Luminal Epithelial Cells Research Articles

Epidemiology and outcome of individuals with intraductal papillary neoplasms of the bile duct.

Intraductal papillary neoplasm of the bile duct (IPNB) is a rare distinct subtype of precursor lesions of biliary carcinoma. IPNB is considered to originate from luminal biliary epithelial cells, typically displays mucin-hypersecretion or a papillary growth pattern, and results in cystic dilatation[1]. IPNB develops anywhere in the intrahepatic and extrahepatic biliary tracts, and can occur in various pathological stages from low-grade dysplasia to invasive carcinoma. IPNBs have similar phenotypic changes in the occurrence and development of all subtypes, and the prognosis is significantly better than that of traditional (non-papillary) cholangiocarcinoma. To evaluate the clinicopathological features of IPNB to provide evidence-based guidance for treatment. Invasive IPNB, invasive intraductal papillary mucinous neoplasm of the pancreas (IPMN), and traditional cholangiocarcinoma data for affected individuals from 1975 to 2016 were obtained from the Surveillance, Epidemiology, and End Results (SEER) database. Annual percentage changes (APCs) in the incidence and incidence-based (IB) mortality were calculated. We identified the independent predictors of overall survival (OS) and cancer-specific survival (CSS) in individuals with invasive IPNB. The incidence and IB mortality of invasive IPNB showed sustained decreases, with an APC of -4.5% (95%CI: -5.1% to -3.8%) and -3.3% (95%CI: -4.1% to -2.6%) (P < 0.001), respectively. Similar decreases in incidence and IB mortality were seen for invasive IPMN but not for traditional cholangiocarcinoma. Both OS and CSS for invasive IPNB were better than for invasive IPMN and traditional cholangiocarcinoma. A total of 1635 individuals with invasive IPNB were included in our prognosis analysis. The most common tumor sites were the pancreaticobiliary ampulla (47.9%) and perihilar tract (36.7%), but the mucin-related subtype of invasive IPNB was the main type, intrahepatically (approximately 90%). In the univariate and multivariate Cox regression analysis, age, tumor site, grade and stage, subtype, surgery, and chemotherapy were associated with OS and CSS (P < 0.05). Incidence and IB mortality of invasive IPNB trended steadily downward. The heterogeneity of IPNB comprises site and the tumor's mucin-producing status.

Bensulide exposure causes cell division cycle arrest and apoptosis in porcine trophectoderm and uterine luminal epithelial cells

As the use of herbicides in agriculture has increased worldwide, the importance of identifying unexpected toxic effects on non-target organisms is emerging. Bensulide is used on various agricultural crops as an organophosphate herbicide; however, it can pose a high risk to non-target organisms because of its long half-life and accumulative potential. Despite its high risk, the hazardous effects of bensulide on implantation and mechanisms in cells have not been reported. Therefore, in this study, intracellular mechanisms and potential risk of implantation failure were identified in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells derived from pigs with human-like molecular mechanisms in implantation. The LC50 values of bensulide were 5.21 mg/L in pTr cells and 6.49 mg/L in pLE cells. Both cell lines were exposed to bensulide at concentrations <5 mg/L in subsequent experiments. Treatment with 5 mg/L bensulide activated ERK1/2 and JNK. Disrupted mitochondrial membrane potentials of both cell types were identified. In addition, mitochondrial Ca2+ concentration increased to 261.24% and 228.04% in pTr and pLE cells, respectively, and cytoplasmic Ca2+ concentrations decreased by approximately 50% in both cell types. The abnormal regulation of various intracellular environments by bensulide causes cell division cycle arrest and apoptosis. Finally, 5 mg/L bensulide inhibited transcription of implantation-related genes. Collectively, our results suggest that bensulide may interrupt implantation during early pregnancy by disrupting maternal-fetal interaction.

Integrated multi-omics analysis reveals insights into Chinese forest musk deer (Moschus berezovskii) genome evolution and musk synthesis.

Among the artiodactyls, male animals belonging to the Family Moschidae have a unique tissue, the musk gland, with the capability of musk synthesis. However, the genetic basis of musk gland formation and musk production are still poorly understood. Here, musk gland tissues from two juvenile and three adult Chinese forest musk deer (Moschus berezovskii) were utilized to analyze genomic evolution events, evaluate mRNA profiles and investigate cell compositions. By performing genome reannotation and comparison with 11 ruminant genomes, three expanded gene families were identified in the Moschus berezovskii genome. Transcriptional analysis further indicated that the musk gland displayed a prostate-like mRNA expression pattern. Single-cell sequencing revealed that the musk gland is composed of seven distinguishable cell types. Among them, sebaceous gland cells and luminal epithelial cells play important roles in musk synthesis, while endothelial cells master the regulation of cell-to-cell communication. In conclusion, our study provides insights into musk gland formation and the musk-synthesizing process.

