Mammary Gland Development Research Articles

Fused in sarcoma (FUS) inhibits milk production efficiency in mammals

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals.

Evaluating breast ultrasonography as a complementary diagnostic method in girls with central precocious puberty.

Assessment of breast development by physical examination can be difficult in the early stages and in overweight girls. To investigate ultrasonography (US) for evaluation of early breast development. In a prospective study, 125 girls (age 7.1 ± 1.5years) with breast development before 8years underwent US breast staging, breast volume, and elastography, in addition to clinical/hormonal evaluation for precocious puberty. Accuracy of US for determining breast development and predicting progression to central precocious puberty was investigated. Physical examination revealed glandular breast enlargement in 100 and predominantly lipomastia in 25. Breast US in the former confirmed glandular breast development in 92 (group 1, physical examination and US positive), but not in 8 (group 2, physical examination positive, US negative). Comparison of the two groups demonstrated lower Tanner and US staging, bone age/chronological age, basal luteinizing hormone (LH), breast volume, and uterine volume in group 2. In the 25 lipomastia patients, US demonstrated no breast tissue in 19 (group 3, physical examination and US negative), but US stage ≥ II in 6 (group 4, physical examination negative, US positive) without differences in clinical parameters. After follow-up of 19.8 ± 4.2months, 46/125 subjects were diagnosed with precocious puberty. US stage, total breast volume, and shear-wave speeds were significantly higher in these 46 patients. Multivariate analyses demonstrated breast volume > 3.4cc had odds ratio of 11.0, sensitivity of 62%, and specificity of 89, in predicting progression to precocious puberty, being second only to stimulated LH for all variables. Breast US is a useful predictive tool for diagnosis of precocious puberty in girls. Higher US stages and higher breast volume on US increased the likelihood of eventual diagnosis of precocious puberty.

Molecular Mechanism of Breast Cancer and Predisposition of Mouse Mammary Tumor Virus Propagation Cycle.

Over the past few decades, women have been troubled by grave diseases such as breast cancer, which are biologically and molecularly classified as hereditary diseases. Even though the risk of other cancers is relatively different and the downstream pathway of genetic mutation differs from breast cancer, the continued transformation of genes such as BRCA1 and BRCA2 leads to breast cancer malignancy. Notably at the molecular level, a parallel connection between the normal growth of breast and the progression of mammary cancer where the breast cancer stem cells play a crucial role in the advancement of mammary carcinoma. Arguably, several significant signaling pathways, for instance, ER signaling, HER2 signaling, and Wnt signaling control the typical breast development as well as breast stem cells, thereby cell proliferation, cell differentiation, and cell motility are involved. Incidentally, the Mouse Mammary Tumor Virus (MMTV) is notable among the unexplained viral components influenced by virus-corrupting mammary carcinomas. According to the genesis, MMTV proviral DNA is integrated into mammary epithelial cells, and genomic lymphoid cells during viral replication and triggers the progression of cellular oncogenesis. This overview reveals the deadliest theories on breast cancer, molecular mechanisms, and the MMTV transmission cycle. To establish prevention therapies that are both acceptable and efficacious, addressing apprehensions related to the toxicity of these interventions must be a preliminary hurdle to overcome.

Hormonal regulation of miRNA during mammary gland development.

The mammary gland is a unique organ as most of its development occurs after birth through stages of proliferation, differentiation and apoptosis that are tightly regulated by circulating hormones and growth factors. Throughout development, hormonal cues induce the regulation of different pathways, ultimately leading to differential transcription and expression of genes involved in this process, but also in the activation or inhibition of post-transcriptional mechanisms of regulation. However, the role of microRNAs (miRNAs) in the different phases of mammary gland remodeling is still poorly understood. The objectives of this study were to analyze the expression of miRNA in key stages of mammary gland development in mice and to determine if it could be associated with hormonal variation between stages. To do so, miRNAs were isolated from mouse mammary glands at stages of adulthood, pregnancy, lactation and involution, and sequenced. Results showed that 490, 473, 419, and 460 miRNAs are detected in adult, pregnant, lactating and involuting mice, respectively, most of them being common to all four groups, and 58 unique to one stage. Most genes could be divided into 6 clusters of expression, including 2 encompassing the highest number of miRNA (cluster 1 and 3) and showing opposite profiles of expression, reaching a peak at adulthood and valley at lactation, or showing the lowest expression at adulthood and peaking at lactation. GO and KEGG analyses suggest that the miRNAs differentially expressed between stages influence the expression of targets associated with mammary gland homeostasis and hormone regulation. To further understand the links between miRNA expression and hormones involved in mammary gland development, miRNAs were then sequenced in breast cells exposed to estradiol, progesterone, prolactin and oxytocin. 38, 4, 24 and 66 miRNAs were associated with estradiol, progesterone, prolactin, and oxytocin exposure, respectively. Finally, when looking at miRNAs modulated by the hormones, differentially expressed during mammary gland development, and having a pattern of expression that could be correlated with the relative levels of hormones known to be found in vivo, 16 miRNAs were identified as likely regulated by circulating hormones. Overall, our study brings a better understanding of the regulation of miRNAs throughout mammary gland development and suggests that there is a relationship with their expression and main hormones involved in mammary gland development. Future studies will examine this role more in detail.

