Expression In Stromal Cells Research Articles

Adipocyte Glucocorticoid Receptor Inhibits Immune Regulatory Genes to Maintain Immune Cell Homeostasis in Adipose Tissue.

Glucocorticoids acting via the glucocorticoid receptors (GR) are key regulators of metabolism and the stress response. However, uncontrolled or excessive GR signaling adversely affects adipose tissue, including endocrine, immune, and metabolic functions. Inflammation of the adipose tissue promotes systemic metabolic dysfunction; however, the molecular mechanisms underlying the role of adipocyte GR in regulating genes associated with adipose tissue inflammation are poorly understood. We performed in vivo studies using adipocyte-specific GR knockout mice in conjunction with in vitro studies to understand the contribution of adipocyte GR in regulating adipose tissue immune homeostasis. Our findings show that adipocyte-specific GR signaling regulates adipokines at both mRNA and plasma levels and immune regulatory (Coch, Pdcd1, Cemip, and Cxcr2) mRNA gene expression, which affects myeloid immune cell presence in white adipose tissue. We found that, in adipocytes, GR directly influences Cxcr2. This chemokine receptor promotes immune cell migration, indirectly affecting Pdcd1 and Cemip gene expression in nonadipocyte or stromal cells. Our findings suggest that GR adipocyte signaling suppresses inflammatory signals, maintaining immune homeostasis. We also found that GR signaling in adipose tissue in response to stress is sexually dimorphic. Understanding the molecular relationship between GR signaling and adipose tissue inflammation could help develop potential targets to improve local and systemic inflammation, insulin sensitivity, and metabolic health.

The importance of CXCR4 expression in tumor stroma as a potential biomarker in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the main causes of cancer mortality in the world. A characteristic feature of this cancer is that a large part of the tumor volume is composed of a stroma with different cells and factors. Among these, we can highlight the cytokines, which perform their function through binding to their receptors. Given the impact of the CXCR4 receptor in the interactions between tumor cells and their microenvironment and its involvement in important signaling pathways in cancer, it is proposed as a very promising prognostic biomarker and as a goal for new targeted therapies. Numerous studies analyze the expression of CXCR4 but we suggest focusing on the expression of CXCR4 in the stroma.MethodsExpression of CXCR4 in specimens from 33 patients with PDAC was evaluated by immunohistochemistry techniques and matched with clinicopathological parameters, overall and disease-free survival rates.ResultsThe percentage of stroma was lower in non-tumor tissue (32.4 ± 5.2) than in tumor pancreatic tissue (67.4 ± 4.8), P-value = 0.001. The level of CXCR4 expression in stromal cells was diminished in non-tumor tissue (8.7 ± 4.6) and higher in tumor pancreatic tissue (23.5 ± 6.1), P-value = 0.022. No significant differences were identified in total cell count and inflammatory cells between non-tumor tissue and pancreatic tumor tissue. No association was observed between CXCR4 expression and any of the clinical or pathological data, overall and disease-free survival rates. Analyzing exclusively the stroma of tumor samples, the CXCR4 expression was associated with tumor differentiation, P-value = 0.05.ConclusionsIn this study, we reflect the importance of CXCR4 expression in the stroma of patients diagnosed with PDAC. Our results revealed a high CXCR4 expression in the tumor stroma, which is related to a poor tumor differentiation. On the contrary, we could not find an association between CXCR4 expression and survival and the rest of the clinicopathological variables. Focusing the study on the CXCR4 expression in the tumor stroma could generate more robust results. Therefore, we consider it key to develop more studies to enlighten the role of this receptor in PDAC and its implication as a possible biomarker.

Immunohistochemical analysis of osteoclastic and osteoblastic activity in ossifying fibroma and juvenile ossifying fibroma: A comparative study.

Cemento-ossifying fibroma (COF) and juvenile ossifying fibroma (JOF) have been considered distinct entities within the category of fibro-osseous lesions. This study aimed to assess osteoblast and osteoclast activity in COF and JOF by investigating bone resorption markers, specifically receptor activator of nuclear factor-kB (RANK), RANK ligand (RANKL), and its inhibitor osteoprotegerin (OPG). A comparative analysis of these markers was performed on all lesions. Immunohistochemistry was employed to evaluate and quantify the expression of these biomarkers in a sample of 20 cases of cemento-ossifying fibroma (COF), 15 cases of psammomatoid juvenile ossifying fibroma (PsJOF), and 10 cases of trabecular juvenile ossifying fibroma (TrJOF). The expression of osteoprotegerin was significantly higher in cemento-ossifying fibroma (33.9±13.0) compared to trabecular juvenile ossifying fibroma (27.3±9.2) and psammatoid ossifying fibroma (25.2±14.9), with the COF showing the highest expression followed by the latter two (p=0.037). There was a higher percentage (80%) of stromal fibroblast cells that showed positive expression of RANKL in cemento-ossifying fibroma (COF) compared to psammomatoid juvenile ossifying fibroma (PsJOF) (33.3%) and trabecular juvenile ossifying fibroma (TrJOF) (30.0%) when considering a positive expression score of 3 (p=0.024). Cemento-ossifying fibroma demonstrated the highest expression of osteoprotegerin and RANKL-positive stromal fibroblast cells, followed by psammomatoid juvenile ossifying fibroma and trabecular juvenile ossifying fibroma. These findings provide valuable insights into the pathogenesis of these lesions.