43 Metabolomic Analysis of Uterine Luminal Fluid During the Peri-Implantation Period of Pregnancy in Pigs

Abstract During the peri-implantation period of pregnancy in pigs, the rapid elongation of conceptuses (embryonic/fetus and its extraembryonic membranes) is highly dependent on the composition of histotroph secreted from uterine luminal (LE) and glandular (GE) epithelial and stroma cells, as well as selective transport of nutrients. However, little is known about the metabolites in the uterine luminal fluid (ULF) associated with the uterine-conceptus communication during early pregnancy in pigs. Thus, this study was conducted to profile the metabolome in porcine ULF between days 10 and 16 of estrus cycle (C) and pregnancy (P). Gilts were observed for estrus and/or bred via artificial insemination at 12 and 24 h after onset of estrus (day 0). On days 10, 12, 14, or 16 of the estrous cycle and pregnancy (n = 6 gilts per day and status), uteri were flushed with 20 mL sterile PBS (pH 7.2) after gilts were subjected to a midventral laparotomy. Pregnancy was confirmed by the presence of one or more morphologically normal conceptuses. Recoverable uterine flushings (i.e., ULF) were then subjected to metabolomic analysis by LC-MS. Overall, 222 metabolites were detected in the ULF of which 102 were altered by status and 63 were altered by days (P &amp;lt; 0.05; fold change&amp;gt;1.5). Comparing to cyclic day 10 (10C), pregnancy stimulated increases in citric acid, lysine, arginine and other 19 metabolites in the ULF at pregnant day 10 (10P). At 12P when porcine conceptus initiates its morphological changes, ornithine, α-D-glucose, phenylalanine and other 13 metabolites were up-regulated as compared with 12C. At 14P when conceptus initiates implantation, 15 metabolites changed in ULF. At 16P, 123 metabolites were altered in ULF as compared with 16C. Interestingly, of 15 altered metabolites in ULF, 12 were downregulated including asparagine, nicotinamide riboside, and citrulline at 14P. Whereas 102 of 123 altered metabolites were upregulated including phosphorylcholine, 6-phosphate fructose and 6-phosphate glucose in ULF at 16P. To determine the absolute amount of amino acids in the ULF, we performed the targeted metabolomic analyses for 19 amino acids. As a result, arginine, asparagine, glutamic acid, glutamine, histidine, leucine/isoleucine, lysine, phenylalanine, proline, and valine were increased between 10P and 16P. They were less expressed in the ULF of estrus cycle as compared with pregnancy, and remained unchanged between 10C and 16C. Pathway analysis based on KEGG database indicating that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis have the highest degree of enrichment. These novel findings provide the foundation for future investigation of metabolomic and functional studies on both endometrium and conceptus required for elongation of porcine conceptus during peri-implantation period of pregnancy. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture

46 Adrenomedullin: A Novel Peptide Hormone for Uterine Receptivity and Conceptus Elongation in Pigs?

Abstract Adrenomedullin (ADM) is an evolutionarily conserved multi-functional peptide hormone in mammalian species. The implantation sites of conceptuses (embryo/fetus and its extra-embryonic membranes) in pregnant Adm+/− female mice are abnormally spaced leading to fetal crowding, intrauterine growth restriction (IUGR) and increases in pregnancy loss. As litter-bearing species, pigs exhibit a high incidence of early embryonic death (30-40%) and naturally occurring IUGR (15-25%), and significant numbers of stillborn piglets (3-9%). Therefore, it is imperative that the roles of ADM in regulation of uterine receptivity, as well as growth and development of conceptus in pigs be established. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components in uteri from cyclic and pregnant gilts, as well as conceptuses during the peri-implantation period of pregnancy. ADM receptor components include: calcitonin receptor-like receptor (CALCRL; G protein-coupled receptor bound by ADM), receptor activity modifying protein (RAMP2; dimerized with CALCRL to form ADM1 receptor) and RAMP3 (dimerized with CALCRL to form ADM2 receptor with lower specificity to ADM) and atypical chemokine receptor 3 (ACKR3; a decoy receptor that serves as a cell-autonomous molecular rheostat to dampen ADM signaling). Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P &amp;lt; 0.05), pregnant status (P &amp;lt; 0.01) and/or day x status (P &amp;lt; 0.05). Within porcine conceptuses, expression of CALCRL, RAMP2 and ACKR3 increased between days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line isolated from elongating porcine conceptuses recovered on day 2 post-breeding, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P &amp;lt; 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein (p-4EBP1) by 6.1- and 4.9-fold (P &amp;lt; 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation and placentation in pigs. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.

Abstract 54: Regulation of luminal epithelial cell identity in the pre-tumorigenic mammary gland

Abstract The transcription factor MYC is frequently altered in a large portion of human breast cancers; the gene is amplified in ~15% of breast cancers and overexpressed in another 30-50%. Mouse mammary tumor models driven by overexpression of MYC, such as MMTV-Myc, also exhibit a rapid onset and high frequency of mammary tumors indicating that MYC overexpression plays an important role in driving breast carcinogenesis. However, the role of MYC in breast cancer initiation and subtype determination is not well understood. In the normal mammary gland, MYC is a critical regulator of and is essential for alveolar luminal epithelial cell differentiation during pregnancy. Aberrant MYC expression can promote improper alveolar development and milk production and is a potent inducer of cellular proliferation. Based on these known functions of MYC during development, we hypothesized that its overexpression may induce early changes in epithelial cell differentiation leading to pre-tumorigenic shifts in cell lineages that contribute to the formation of luminal breast tumors. To investigate this, we analyzed the mammary glands of MMTV-Myc (MYC) mice prior to any signs of tumor formation. As early as 5 weeks of age, MYC overexpression led to alterations at both the histological and cellular levels. Interestingly, pre-tumorigenic MYC glands phenotypically resembled mid-pregnant glands of WT mice. Specifically, pre-tumorigenic MYC glands demonstrated budding along the mammary ducts and expansion of a Sca1−/CD49b− luminal epithelial cell subset not typically seen in nulliparous mice. RNA-seq was performed on luminal epithelial cells isolated from WT or pre-cancerous MYC mammary glands to define the luminal differentiation states upon MYC overexpression. While there was an overlap in twenty-nine genes that control milk production and cellular metabolism between pre-cancerous MYC luminal cells and those from mid-pregnant WT mice, luminal cells from MYC mice also exhibited a unique fingerprint of forty-one genes that overlap with cell states found in breast cancer. These uniquely expressed luminal cell genes may be important for MYC-regulated tumorigenesis. Ongoing and future experiments will investigate the role of specific MYC-target genes in mammary gland development versus tumor formation to clarify the role of MYC in these divergent processes. Citation Format: Jerrica Breindel, Kayla M. Gross, Charlotte Kuperwasser. Regulation of luminal epithelial cell identity in the pre-tumorigenic mammary gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 54.