130 Parity affects mammary development in late-pregnant swine

Abstract This project was undertaken to determine if mammary development at the end of gestation differs between primiparous and multiparous sows. During gestation, primiparous (n = 19) and multiparous (parities 2 and 3, n = 17) Yorkshire × Landrace sows were fed one daily meal of a conventional corn-based diet. Amounts fed were based on body weight (BW) and backfat thickness at mating. On d 110 ± 1 of gestation, a jugular blood sample was obtained from all sows to measure IGF-1, glucose, FFA and urea. Sows were then weighed and their backfat thickness measured before euthanasia. Mammary glands from one side of the udder (7 glands) were dissected for compositional analyses. The fifth gland of the contralateral row of mammary gland was sampled for histology and immunohistochemical localization of Ki67. There was less total parenchyma (1,437.4 vs 2,004.7 ± 127.1 g) and total extra-parenchymal tissue (1,691.0 vs 2,407.0 ± 125.3 g; P < 0.001) in mammary gland of primiparous compared with multiparous sows. When these values were expressed per kg BW (respective means of 226.0 and 284.0 ± 2.7 kg for primiparous and multiparous sows), there was no difference for either parenchyma or extra-parenchymal tissue across parity groups (P > 0.05). Parity altered all components within the parenchyma (P < 0.001). Specifically, parenchymal tissue from primiparous sows had greater fat and DM percentages, decreased protein percent, and reduced concentrations of DNA (6.59 vs 9.35 ± 0.53 mg/g DM) and RNA (7.76 vs 12.33 ± 0.70 mg/g DM) than that from multiparous sows. On the other hand, the circumference of alveolar lumens was greater in primiparous than multiparous sows (P < 0.001), and the percentage of epithelial cells that were positive for Ki67, an indicator of cell proliferation, also tended to be greater in primiparous sows (P = 0.07). Circulating concentrations of IGF-1 were greater in primiparous than multiparous sows (45.0 vs 27.3 ± 2.8 ng/mL, P < 0.001). None of the other blood variables were affected by parity (P > 0.10). Results show a marked effect of parity on mammary gland development in swine. At the end of gestation, primiparous sows had less developed mammary parenchyma with a tendency for a greater rate of proliferation than multiparous sows. Such a difference could affect the response of mammary tissue to various nutritional or endocrine signals. This information is crucial for developing optimal management strategies that will maximize sow milk yield.

PSIII-12 Influence of early gestational heat stress on biomarkers of mammary gland development in replacement gilts genomically selected for thermotolerance or thermosensitivity

Abstract Heat stress (HS) alters physiological and metabolic processes in lactating sows leading to decreased milk production, which is a major factor limiting growth and survivability of piglets. While genomic selection for thermotolerance may be a viable solution to alleviate the negative effects of HS on pig welfare, it may be linked to a reduction in milk production and subsequently, litter growth performance. Therefore, the study objective was to evaluate the effects of genomic selection for thermotolerance and its interaction with early gestational HS on biomarkers of mammary gland development in replacement gilts. We hypothesized that HS exposure as well as genomic selection for thermotolerance would negatively affect mammary epithelial proliferation rate. A total of 36 Landrace (33%) × Large White (67%) crossbred gilts divergently selected for thermotolerance (TOL; n = 18) or thermosensitivity (SEN; n = 18) were balanced by body weight, bred to a single Duroc sire, and then exposed to either thermoneutral (TN; constant 17 to 22°C) or cyclical HS (26 to 36°C) conditions until d 65 of gestation. From d 66 of gestation until farrowing, all pregnant gilts were exposed to TN conditions. Of the 36 total gilts bred, only 28 became pregnant yielding 15 HS gilts (n = 8 SEN and 7 TOL) and 13 TN gilts (n = 7 SEN and 6 TOL). On d 105 of gestation, a mammary biopsy was taken from all gilts, mammary tissue was placed into 10% buffered formalin, and KI67 immunohistochemical staining was performed to identify proliferating mammary epithelial cells (MEC). Proliferating and non-proliferating MEC populations were counted using ImageJ tool. Data were analyzed using PROC GLM in SAS 9.4 with individual gilt as the experimental unit. Overall, early gestational HS decreased (P < 0.01; 16.53 ± 2.12%) the percent of proliferating MEC relative to those kept under TN conditions (26.42 ± 2.39%). However, no effect of genomic line divergence was detected with any comparison (P > 0.05). In conclusion, early gestation HS, but not genomic selection for thermotolerance, had a negative impact on biomarkers of mammary gland development in replacement gilts. These data may suggest that early gestation HS could have a reductive effect on milk production capacity, which may negatively affect litter growth.