System analysis based on glutamine catabolic-related enzymes identifies GPT2 as a novel immunotherapy target for lung adenocarcinoma

BackgroundIn recent years, targeting glutamine metabolism has gained attention as a promising therapeutic approach. Glutamine catabolic-related enzymes play a crucial role in modulating glutamine metabolism and influencing immune responses in the tumor immune microenvironment (TME). However, current literature on the function of glutamine catabolic enzymes in lung adenocarcinoma (LUAD) is limited. MethodsWe validated the glutamine dependency of LUAD cells in vitro, followed by transcriptome data to identify differentially expressed genes (DEGs), with transcriptome and single-cell data analysis utilized to explore the role of such genes within the tumor immune microenvironment. We performed employed subcutaneous injection of lewis lung carcinoma cells in C57BL/6 mice to confirm the role of candidate genes in tumor growth and anti-tumor immunity. ResultsOur study revealed that glutamine is essential for the growth of LUAD cells. Subsequently, we identified four DEGs - glutamate pyruvate transaminase 1 (GPT1), glutamate pyruvate transaminase 2 (GPT2), glutamic-oxaloacetic transaminase 1 (GOT1), and glutamic-oxaloacetic transaminase 2 (GOT2) - in LUAD patients, which were highly expressed in tumor tissue and associated with an immunosuppressive TME. Single-cell sequencing analysis detected high expression levels of GOT1 and GOT2 in immune and stromal cell subpopulations, while GPT1 and GPT2 showed relatively lower expression. Based on the lower immune score and lower expression in immune and stromal cells, we validated the role of GPT2 in vivo for modulating the TME and tumor growth. Inhibition of GPT2 resulted in suppressed tumor growth and increased the expression of CD4 and CD8. Additionally, GPT2 inhibitors induced a stronger antitumor immunity when used in combination with anti-programmed cell death ligand 1. ConclusionThis study is the first to show the critical role of glutamine catabolic-related enzymes in the TME, and identified GPT2 as a promising therapeutic target for inhibiting tumor growth and improving anti-tumour immune responses for LUAD. Additional studies will be required to define the roles glutamine catabolic-related enzymes play in LUAD.

Tissue expression of LC3B autophagy marker as a potential biomarker of prostate cancer recurrence after treatment with high-intensity focused ultrasound (pilot study)

Background. The role of autophagy markers in prostate tumor recurrence has not been sufficiently investigated. We hypothesized that autophagy activation may be one mechanism by which prostate cancer cells survive exposure to high-intensity focused ultrasound (HIFU).Aim. To compare tissue expression of autophagic LC3B marker in prostate biopsies before and after treatment of localized prostate cancer by HIFU ablation.Materials and methods. 45 patients with localized morphologically confirmed prostate cancer were examined: group 1 – 25 patients of 65.6 ± 8.4 years without signs of recurrence or progression of the disease; group 2 – 20 patients of 67.5 ± 7.9 years with tumor recurrence proven during morphological examination. Immunohistochemical examination was performed by streptavidin-biotin method. In all cases, Anti-LC3B antibody ab48394 was used. The reaction results were quantified using the Histochemical score (Hs) system.Results. Prior to treatment, all patients of group 1 showed moderate cytoplasmic expression (Hs = 111 [111; 115]) of antibodies against LC3B in prostate adenocarcinoma cells, 5 % of patients – weak cytoplasmic expression in muscle connective stromal cells (Hs = 47 [43; 50]), 10 % of patients – weak positive LC3B reaction in the vessel wall (Hs = 28 [20; 35]). After treatment, the expression of LC3B in adenocarcinoma cells became negative, in the cytoplasm of muscle connective stromal cells weak (Hs = 75 [67.5; 80.0]), in the endothelium of the vascular wall even weaker (Hs = 55 [45.5; 60.0]) (p <0.001). Prior to treatment in group 2, LC3B expression in tumor tissue was moderate in 89 % of patients (Hs = 151.5 [137.5; 160.0]), weak in muscle connective stromal cells in 12 % of patients (Hs = 44 [35; 51.5]), and weak in the vascular wall in 5 % of patients (Hs = 30 [25; 35]). After treatment, LC3B expression in adenocarcinoma cells became pronounced (Hs = 260 [250; 285]), in muscle connective stromal cells – moderate (Hs = 118 [100; 130]), in the vascular wall – weak (Hs = 45 [30; 55]) (p <0.001). There was a significant correlation between tumor recurrence and LC3B overexpression (r = 0.51; p <0.001).Conclusion. The development of prostate cancer recurrence is associated with increased expression of autophagic LC3B protein. Increased LC3B expression, which is interpreted as evidence of autophagy activation and correlates with the risk of disease progression, is used by the tumor as an oncogenic advantage.

