Nerve Loss in Female Mice.

Aging impacts the ocular surface and reduces intraepithelial corneal nerve (ICN) density in male and female mice. Many researchers use retired breeders to study naturally aged female mice. Yet, the impact of parity and the length of time since breeders were retired on age-related changes in the intraepithelial corneal nerves is not known. Here we study 2 month (M) nulliparous (NP) females as well as 9M, 10M, and 11M NP and multiparous (MP) female mice to determine whether parity impacts the age-related decline seen in corneal axon density; 9M male mice are also included in these assessments. After showing that parity attenuates age-related loss in axon density, we also assess the impact of parity on corneal epithelial cell proliferation and find that it impacts cell proliferation and axon density normalized by cell proliferation. Stromal nerve arborization is also impacted by aging with parity enhancing stromal nerves in older mice. qPCR was performed on 20 genes implicated in ICN density using corneal epithelial RNA isolated from 10M NP and MP mice and showed that NGF expression was significantly elevated in MP corneal epithelium. Corneal sensitivity was significantly higher in 9M MP mice compared to NP mice and increased sensitivity in MP mice was accompanied by increased nerve terminals in the apical and middle cell layers. Together, these data show that parity in mice attenuates several aspects of the age-related decline seen on the ocular surface by retaining sensory axons and corneal sensitivity as mice age.