Abstract PO4-24-06: The function of GATA3-PARP1 complex in breast cancer during GATA3-mediated cellular reprogramming

Abstract In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins. Chromatin acts as a physical barrier for many transcription factors, preventing them from efficiently binding to their recognition sequences within the nucleosome. Pioneer factors are a class of transcription factors that bind to nucleosomes and activate silent chromatin. Pioneer factors are frequently involved in various immune cell pathways, tissue development, and homeostasis, thereby, underscoring their important roles in development and disease. Previous research found that pioneer factors have at least three fundamental properties: However, the molecular mechanisms of pioneer factor-mediated cellular reprogramming (mesenchymal-to-epithelial transition (MET)) are largely unknown. For this study, I will use GATA3-mediated MET to study the pioneer factor-mediated cellular reprogramming. GATA3 is a transcription factor that plays a pivotal role in mammary gland development, luminal epithelial differentiation, and cellular reprogramming. Additionally, it has been implicated in various cellular processes such as proliferation, migration, and invasion and is a critical regulator in breast cancer, including triple-negative breast cancer (TNBC). In TNBC, GATA3 expression is often downregulated, correlating with poor prognosis. Our lab and others have shown that GATA3 functions as a pioneer factor that actively changes the chromatin state from closed to open. In luminal breast cancer cells, ER-alpha and FOXA1 are well-known GATA3 co-factors. In mesenchymal breast cancer cells, GATA3 can suppress tumor metastasis by inducing MET in the absence of well-known GATA3 co-factors FOXA1 and ER-alpha1. These findings suggest that GATA3 works with additional, unknown co-factors during MET. We have been studying how GATA3 activates silent chromatin. More than 7 million GATA3 motifs exist in the human genome, yet the experimental data from GATA3 ChIP-seq analysis indicates less than 1% of the motifs are occupied by the pioneer factor, GATA3. In addition to binding selectivity, we have shown that the pioneer factor action is site-specific (context-dependent) and only induces chromatin opening and enhancer formation at a subset of binding sites. To identify a novel co-factor that is involved in GATA3-mediated MET, we performed a rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) assay, which identified the chromatin-modification enzyme, Poly ADP-ribose polymerase 1 (PARP1), as a potential GATA3 co-factor. PARP1 is essential for gene expression regulation and initiating DNA repair. Previous studies have shown PARP1 as a co-factor to the pioneer factor, SOX2. Our genomics data strongly suggest that PARP1 is involved in such context-dependent action of GATA3. Our overall goal is to investigate GATA3-PARP1 interaction during MET to understand the role of pioneer factors in cellular reprogramming. To do so, we are dissecting the GATA2-PARP1 function in two distinct steps: Citation Format: Mikhala Cooper, Motoki Takaku. The function of GATA3-PARP1 complex in breast cancer during GATA3-mediated cellular reprogramming [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-06.

Abstract PO4-24-05: Fluid forces and hormone levels during mammary gland development drive changes in breast epithelium that are relevant to the progression of postpartum breast cancer

Abstract Postpartum breast cancer (PPBC), diagnosed in the 5-10 years after childbirth, has an elevated risk of metastasis and death. Poor outcomes are thought to be due to factors involved in mammary gland involution, an important stage of mammary gland development. Involution functions to return the mammary gland to the normal post-lactation state and involves activation of multiple processes such as inflammation, wound healing, and lymphangiogenesis. Previously, these processes have been linked to mechanical forces induced by fluid flowing past cells, known as fluid shear stress (FSS). We and others have shown FSS impacts processes associated with lactation and breast cancer metastasis. Herein, we describe the results of a cell model for mimicking lactation and the cessation of lactation as an investigative tool for probing the mechanisms involved in PPBC development and progression. We identified changing levels of FSS as a potential physiologic biomarker through which pathways associated with the progression of PPBC could be identified. The role of fluid shear stress in the progression of cancer remains relatively unexplored, mostly due to practical research barriers. Since FSS cannot be accurately measured in vivo, most research to date has relied on in vitro modelling. In contrast, the role of the involuting mammary gland on the progression of breast cancer has often been studied using rodent models. As a result, studying the interactions between fluid shear stress and involution poses a novel engineering challenge. To address this problem, we developed a bioreactor cell model to enable cell exposure to fluid flow, mimicking forces experienced during lactation, in presence of lactogenic hormones (dexamethasone, insulin, prolactin). Previous work has induced FSS using parallel plate flow chambers, and induced lactation in mammary epithelial cells grown in vitro using lactogenic hormone treatment. To our knowledge, this is the first time these experimental conditions have been studied in combination. We established three distinct stages of treatment, consistent with the fluid shear stress and hormonal levels expected in (1) lactation, (2) cessation of lactation, and (3) involution. To determine whether the model mimicked expected in vivo conditions in the postpartum mammary gland, we used morphological markers and measured the protein expression of β-casein (CSN2), a milk protein that is an established marker of lactation. Each stage was validated and analyzed using proteomic and genomic sequencing. Generated datasets were further analyzed using unsupervised clustering, combining publicly available datasets with our data. We determined that β-casein levels were highest when protein was collected after the lactation stage, followed by an initial drop-off in β-casein expression when fluid shear stress were lowered during the cessation period. The β-casein levels continued at a constant, low level during the involution period, despite a reintroduction of fluid shear stress. This suggests that breast cancer cells are initially responsive to shear stress stimulation, consistent with what would be expected in the postpartum mammary gland in vivo. Proteomic and genomic datasets generated from cells exposed to FSS demonstrated that multiple pathways associated with metastasis are upregulated, as compared to static controls. When combined with publicly available involution datasets, pathways involved in extracellular matrix remodeling, inflammation, and lymphangiogenesis emerged. Multiple targets identified through this analysis have been previously linked to metastatic activities during breast cancer, suggesting that FSS and involution are relevant physiologic biomarkers in the context of PPBC. Work to validate hormonal and fluid shear stress conditions in combination is ongoing. Future work will use the validated model to test relevant targets identified from analysis of generated datasets. Citation Format: Maya Stibbards-Lyle, Kristina Rinker, Laura Hall, Seleem Badawy, Kathy Zhan. Fluid forces and hormone levels during mammary gland development drive changes in breast epithelium that are relevant to the progression of postpartum breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-05.