Abstract 2657: Preclinical model to evaluate the safety and efficacy of PODO83: A monoclonal antibody against podocalyxin that targets tumor growth and metastasis

Abstract Podocalyxin (PODXL) is a highly glycosylated transmembrane sialomucin normally found on the apical free surface of mesothelial, vascular endothelial, luminal mammary epithelial cells, kidney podocytes and pluripotent and hematopoietic stem cells. We were the first to show that PODXL is upregulated on human cancers and an independent predictor of poor prognosis in breast and high-grade serous ovarian carcinoma (HGSOC). We also demonstrated that PODXL expression is crucial for primary tumor growth, local invasion, and experimental metastasis to distant organs. Subsequently, these results were extended to a wide range of other tumor types. We next developed a large series of high-affinity monoclonal antibodies (mAb) to human PODXL and evaluated these for therapeutic activity against the PODXL+ metastatic breast cancer cell line, MDA-MB-231, in xenografted NOD-SCID IL2Rγ-/- (NSG) mice. One of these, PODO83, potently attenuated primary tumor growth and formation of distal metastases. The PODO83 reactive epitope maps to the juxtamembrane stalk domain (non-glycosylated region) of the PODXL core protein. As a promising therapeutic it is imperative to perform toxicity studies and determine if PODO83 adversely affects the function of PODXL on normal tissues. Accordingly, we generated a transgenic mouse strain (C57Bl/6 congenic) replacing 4 of the 8 mouse PODXL-encoding exons with the orthologous human exons (including those encoding the PODO83 epitope). These mice were healthy and we confirmed by immunohistochemistry that they exhibit appropriate PODO83 immunoreactivity in all expected tissues (podocytes, endothelia, etc). Strikingly, administration of PODO83 in vivo at high doses (4 treatments of 5 mg/kg over 14 days) resulted in no overt signs of toxicity. To now fully assess the safety and efficacy profile of the PODO83 mAb in this preclinical humanized mouse model we have developed mouse metastatic tumor cell line expressing human PODXL and will evaluate the ability of PODO83 to inhibit tumor growth and metastasis while sparing normal tissues in vivo. The results from this study will help us advance PODO83 mAb as a safe and effective approach to treating the wide variety of cancer subtypes that express PODXL. Citation Format: Julyanne Brassard, Michael R. Hughes, Pamela Dean, Diana Canals Hernaez, Janella Schwab, Yasmine Lau, Calvin D. Roskelley, Kelly M. McNagny. Preclinical model to evaluate the safety and efficacy of PODO83: A monoclonal antibody against podocalyxin that targets tumor growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2657.

Abstract 5458: TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast cancer

Abstract Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to lack of an appropriate isogenic model system. To address this technical challenge, we utilized primary breast luminal epithelial cells propagated from healthy donors of different genetic ancestry and their hTERT-immortalized counterparts to identify functional gene expression changes associated with immortalization. We identified elevated expression of TONSL (Tonsoku Like, DNA Repair Protein) as one of the earliest events during immortalization. TONSL is located on chromosome 8q24.3 and amplified in ~20% of breast cancers with significantly higher amplification in metastatic tumors. TONSL forms a complex with FACT and MMS22L1 to modulate multiple cellular pathways including DNA replication, repair through homologous recombination (HR) and functions as a post replicative histone/chromatin reader. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity. While TONSL overexpression alone was insufficient for neoplastic transformation, TONSL-immortalized primary cells modified to overexpress defined oncogenes generated estrogen receptor-positive adenocarcinomas in NSG mice. Analysis of breast tumor microarray with ~500 tumors revealed poor overall and progression free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors including NF-κB and limited access to the tumor suppressor p53. Most importantly, TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in HR and Fanconi Anemia pathways. Consistent with the effects of TONSL on HR-associated genes, TONSL overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was an essential gene for growth of TONSL-amplified breast cancer cell lines in vivo. Breast cancer cell lines with TONSL/chr8q24.3 amplification were sensitive to TONSL-FACT complex inhibitor CBL0137, both in vitro and in vivo. To our knowledge, TONSL is the only gene other than telomerase with immortalizing function and represents a new therapeutic target for breast cancer with chr8q24.3 amplification. Citation Format: Aditi S. Khatpe, Rebecca Dirks, Poornima Bhat-Nakshatri, Henry Mang, Katie Batic, Sarah Swiezy, Jacob Olson, Xi Rao, Yue Wang, Hiromi Tanaka, Sheng Liu, Jun Wan, Duojiao Chen, Yunlong Liu, Fang Fang, Sandra Althouse, Emily Hilsey, Maggie M. Granatir, Rebekah Addison, Constance J. Temm, George Sandusky, Audrey Lee-Gosselin, Kenneth Nephew Nephew, Kathy D. Miller, Harikrishna Nakshatri. TONSL is an immortalizing oncogene of the chromosome 8q24.3 amplicon and new therapeutic target in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5458.