Abstract PO4-28-06: A Singleminded-2s/Sirtuin interac4on in the regula4on of mitochondrial dynamics across normal mammary development and ER+ BC progression

Abstract Characterizing the mechanisms underlying the regulation of cellular differentiation and mitochondrial dynamics are critical to understanding normal development and breast cancer in the mammary gland. Our previous studies have highlighted the significance of Singleminded-2s (Sim2s), a member of the bHLH/PAS family, in regulating mitochondrial dynamics during mammary gland development and the progression of estrogen receptor-positive (ER+) breast cancer. Sim2s is temporally regulated, with maximal expression occurring during mid-lactation. Cross-fostered pups nursed by miceover-expressing Sim2s under the mouse mammary tumor virus (MMTV-Sim2s) display significantly higher weights by mid-lactation compared to pups nursed by control dams. Overexpression of Sim2s leads to alterations in mitochondrial morphology and dynamics, characterized by increased OPA1 expression and decreased DRP1 levels, resulting in enhanced mitochondrial fusion and elongation. Furthermore, we have extended our investigations to an ER+ breast cancer cell line (MCF7). We have identified SIM2s as a tumor suppressor expressed in mammary epithelial cells, known to inhibit epithelial-mesenchymal transition (EMT) and metastasis. In our previous studies, we discovered that loss of SIM2s expression in the MCF7 cell line promotes mitochondrial fragmentation, as evidenced by a decrease in OPA1 expression and an increase in DRP1 levels. In conjunction with these findings, literature suggests that sirtuins also play a role in the regulation of mitochondrial dynamics similar to Sim2s. Specifically, there is an upregulation of Sirtuin 1 in cancer when Sim2 is lost, while Sirtuin 3 increases during differentiation and normal development progression. Therefore, our aim is to elucidate the potential interaction between Singleminded-2s and sirtuins, a family of NAD+-dependent deacetylases, in the regulation of mitochondrial dynamics. We hypothesize that Sim2s may modulate mitochondrial function through its association with sirtuins, thereby influencing normal mammary gland development and breast cancer progression. Citation Format: Hannah Carter. A Singleminded-2s/Sirtuin interac4on in the regula4on of mitochondrial dynamics across normal mammary development and ER+ BC progression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-06.

Pubertal Suppression in Early Puberty Followed by Testosterone Mildly Increases Final Height in Transmasculine Youth

Abstract Purpose Treatment for transmasculine youth (TMY) can involve testosterone (T) treatment and is sometimes preceded by gonadotropin-releasing hormone agonist (GnRHa) treatment for puberty blockade. GnRHa can increase final height in birth-assigned females with central precocious puberty. Maximizing final adult height (FAH) is an important outcome for many TMY. Our objective was to determine how GnRHa treatment before testosterone impacts FAH. Methods Retrospective cohort study at 5 U.S. transgender health clinics. Participants were 32 TMY treated with GnRHa in early-to-mid-puberty before testosterone (GnRHa+T group) and 62 late/post-pubertal TMY treated with only testosterone (T-only group). Results The difference between FAH minus mid-parental target height (MPTH) was +2.3 ± 5.7cm and -2.2 ± 5.6cm in the GnRHa+T and T-only groups, respectively (p<0.01). In the GnRHa+T group, FAH was 1.8 ± 3.4cm greater than predicted adult height (PAH) (p<0.05) and FAH vs. initial height (IH) z-score was 0.5 ± 1.2 vs. 0.16 ± 1.0 (p<0.05). After adjusting for patient characteristics, each additional month of GnRHa monotherapy increased FAH by 0.59cm (95% CI: 0.31,0.9 cm), stage 3 breast development at start of GnRHa was associated with 6.5cm lower FAH compared to stage 2 (95% CI: -10.43, -2.55), and FAH was 7.95cm greater in the GnRHa+T group compared to T-only group (95% CI: -10.85, -5.06). Conclusions Treatment with GnRHa in TMY in early puberty before testosterone increases FAH compared with MPTH, PAH, IH, and TMY who only received testosterone in late/post-puberty. TMY considering GnRHa should be counseled that GnRHa may mildly increase their FAH if started early.

Abstract PO1-24-06: Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in Breast Cancer

Abstract The premise of differentiation therapy in cancer is a strategy that aims at engaging-forward differentiation and cellular reprograming restricting the proliferative, tumor repopulation, stemness, EMT and metastatic capacities of tumor cells leading to the cessation of the aggressive tumor phenotype and offering the cancer patients improved survival for decades. Therefore, deciphering molecular mechanisms deriving differentiation in normal mammary and breast cancer cells may allow the discovery of innovative differentiation-based biomarkers and therapeutics in breast cancer. Lactation is an intricate process that results in the ability of the breast cells to produce a complex nutritious biological fluid to nurse the new-born. While the beneficial effects of breastfeeding to the infant is irrefutable, its effects on maternal health are less investigated especially in relation to breast cancer that still affects 1 in 8 women in high income countries and 1 in 20 globally. Importantly, epidemiological studies have linked breastfeeding to reduced risk of breast cancer by promoting terminal differentiation of the breast epithelial cells. While breastfeeding process is regulated by multiple hormones, growth factors, and transcriptional regulators, the lactation hormone prolactin (PRL) is known to be directly implicated in the hormonal control of breastfeeding. Extensive research including our own work has shown that PRL/PRL receptor (PRLR) pathway derives mammary gland development and importantly mammary epithelial cell terminal differentiation allowing successful lactation. In contrast the role of PRL in breast cancer is still to be fully defined. Importantly, we have accumulated compelling evidence using in-silico publicly available patient data sets and molecular data implicating this pathway as a clinically relevant pro-differentiation pathway driving differentiation and cellular reprograming suppressing stemness, EMT, metastasis and tumorigenesis in breast cancer. To better characterize PRL signaling mediating its pro-differentiation effects, we used large-scale unbiased proteomics analysis of PRLR/interacting proteins in breast cancer cells. Our analyses revealed several novel PRLR-interactors. We have further confirmed these interactions through co-immunoprecipitations/western blotting analyses in breast cancer cells representative the different breast cancer subtypes. We also examined their functional contributions to mammary differentiation and tumorigenesis using in vitro and in vivo assays. Moreover, we defined their potential clinical relevance using bioinformatics in silico databases of breast cancer. Together our study delineates novel PRL/PRLR-downstream signaling mediators important for mammary differentiation and tumorigenesis and may thus be useful as biomarkers and/or therapeutic targets in breast cancer. Citation Format: Dana Hamam, Suhad Ali. Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-06.

Abstract PO5-25-02: Microenvironment based co-culture dependence of breast cancer and immune cell interactions on functional outcomes

Abstract The tumour microenvironment (TME) includes physical forces from interstitial fluid flow and cell-cell interactions that modulate cancer development and progression through activation of multiple oncogenic pathways leading to tumor growth and metastatic spreading. We previously reported on the role of physical forces on epithelial to mesenchymal transition (EMT) and S100 gene family expression and cell to cell adhesion properties with implications to normal breast development and cancer. Signals from the tumor exit the local tumor environment through interstitial fluid transport and cell migration where they can be found in the blood stream. We developed a whole blood RNA gene expression biomarker panel coupled with proprietary software capable of identifying the presence of an active breast cancer signature at early stages of disease with clinical study results previously reported. Here we report on results from transcriptomic and function studies with cell interaction models. Cancer cells can influence immune cells through direct contact or via secreted molecules causing the immune cells to undergo functional changes. To investigate mechanisms involved in the induction of these alterations in a model system, we performed co-culture studies with human monocytic cells and breast cancer cells. Interaction of human monocytic cells with the breast cancer cells caused significant changes in both behavior and transcriptomes. Moreover, using RNAseq and pathway analysis we demonstrated that the changes induced through the exposure to the more aggressive triple negative (TNBC) phenotype were distinct from the alterations triggered upon contact with an ER+ breast cancer cell line. We also show that cancer-immune crosstalk triggers EMT as well as activation of multiple oncogenic pathways associated with cell migration and invasion, cell chemotaxis and cell-to-cell interactions. As the interplay between cancer and immune cells plays an important role in cancer progression, these findings provide important insight into key mechanisms controlling these outcomes. Citation Format: Karen Norek, Jacob Kennard, Kenneth Fuh, Robert Shepherd, Kristina Rinker, Olesya Kharenko. Microenvironment based co-culture dependence of breast cancer and immune cell interactions on functional outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-02.

Pubertal Timing Across Asian American, Native Hawaiian, and Pacific Islander Subgroups.