Abstract 1270: ECHS1 mediates metabolic disruption in hormone receptor-positive breast tumor microenvironment

Abstract Unchecked hyperadiposity causes systemic metabolic perturbations and subclinical chronic inflammation, promoting hormone receptor positive (HR+) breast cancer. Murine models of high-fat diet-induced obesity have shown alterations in proteins involved in fatty acid binding and mitochondrial beta-oxidation, including enoyl-CoA hydratase short chain 1 (ECHS1), which promote increased uptake of fatty acids by primary tumor cells. This creates a metabolic tug of war between tumor and immune cells in the tumor microenvironment, thereby depriving cytotoxic immune cells of the metabolic reprogramming for anti-tumor functionality. Nonetheless, metabolic gene expression changes in breast tumor microenvironment of obese individuals remain elusive. We hypothesize that increased expression of ECHS1 leads to immune dysregulation in breast tumor microenvironment and increases risk of cancer progression. Proteomic and genomic expression and survival characteristics of ECHS1 in invasive breast cancer was explored in The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium. We the modeled the effects of ECHS1 expression on immune infiltration in the tumor microenvironment using a regression framework. We also performed single cell RNA sequencing analysis for differential expression of ECHS1 in various human breast cells. Additionally, expression level of ECHS1 was validated in silico in adipose tissue to elucidate increased immune dysregulation and risk of progression of breast carcinoma in obese individuals. We found a significantly increased expression of ECHS1 at both RNA and protein levels in HR+ breast cancer (p&amp;lt;0.001 for both), compared to Herceptin 2 receptor-positive or triple negative breast cancer. High expression of ECHS1 in female breast cancer is also associated with significantly decreased survival based on the TCGA data. Further, we found a significantly negative correlation of CD8+ T cells, neutrophils, and macrophages, and a significantly positive correlation of regulatory T cells with ECHS1 expression in the breast tumor microenvironment. We discovered increased expression of ECHS1 in luminal epithelial cells compared to myoepithelial cells based on single cell RNA sequencing. Lastly, high expression of ECHS1 protein expression based on immunohistochemistry was confirmed in human adipocytes. Collectively, our observations support the hypothesis that preferential uptake of free fatty acid through increased expression of ECHS1 in HR+ breast cancer impairs cytotoxic and anti-tumor effects of CD8+ T cells in the tumor microenvironment. Immune dysregulation is further amplified in obese individuals given increased levels of adipose cells and higher ECHS1 expression. Altogether, ECHS1 is a putative biomarker and potential therapeutic target as its downregulation may improve survival in obese patients with HR+ breast cancer. Citation Format: Tina Bharani, Divyansh Agarwal, Estefania Roldan-Vasquez, Jessalyn Ubellacker, Ted A. James. ECHS1 mediates metabolic disruption in hormone receptor-positive breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1270.

MP60-20 ROLE OF SHH PATHWAY IN URINARY CONTINENCE AND ITS IMPACT ON CELLULAR FATE IN MOUSE URETHRA

You have accessJournal of UrologyCME1 Apr 2023MP60-20 ROLE OF SHH PATHWAY IN URINARY CONTINENCE AND ITS IMPACT ON CELLULAR FATE IN MOUSE URETHRA Walid Farhat, Priyank Yadav, Marcela Ambrogi, Audrey Spiegelhoff, Chad Vezina, and Kimberly Keil Stietz Walid FarhatWalid Farhat More articles by this author , Priyank YadavPriyank Yadav More articles by this author , Marcela AmbrogiMarcela Ambrogi More articles by this author , Audrey SpiegelhoffAudrey Spiegelhoff More articles by this author , Chad VezinaChad Vezina More articles by this author , and Kimberly Keil StietzKimberly Keil Stietz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003318.20AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sonic hedgehog (Shh) signaling is critical in many developing organs and subject to disruption through gene-environment interactions. Urinary continence is dependent on appropriate development of bladder neck and urethra driven by epithelial-mesenchymal interactions mediated in part by SHH. To further understand the impact of Shh pathway disruption on lower urinary tract and continence, we examined voiding physiology and histology in compound mutant GLI family zinc finger Gli2+/−; Gli3Δ699/+ mice, which have hypomorphic hedgehog signaling. METHODS: Voiding physiology was tested in young adult female mice (n=5-8/genotype) using void spot assay, uroflowmetry and ex vivo bladder bath assays. Urethra and bladder (n=3/genotype) were collected for immunostaining using antibodies against smooth muscle alpha actin to visualize urethral smooth muscle thickness, or uroplakin, transformation related protein 63, and keratin 5 (combinatorial staining resolves basal, intermediate and luminal epithelial cells). RESULTS: Gli2+/−; Gli3Δ699/+ female mice void more urine spots, void more spots of smaller diameter and void a greater percentage of urine in the cage center than wild type mice. Voids from Gli2+/−; Gli3Δ699/+ mice, measured by uroflowmetry, weigh less (indicating smaller volume) and are shorter in duration than wild type mice. Gli2+/−; Gli3Δ699/+ female mouse bladders, isolated in ex vivo bath preparations, are less responsive to electrical field stimulation and potassium chloride than bladders from wild type mice. Our results are consistent with detrusor under activity and potential stress incontinence in Gli2+/−; Gli3Δ699/+ female mice. In search for a physiological mechanism, we conducted histological analysis to reveal a clear anatomic boundary between wild type mouse bladder neck and urethra. Umbrella shaped uroplakin+ cells populate the bladder neck and cuboidal shaped uroplakin- superficial cells populate the urethra. The bladder neck epithelium of Gli2+/−; Gli3Δ699/+ mice does not appreciably differ from wild type, but urethral epithelial cells are uroplakin+, suggesting possible segmental deficits during urethral development that may account in part for incontinence in Gli2+/−; Gli3Δ699/+ mice. CONCLUSIONS: Our data suggest that establishing continent voiding function in mice is dependent on appropriate urethra epithelial cell fate. Future work to understand mechanisms underlying decreased bladder contractile properties, and urethral cell fate will enhance understanding of the continence mechanism. Source of Funding: DK104310 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e851 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Walid Farhat More articles by this author Priyank Yadav More articles by this author Marcela Ambrogi More articles by this author Audrey Spiegelhoff More articles by this author Chad Vezina More articles by this author Kimberly Keil Stietz More articles by this author Expand All Advertisement PDF downloadLoading ...

Defining morphologic features of invasion in pulmonary non-mucinous adenocarcinoma with lepidic growth - A proposal by the IASLC Pathology Committee.

Since the 8th edition of UICC/AJCC TNM classification system the primary tumor pT stage is determined based on presence and size of the invasive components. The aim of this study was to identify histological features in tumors with lepidic growth pattern that may be used to establish criteria for distinguishing invasive from non-invasive areas. A Delphi approach was used with two rounds of blinded anonymized analysis of resected non-mucinous lung adenocarcinoma cases with presumed invasive and non-invasive components, followed by one round of reviewer de-anonymized and unblinded review of cases with known outcomes. A digital pathology platform was used for measuring total tumor size and invasive tumor size. The mean coefficient of variation for measuring total tumor size and tumor invasive size was 6.9% (range 1.7-22.3%) and 54% (range 14.7-155%), respectively, with substantial variations in interpretation of the size and location of invasion among pathologists. Following the presentation of the results and further discussion among members at large of the IASLC Pathology Committee, extensive epithelial proliferation (EEP) in areas of collapsed lepidic growth pattern is recognized as a feature likely to be associated with invasive growth. EEP is characterized by multilayered luminal epithelial cell growth, usually with high grade cytological features in several alveolar spaces. Collapsed alveoli and transition zones with EEP were identified by the Delphi process as morphologic features that were a source of interobserver variability. Definition criteria for collapse and EEP are proposed to improve reproducibility of invasion measurement.

PD02-08 ESR1+ LUMINAL EPITHELIAL CELLS DRIVE THE PROSTATE GROWTH AND SURVIVAL IN THE ABSENCE OF SRD5A2

You have accessJournal of UrologyCME1 Apr 2023PD02-08 ESR1+ LUMINAL EPITHELIAL CELLS DRIVE THE PROSTATE GROWTH AND SURVIVAL IN THE ABSENCE OF SRD5A2 Christina Sharkey, Xingbo Long, Ra'ad Al-Faouri, Aria F. Olumi, and Zongwei Wang Christina SharkeyChristina Sharkey More articles by this author , Xingbo LongXingbo Long More articles by this author , Ra'ad Al-FaouriRa'ad Al-Faouri More articles by this author , Aria F. OlumiAria F. Olumi More articles by this author , and Zongwei WangZongwei Wang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Steroid 5α reductase 2 (SRD5A2) is the predominant enzyme responsible for prostatic development and growth. 5α reductase inhibitors (5ARI) are the only class of benign prostate hyperplasia-related medications that reduce prostate size. However, why the patients respond variably to 5ARI therapies is still largely unknown. We previously demonstrated that 30% of adult human prostatic tissues do not express the SRD5A2 gene and protein via epigenetic modifications. We established a SRD5A2 null (Srd5a2-/-) mouse model and through single cell RNA sequencing, we identified LE2, a sub-cell population of ESR1+ luminal epithelial that is upregulated in Srd5a2-/- mice. Our goal in this study is to characterize the spatial expression of LE2 markers and its functional phenotype when SRD5A2 is absent. METHODS: Homozygous Srd5a2-/- mice and littermate control heterozygous SRD5A2+/- mice were generated and differentially expressed gene profiles of LE2 were obtained from single-cell RNA sequencing (scRNA seq). Spatial expression of LE2 markers was evaluated by RNA fluorescence in-situ hybridization (RNA-FISH). Cell proliferation was detected by Ki67 and BrdU staining. The Cell Death and ER signaling pathways were evaluated with RT2 PCR profiler arrays (84 genes per array) and downstream signaling pathways were analyzed by western blot. Prostate biopsies from the clinical trial of 5ARI for Medical Therapy of Prostatic Symptoms (MTOPS) were used for immunohistochemistry. RESULTS: The prostate weight of Srd5a2-/- mice was significantly decreased compared to Srd5a2+/- littermate control mice (p<0.001). The prostate cell proliferation was suppressed in Srd5a2-/- mice (p=0.05). The cell death signaling akt1, caspase 7, caspase 9, and ER target genes cyp1a1 and vegfa were downregulated (p<0.05). On the other hand, in the anterior lobe of prostate of Srd5a2-/- mice, the cell proliferation was higher than that in control. scRNA seq showed that ESR1 was expressed mainly in the prostate anterior lobe and the number of ESR1+ LE2 cells was significantly increased upon SRD5A2 absence. This was confirmed by RNA-FISH using the probes of LE2 cell markers ESR1 and PVT1. Co-staining of LE2 cell markers ESR1 and PKCa in prostate biopsies of MTOPS further confirmed the existence of an equivalent LE2 cell population in the human prostate with BPH. CONCLUSIONS: Our data demonstrate that LE2, the subpopulation of prostate cells may play an important role in alternative pathways for prostate growth and survival in absence of SRD5A2 and androgen deprivation. Understanding the underlying androgen-independent pathways contributing to prostate growth can help identify novel therapeutic targets for the management of prostatic diseases. Source of Funding: NIH/R01 DK124502 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e71 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christina Sharkey More articles by this author Xingbo Long More articles by this author Ra'ad Al-Faouri More articles by this author Aria F. Olumi More articles by this author Zongwei Wang More articles by this author Expand All Advertisement PDF downloadLoading ...