Earlier puberty is associated with adverse health outcomes, such as mental health issues in adolescence and cardiometabolic diseases in adulthood. Despite rapid growth of the Asian American, Native Hawaiian, and Pacific Islander populations in the US, limited research exists on their pubertal timing, potentially masking health disparities. To examine pubertal timing among Asian American, Native Hawaiian, and Pacific Islander children and adolescents by disaggregating ethnic subgroups. This retrospective cohort study included Asian American, Native Hawaiian, and Pacific Islander youths aged 5 to 18 years assessed for pubertal development at Kaiser Permanente Northern California, a large, integrated health care delivery system. Follow-up occurred from March 2005, through December 31, 2019. Data were analyzed in October 2023. Race and ethnicity, categorized into 11 ethnic subgroups: Asian Indian, Chinese, Filipino, Japanese, Korean, Native Hawaiian and Pacific Islander, Other South Asian, Other Southeast Asian, Vietnamese, multiethnic, and multiracial. Pubertal timing was determined using physician-assessed sexual maturity ratings (SMRs). Outcomes included the median age at transition from SMR 1 (prepubertal) to SMR 2 or higher (pubertal) for onset of genital development (gonadarche) in boys, breast development (thelarche) in girls, and pubic hair development (pubarche) in both boys and girls. In this cohort of 107 325 Asian American, Native Hawaiian, and Pacific Islander children and adolescents (54.61% boys; 12.96% Asian Indian, 22.24% Chinese, 26.46% Filipino, 1.80% Japanese, 1.66% Korean, 1.96% Native Hawaiian and Pacific Islander, 0.86% Other South Asian, 3.26% Other Southeast Asian, 5.99% Vietnamese, 0.74% multiethnic, and 22.05% multiracial), the overall median ages for girls' pubarche and thelarche were 10.98 years (95% CI, 10.96-11.01 years) and 10.13 years (95% CI, 10.11-10.15 years), respectively. For boys' pubarche and gonadarche, median ages were 12.08 years (95% CI, 12.06-12.10 years) and 11.54 years (95% CI, 11.52-11.56 years), respectively. Differences between subgroups with earliest and latest median age at onset were 14 months for girls' pubarche, 8 months for thelarche, 8 months for boys' pubarche, and 4 months for gonadarche. In general, Asian Indian, Native Hawaiian and Pacific Islander, and Other South Asian subgroups had the earliest ages at onset across pubertal markers, while East Asian youths exhibited the latest onset. Restricting to those with healthy body mass index did not substantially change the findings. In this cohort study of Asian American, Native Hawaiian, and Pacific Islander children and adolescents, pubertal timing varied considerably across ethnic subgroups. Further investigation is warranted to assess whether these differences contribute to observed health disparities in adulthood, such as type 2 diabetes and cardiovascular diseases.

Is bovine somatotropin an alternative strategy to overcome the detrimental effects of high-gain diets on prepubertal Holstein × Gyr heifers?

Feeding high-gain diets and an inadequate energy and protein ratio during pre-puberty may lead to impaired growth and mammary gland development of heifers. Thus, frequent application of bovine somatotropin (bST) may prevent future losses in productivity, improve mammary development and animal performance. We aimed to evaluate the effects of bST on digestibility, performance, blood metabolites, mammary gland development, and carcass composition of high-performance prepubertal Holstein × Gyr heifers. Thirty-four Holstein × Gyr heifers with an average initial body weight of 218 ± 49 kg and 14 ± 4 months of age were submitted to an 84-day trial evaluating the effects of no bST or bST injections. Treatments were randomly assigned to each animal within one of the tree blocks. The bST did not influence digestibility or performance parameters. Regarding blood results, IGF1 concentration presented an interaction between treatment and day, where bST heifers had the highest IGF1 concentration. Heifers receiving bST also showed increased ribeye area; however, only an experimental day effect for backfat thickness was observed, with greater accumulation of carcass fat on day 84. Heifers receiving bST had lower pixels/mm² on parenchyma, characteristic of greater parenchymal tissue. Moreover, heifers on bST treatment also had reduced pixels/mm2, characteristic of reduced fat pad tissue. Lastly, bST injections did not influence liver and muscle gene expression, nor most genes evaluated in mammary gland tissue, except for IGFBP3 expression, which was greater for bST heifers. In summary, we confirm the efficacy of bST injections to overcome the detrimental effects of high-gain diets on mammary gland growth and to improve lean carcass gain of prepubertal Holstein × Gyr heifers.

Association analysis of transcriptome and quasi-targeted metabolomics reveals the regulation mechanism underlying broiler muscle tissue development at different levels of dietary guanidinoacetic acid.

The development and characteristics of muscle fibers in broilers are critical determinants that influence their growth performance, as well as serve as essential prerequisites for the production of high-quality chicken meat. Guanidinoacetic acid (GAA) is a crucial endogenous substance in animal creatine synthesis, and its utilization as a feed additive has been demonstrated the capabilities to enhance animal performance, optimize muscle yield, and augment carcass quality. The objective of this study was to investigate the regulation and molecular mechanism underlying muscle development in broilers at different levels of GAA via multiple omics analysis. The 90 Cobb broilers, aged 1 day, were randomly allocated into three treatments consisting of five replicates of six chickens each. The control group was provided with a basal diet, while the Normal GAA and High GAA groups received a basal diet supplemented with 1.2 g/kg and 3.6 g/kg of GAA, respectively. After a feeding period of 42 days, the pectoralis muscles were collected for histomorphological observation, transcriptome and metabolomic analysis. The results demonstrated that the addition of 1.2 g/kg GAA in the diet led to an augmentation in muscle fiber diameter and up-regulation of IGF1, IHH, ASB2, and ANKRD2 gene expression. However, a high dose of 3.6 g/kg GAA in the diet potentially reversed the beneficial effects on chicken breast development by excessively activating the TGF-β signaling pathway and reducing nucleotide metabolite content. These findings would provide a theoretical foundation for enhancing the performance and meat quality of broilers by incorporating GAA as a feed additive.