Endoplasmic reticulum stress response and the regulation of endometrial interferon-beta production

To gain an understanding of the potential role of endoplasmic reticulum (ER) stress in the endometrial compartment during early pregnancy, a highly understudied area. This study examined the regulation of interferon-β (IFNβ) in response to ER stress in human decidualized and nondecidualized endometrial cells (human endometrial stromal cells [HESCs]) invitro. Invivo, we examined ER stress and the IFNβ levels locally in the mouse endometrium before and after implantation at embryonic day (E)1, E3, and E6. The study was performed in a reproductive sciences laboratory for Human Growth and Development. None. None. Quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis allowed us to test the action of endogenous ER stress activation in the endometrial compartment likely triggered by implantation and its ability to increase the endometrial IFNβ levels. Invitro, we observed a significant difference in the IFNβ levels in HESCs, in response to ER stress activation, where decidualized HESCs exhibited a threefold increase in the IFNβ levels compared with nondecidualized HESCs. Apoptotic caspase-3 activation was also isolated to the decidualized cells as a result of ER stress-dependent suppression of nuclear factor-kappa beta-regulated antiapoptotic factors, XIAP and MCL-1. Invivo, mouse endometrial IFNβ was present in F4/80-positive macrophages at all time points examined. After implantation (E6), the mouse luminal epithelial cells robustly coexpressed both IFNβ and the ER stress marker immunoglobulin heavy chain binding protein (BiP). These analyses demonstrate that both invivo and invitro, differentiated and decidualized endometrial cells undergoing ER stress have the capacity to produce increased IFNβ levels; therefore, ER stress activation in the endometrial compartment may play a vital role in promoting successful implantation events.

Protective Role of Kelulut Honey against Toxicity Effects of Polystyrene Microplastics on Morphology, Hormones, and Sex Steroid Receptor Expression in the Uterus of Rats.

Microplastics (MPs) are an emerging global pollutant. Previous studies have revealed that chronic exposure to MPs can affect animal and human reproductive health, particularly by impairing the reproductive system's normal functions, which may increase the risk of infertility in both males and females. Kelulut honey (KH), an excellent source of antioxidants, has been used to counteract the disruptive effects of Polystyrene microplastics (PS-MPs) in the rat uterus. Thus, this study aimed to investigate the potential protective effects of Kelulut honey against PS-MPs-induced uterine toxicity in pubertal rats. Prepubertal female Sprague Dawley rats were divided into four groups (n = 8): (i) normal control group (NC: treated with deionized water), MPs-exposed group (M: exposed to PS-MPs at 2.5 mg/kg), (iii) Kelulut honey group (DM: pretreated with 1200 mg/kg of KH 30 minutes before they were administered with PS-MPs at 2.5 mg/kg), and (iv) Kelulut honey control group (DC: only treated with KH at 2.5 mg/kg). The rats were treated orally once daily for six consecutive weeks. Uterine abnormalities in PS-MPs-exposed rats were significantly improved after concurrent treatment with Kelulut honey. Morphology improvement was observed and luminal epithelial cells seemed thicker with more goblet cells, glandular cells had a more regular and circular shape, stromal cell increased in size, interstitial gaps between stromal cells expanded, and the myometrium layer was thicker. Kelulut honey treatment also effectively normalized the suppressive effect of PS-MPs on the expression and distribution of sex steroid receptors (ERα and ERβ), as well as the level of serum gonadotropin (LH and FSH) and sex steroid (estradiol and progesterone) hormones. Kelulut honey can protect the female reproductive system against the disruptive effects of PS-MPs. The phytochemical properties of Kelulut honey might be responsible for these beneficial benefits. However, future studies are warranted to identify the mechanisms involved.

Hormonal profile prior to luteolysis modulates the uterine luminal transcriptome in the subsequent cycle in beef cross-bred cows§.