Central precocious puberty secondary to postoperative craniopharyngioma: two case reports and a literature review.

Craniopharyngioma is a common intracranial tumour in children. Clinical manifestations are related to hypothalamic/pituitary deficiencies, visual impairment, and increased intracranial pressure. Defects in pituitary function cause shortages of growth hormone, gonadotropin, corticotropin, thyrotropin, and vasopressin, resulting in short stature, delayed puberty, feebleness, lethargy, polyuria, etc. However, manifestations involving precocious puberty (PP) are rare. In both patients, surgical resection was performed after the diagnosis of craniopharyngioma, and breast development occurred postoperatively at one month in one patient and at one year and three months in the other patient. Central precocious puberty (CPP) was diagnosed via relevant examinations. Leuprorelin was injected subcutaneously every 28days, and changes in height, weight, bone age, gonadal ultrasound and sex hormones were recorded. During the follow-up of the two children, the sex hormone levels were significantly reduced, and significant acceleration in bone age was not observed. CPP was induced by craniopharyngioma surgery, and treatment with gonadotropin-releasing hormone analogues (GnRHa) inhibited sexual development and bone age progression. More attention should be given to monitoring for CPP during long-term follow-up of craniopharyngiomas in the clinic.

[Current status of pubertal sexual characteristics development of 2 704 girls aged 6-18 years in Tongzhou District of Beijing].

Objective: To understand the current status of pubertal sexual characteristics development of girls aged 6-18 years in Tongzhou District of Beijing and to compare the differences in sexual characteristics development among girls characterized as thin, normal, overweight, and obese. Methods: A cross-sectional survey was conducted among 2 844 girls aged 6-18 years in Tongzhou District of Beijing from September 2022 to July 2023. The developmental stages of breast and pubic hair were assessed on site, and menarche status was inquired. Weight and height were measured. The girls were subsequently characterized into thin, normal, overweight and obese groups. Basic information (including family and personal history) was obtained through questionnaires. Probit probability unit regression was applied to calculate the age of each Tanner stage of sexual characteristics development and the age of menarche. The χ2 test was applied to compare the counting data between two or multiple groups. Results: A total of 2 844 girls were surveyed and 2 704 girls met the inclusion criteria, resulting in a valid response rate of 95.1%. Among these girls, 1 105 (40.9%) were aged 6-9 years, 1 053 (38.9%) were aged 10-13 years, and 546 (20.2%) were aged 14-18 years. The of height-for-age Z-score (HAZ), weight-for-age Z-score (WAZ), and body mass index-for-age Z-score (BAZ) were 0.46(-0.23,1.16), 0.69(-0.16,1.67), and 0.67(-0.27,1.73) respectively. The prevalences of thin, overweight, and obesity were respectively 1.7% (45/2 704), 17.3% (467/2 704), and 19.9% (538/2 704), respectively. There were 45 girls in the thin group, 1 654 girls in the normal weight group, 1 005 girls in the overweight and obesity group. The age of Tanner stage breast 2 (B2), Tanner stage pubic hair 2 (P2), and menarche was 9.0 (95%CI 8.9-9.1), 10.5 (95%CI 10.4-10.6), and 11.4 (95%CI 11.3-1.5) years, respectively. The current status of breast and pubic hair maturity in girls with pubertal development shows that 64.6% (1 211/1 874) of these girls had breast development preceding pubic hair development, 32.4% (607/1 874) had concurrent breast and pubic hair development, and 3.0% (56/1 874) had pubic hairs development preceding breast development. The interval age between B2 and B5 was 4.7 (95%CI 4.6-4.8) years, between P2 and P5 was 4.5 (95%CI 4.4-4.6) years, and between B2 and menarche was 2.4 (95%CI 2.3-2.5) years. The ages of sexual characteristics development in overweight and obese groups were earlier than that in normal and thin groups. The ages of B2 in thin, normal, overweight, and obese groups were 10.0 (95%CI 9.5-10.6), 9.3 (95%CI 9.2-9.4), and 8.6 (95%CI 8.4-8.7) years, respectively. The age of menarche in thin, normal, overweight, and obese groups were 13.1 (95%CI 12.4-13.7), 11.6 (95%CI 11.4-11.7), and 11.1 (95%CI 11.0-11.2) years, respectively. The interval ages between B2 and B5 and between P2 and P5 was 4.5 and 4.1 years, respectively in the overweight and obese groups, and those in normal group and thin group was 4.7 and 4.5 years, 4.6 and 4.7 years, respectively. Conclusions: The ages of sexual characteristics development and menarche tend in Tongzhou District of Beijing to be earlier than that being reported of Beijing's survey 20 years ago. Girls characterized as overweight and obese not only start puberty at an earlier age than girls of normal weight, but also have a shorter developmental process.