Sex steroid concentrations modulate endometrial function and fertility in cattle. Our objective was to compare the post-estrus luminal transcriptome of cows that were exposed to contrasting concentrations of progesterone (P4) before luteolysis that displayed estrus and ovulated spontaneously. Cross-bred beef cows received either 1) a new CIDR and GnRH (d - 9; high progesterone treatment; HP4; n = 16) or 2) a previously used CIDR, PGF2α, and GnRH (low progesterone treatment; LP4; n = 24). All cows received PGF2α at CIDR removal (d - 2). Ovarian ultrasonography and blood collections were performed on d - 9, d - 2, d - 0.5, d 0 (day of observed estrus), d 4, d 7, and d 14 for measurement of ovarian structures, P4, and estradiol (E2). Luminal epithelial cells were collected using a cytology brush on d 4, d 7, and d 14 for RNAseq. On d - 2, CL area and concentrations of P4 were greater, while on d - 0.5 concentrations of E2 were decreased in HP4. Ovarian structures and hormonal concentrations were similar on d 4, d 7, or d 14 (P > 0.05). There were enriched pathways in HP4 related to activation and signaling of the innate immune system at d 4, downregulation in the network involved in the extracellular matrix remodeling at d 7, and exacerbated inflammatory response as well as differentiation and activation of macrophages at d 14 (Benjamini - Hochberg P-value ≤0.05). In conclusion, manipulation of pre-luteolysis sex steroid concentrations altered the post-estrus luminal transcriptome even though all cows showed estrus and ovulated spontaneously.

Abstract P5-03-09: Cell composition changes in healthy breast tissue is associated with advancing age and epigenetic age acceleration

Abstract Introduction: Risk factors for breast cancer include advancing age, lifetime estrogen exposure, and breast density. DNA methylation-based estimates of age are elevated in breast tissue of healthy women compared with paired blood samples, and the degree of age acceleration is associated with lifetime estrogen exposure. However, no prior work has examined cell compositional changes associated with breast epigenetic aging. In this study we estimated the abundance of different cell types in healthy breast, computed using gene expression data, and investigate cell composition changes that accompany advancing chronologic age and breast epigenetic age acceleration. Methods: DNA/RNA were extracted (AllPrep, Qiagen) from breast tissue specimens from 192 healthy women aged 19-90 years who donated breast tissue to the Susan G. Komen Tissue Bank at the Indiana University Simon Comprehensive Cancer Center. Transcriptome analysis was performed using the QuantSeq 3’mRNA-SeqFWD kit to generate RNA sequencing libraries. DNA methylation age was estimated using beta-values from Illumina EPIC 850K array platform. Age acceleration is defined using the residual of a linear regression of methylation age on chronologic age. Cell deconvolution was performed using CIBERSORTx to estimate the abundance of adipocytes, luminal epithelial cells, basal myoepithelial cells, vascular endothelial cells, lymphatic endothelial cells, immune cells (dendritic cells &amp; macrophages), pericytes &amp; smooth muscle cells, and fibroblasts. We examined cell composition changes with chronologic age, and with age-adjusted measures of acceleration in 6 epigenetic clocks: the Horvath Pan-tissue, Hannum, Phenotypic, Grim, Skin &amp; Blood, and Epigenetic Pacemaker clocks, as well as the DNA methylation-based estimate of telomere length, DNAmTL. Results: Advancing chronologic age was associated with an increase in the imputed proportion of adipocytes (R=0.40, p&amp;lt; 0.0001), vascular endothelial cells (R=0.23, p=0.0033), and immune cells (dendritic cells &amp; macrophages) (R=0.29, p=0.00016), and a decrease in the proportion of luminal epithelial cells (R=-0.43, p&amp;lt; 0.0001), and basal myoepithelial cells (R=-0.27, p=0.00047). Epigenetic age acceleration was significantly associated with increases in proportions of luminal epithelial cells (p&amp;lt; 0.0001 for age-adjusted Hannum, Phenotypic, Grim, and Skin &amp; Blood clocks, p&amp;lt; 0.05 for Pan-tissue) and basal myoepithelial cells (p&amp;lt; 0.0001 for Phenotypic and Skin &amp; Blood clocks), and decreased proportions of adipocytes and vascular endothelial cells (p&amp;lt; 0.0001 for Hannum, Phenotypic, Grim, and Skin &amp; Blood clocks, p&amp;lt; 0.05 for Pan-tissue). Conclusion: Using gene expression data in healthy female breast tissue, we identified significant changes in cell-type-specific abundance that accompany advancing chronologic age, with an increase in adipocytes and immune cells, and a decline in luminal epithelial cells and basal myoepithelial cells. By contrast, epigenetic age acceleration in breast tissue is associated with decreasing proportions of adipocytes and vascular endothelial cells and a rise in basal myoepithelial cells and luminal epithelial cells. Our findings suggest distinct patterns of cellular composition changes that accompany normal aging compared with accelerated aging in breast tissue. Citation Format: Mary E. Sehl, Wenbin Guo, Collin Farrell, Natascia Marino, Jill Henry, Anna Maria Storniolo, Jeanette Papp, Jingyi Li, Steve Horvath, Matteo Pellegrini, Patricia A. Ganz. Cell composition changes in healthy breast tissue is associated with advancing age and epigenetic age acceleration [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-09.

Lineage plasticity enables low-ER luminal tumors to evolve and gain basal-like traits

Stratifying breast cancer into specific molecular or histologic subtypes aids in therapeutic decision-making and predicting outcomes; however, these subtypes may not be as distinct as previously thought. Patients with luminal-like, estrogen receptor (ER)-expressing tumors have better prognosis than patients with more aggressive, triple-negative or basal-like tumors. There is, however, a subset of luminal-like tumors that express lower levels of ER, which exhibit more basal-like features. We have found that breast tumors expressing lower levels of ER, traditionally considered to be luminal-like, represent a distinct subset of breast cancer characterized by the emergence of basal-like features. Lineage tracing of low-ER tumors in the MMTV-PyMT mouse mammary tumor model revealed that basal marker-expressing cells arose from normal luminal epithelial cells, suggesting that luminal-to-basal plasticity is responsible for the evolution and emergence of basal-like characteristics. This plasticity allows tumor cells to gain a new lumino-basal phenotype, thus leading to intratumoral lumino-basal heterogeneity. Single-cell RNA sequencing revealed SOX10 as a potential driver for this plasticity, which is known among breast tumors to be almost exclusively expressed in triple-negative breast cancer (TNBC) and was also found to be highly expressed in low-ER tumors. These findings suggest that basal-like tumors may result from the evolutionary progression of luminal tumors with low ER expression.

Abstract P2-26-08: Influence of genetic ancestry on breast stromal cells provides biologic basis for increased incidence of metaplastic breast cancer in women of African descent

Abstract The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to the ductal epithelial cells in the normal breast of women of African Ancestry (AA) compared to European Ancestry (EA). By establishing and characterizing cell lines corresponding to these cells and validating in vitro findings with tissue microarrays of healthy breast tissue from AA, EA and Latina Ancestry (LA) women, we demonstrate that these cells have the properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFR. PROCR+/ZEB1+/PDGFR+ cells, hence renamed as PZP cells, are enriched in the normal breast tissues of AA compared to EA or LA women. In vitro, PZP cells trans-differentiated into adipocytes or osteocytes. In co-culture conditions, PZP:epithelial cell communication resulted in luminal epithelial cells acquiring basal/stem cell characteristics and increased expression of IL-6 suggesting the impact of this communication on the microenvironment and breast epithelial hierarchy. Consistent with this possibility, the level of phospho-STAT3, which is a downstream target of IL-6, was higher in the normal and cancerous breast tissues of AA compared to EA women. PZP cells transformed with HRasG12V ± SV40-T/t antigens generated metaplastic carcinoma in NSG mice suggesting that these cells could be the cell-of-origin of metaplastic breast cancers. Collectively, these results identify a stromal cell component that could influence the biology of breast cancer in AA women. Citation Format: Brijesh Kumar, Katie Batic, Poornima Bhat-Nakshatri, Maggie Granatir, Rebekah Addison, Megan Szymanski, Lee Ann Baldridge, Constance Temm, George Sandusky, Sandra Althouse, Anna Maria Storniolo, Harikrishna Nakshatri. Influence of genetic ancestry on breast stromal cells provides biologic basis for increased incidence of metaplastic breast cancer in women of African descent [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-08.

Pendimethalin exposure disrupts mitochondrial function and impairs processes related to implantation.

Pendimethalin as a dinitroaniline herbicide is used to eliminate weeds during the cultivation of various crops such as grains, fruits, and vegetables. This study reveals that pendimethalin exposure at various concentrations led to disruption in Ca2+ homeostasis and mitochondrial membrane potential, as well as dysregulation of the mitogen-activated protein kinase signaling pathway and implantation-related genes in porcine trophectoderm and uterine luminal epithelial cells. The use of herbicides is a major control method in agriculture. Pendimethalin (PDM) has been increasingly used as a herbicide for approximately 30 years. PDM has been reported to cause various reproductive problems, but its toxicity mechanism in the pre-implantation stage has not been investigated in detail. Herein, we studied the effects of PDM on porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells and identified a PDM-mediated anti-proliferative effect in both cell types. PDM exposure generated intracellular reactive oxygen species, induced excessive Ca2+ influx into mitochondria, and activated mitogen-activated protein kinase signaling pathway. Ca2+ burden resulted in the dysfunction of mitochondria and eventual disruption of Ca2+ homeostasis. Further, PDM-exposed pTr and pLE cells showed cell cycle arrest and programmed cell death. In addition, a decrease in migration ability and dysregulated expression of genes related to the functioning of pTr and pLE cells was evaluated. This study provides insight into time-dependent transitions within the cell environment after PDM exposure and elucidates a detailed mechanism of induced adverse effects. These results imply that PDM exposure can potentially cause toxic effects on the implantation-related process in pigs. Moreover, to the best of our knowledge, this is the first study to describe the mechanism by which PDM induces these effects, enhancing our understanding of the toxicity of this herbicide.

Picolinafen exposure induces ROS accumulation and calcium depletion, leading to apoptosis in porcine embryonic trophectoderm and uterine luminal epithelial cells during the peri-implantation period

The global use of herbicides accounts for more than 48% of total pesticide usage. Picolinafen is a pyridine carboxylic acid herbicide that is predominantly used to control broadleaf weeds in wheat, barley, corn, and soybeans. Despite its widespread use in agriculture, its toxicity in mammals has rarely been studied. In this study, we first identified the cytotoxic effects of picolinafen on porcine trophectoderm (pTr) and luminal epithelial (pLE) cells, which are involved in the implantation process during early pregnancy. Picolinafen treatment significantly decreased the viability of pTr and pLE cells. Our results demonstrate that picolinafen increased the number of sub-G1 phase cells and early/late apoptosis. In addition, picolinafen disrupted mitochondrial function and resulted in the accumulation of intracellular ROS, leading to a reduction in calcium levels in both the mitochondria and cytoplasm of pTr and pLE cells. Moreover, picolinafen was found to significantly inhibit the migration of pTr. These responses were accompanied by the activation of the MAPK and PI3K signal transduction pathways by picolinafen. Our data suggest that the deleterious effects of picolinafen on the viability and migration of pTr and pLE cells might impair their implantation potential.