Diagnostic Conundrum of a Sertoli Cell Tumor in a 2-Year-Old Girl with Peripheral Precocious Puberty and a Café-Au-Lait Macule: A Case Report

Introduction: Ovarian Sertoli cell tumors represent a subset of sex cord-stromal tumors and are exceedingly rare in prepubertal children. Here we report a girl with vaginal bleeding due to a Sertoli cell tumor who was originally thought to have McCune-Albright syndrome (MAS). Case presentation: A previously healthy girl presented at age two years six months with breast development and vaginal bleeding. On exam, she had Tanner 4 breasts, Tanner 1 pubic hair, estrogenized vaginal mucosa, and a café-au-lait macule. Laboratory studies revealed an elevated estradiol with suppressed gonadotropins and negative tumor markers. Her bone age was advanced by more than three years. Pelvic ultrasound (US) revealed an enlarged uterus and a slightly larger left compared to right ovary. She was started on tamoxifen for presumed MAS. A repeat pelvic US one month later showed a heterogenous mass in the left ovary which was subsequently resected. Pathology revealed a Sertoli cell tumor, lipid-rich variant. Germline sequencing revealed a pathogenic STK11 variant, diagnostic for Peutz-Jeghers syndrome (PJS). Conclusion: The findings in our patient were strikingly similar to those encountered in MAS. To our knowledge, our patient is the youngest ever reported to present with precocious puberty due to a Sertoli cell tumor in the setting of PJS.

Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells.

Adipose tissue is an active endocrine gland, synthesizing and secreting multiple signaling molecules termed adipokines. Following the detection of adipokines and their receptors in the mammary tissue of various species, it is indicated that adipokines play a role in the development of the mammary gland. The aim of the present study was to determine the concentration-dependent influence of three adipokines, leptin, adiponectin, and chemerin, on the viability, apoptosis, and secretory activity of BME-UV1 bovine mammary epithelial cells. The study confirmed that BME-UV1 cells contain the leptin receptor (Ob-R) protein, and express transcripts of adiponectin (ADIPOR1 and ADIPOR2) and chemerin (CMLKR1 and GPR1) receptors. Regardless of the administered dose, none of the three tested adipokines had an effect on the viability of BME-UV1 cells, and the number of apoptotic cells remained unchanged. However, chemerin (100 ng/mL) stimulated BME-UV1 cells to synthesize and secrete αS1-casein, the major protein component of milk. These results indicate that chemerin may be a potent regulator of the bovine mammary epithelial cells' functional differentiation, contributing, along with the major systemic hormones and local growth factors, to the development of the bovine mammary gland.

Carry-over effects of maternal late-gestation heat stress on granddaughter's growth and mammary gland development

Maternal (F0) exposure to late-gestation heat stress reduces their daughter's (F1) mammary gland fat pad mass (FP), parenchyma (PAR) mass, and epithelial cell proliferation when evaluated at birth and weaning, and go on to produce less milk in their first lactation. Herein, we investigated the effect of maternal late-gestation heat stress on whole-body growth and mammary development of their granddaughters (F2). Multiparous F0 cows had access to heat abatement (n = 41, shade, and active cooling via fans and water soakers) or not (n = 41, shade only) for the last 56 d of gestation during a subtropical summer. Consequently, the F1 daughters, born to F0 cows, were heat-stressed (HTF1, n = 36) or cooled (CLF1, n = 37) in utero during the last 2 mo of gestation. All F1 heifers were raised as an identically managed cohort until first calving. The F2 granddaughters, born to HTF1 (HTF2, n = 12) or CLF1 (CLF2, n = 17), were raised as an identically managed cohort until 70 d of age. Dry matter intake (DMI), body weight, hip height, wither height, chest girth, head circumference, mammary gland teat length, and left-right and front-rear teat distances were measured. Average daily gain (ADG) was calculated for the pre-weaned period (0-49 d). Mammary ultrasounds were performed on d 21, 49, and 70 (n = 9/group) on the rear left and right quarters to quantify PAR and FP areas. Mammary biopsies were collected for histological evaluation of epithelial structures (H&E staining), and to quantify cells positive for ERα (estrogen receptor, α subunit), cell proliferation (Ki67), and apoptosis (TUNEL). Heifer growth from birth to d 49 was similar between CLF2 and HTF2 for all parameters evaluated. Distances between teats and teat length were not different between groups. On d 70, CLF2 tended to have a greater average PAR (right and left quarters) relative to HTF2. Although the left FP was smaller in HTF2 relative to CLF2, the average FP was not different. The lumenal and non-lumenal epithelial structures in the PAR of HTF2 were significantly smaller than those of CLF2. In addition, HTF2 had a reduced percentage of proliferating cells in the epithelial and stromal compartments and a greater percentage of apoptotic cells, particularly in the stroma. The percentage of ERα positive cells was significantly reduced in HTF2. In summary, although HTF2 heifer's DMI was similar and they grew at the same rate as CLF2 heifers throughout the pre-weaning phase, their mammary glands had smaller PAR areas with fewer epithelial structures characterized by reduced cell turnover and lower ERα expression. These early changes in the microstructure and cellular turnover of the mammary gland may partly explain the reduction in lactation performance relative to CLF2 counterparts at maturity.