Colocalization of corneal resistance factor GWAS loci with GTEx e/sQTLs highlights plausible candidate causal genes for keratoconus postnatal corneal stroma weakening.

Background: Genome-wide association studies (GWAS) for corneal resistance factor (CRF) have identified 100s of loci and proved useful to uncover genetic determinants for keratoconus, a corneal ectasia of early-adulthood onset and common indication of corneal transplantation. In the current absence of studies to probe the impact of candidate causal variants in the cornea, we aimed to fill some of this knowledge gap by leveraging tissue-shared genetic effects. Methods: 181 CRF signals were examined for evidence of colocalization with genetic signals affecting steady-state gene transcription and splicing in adult, non-eye, tissues of the Genotype-Tissue Expression (GTEx) project. Expression of candidate causal genes thus nominated was evaluated in single cell transcriptomes from adult cornea, limbus and conjunctiva. Fine-mapping and colocalization of CRF and keratoconus GWAS signals was also deployed to support their sharing causal variants. Results and discussion: 26.5% of CRF causal signals colocalized with GTEx v8 signals and nominated genes enriched in genes with high and specific expression in corneal stromal cells amongst tissues examined. Enrichment analyses carried out with nearest genes to all 181 CRF GWAS signals indicated that stromal cells of the limbus could be susceptible to signals that did not colocalize with GTEx's. These cells might not be well represented in GTEx and/or the genetic associations might have context specific effects. The causal signals shared with GTEx provide new insights into mediation of CRF genetic effects, including modulation of splicing events. Functionally relevant roles for several implicated genes' products in providing tensile strength, mechano-sensing and signaling make the corresponding genes and regulatory variants prime candidates to be validated and their roles and effects across tissues elucidated. Colocalization of CRF and keratoconus GWAS signals strengthened support for shared causal variants but also highlighted many ways into which likely true shared signals could be missed when using readily available GWAS summary statistics.

Distinct Requirements for Adaptor Proteins NCK1 and NCK2 in Mammary Gland Development

The adaptor proteins NCK1 and NCK2 are well-established signalling nodes that regulate diverse biological processes including cell proliferation and actin dynamics in many tissue types. Here we have investigated the distribution and function of Nck1 and Nck2 in the developing mouse mammary gland. Using publicly available single-cell RNA sequencing data, we uncovered distinct expression profiles between the two paralogs. Nck1 showed widespread expression in luminal, basal, stromal and endothelial cells, while Nck2 was restricted to luminal and basal cells, with prominent enrichment in hormone-sensing luminal subtypes. Next, using mice with global knockout of Nck1 or Nck2, we assessed mammary gland development during and after puberty (5, 8 and 12 weeks of age). Mice lacking Nck1 or Nck2 displayed significant defects in ductal outgrowth and branching at 5 weeks compared to controls, and the defects persisted in Nck2 knockout mice at 8 weeks before normalizing at 12 weeks. These defects were accompanied by an increase in epithelial cell proliferation at 5 weeks and a decrease at 8 weeks in both Nck1 and Nck2 knockout mice. We also profiled expression of several key genes associated with mammary gland development at these timepoints and detected temporal changes in transcript levels of hormone receptors as well as effectors of cell proliferation and migration in Nck1 and Nck2 knockout mice, in line with the distinct phenotypes observed at 5 and 8 weeks. Together these studies reveal a requirement for NCK proteins in mammary gland morphogenesis, and suggest that deregulation of Nck expression could drive breast cancer progression and metastasis.

Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells.

The abundance of circRNAs varies systematically between subcompartments of solid tumors and adjacent tissues, implying that differentially expressed circRNAs discovered in bulk tissue analyses may reflect differences in cell type composition between samples.

The SLC38A5/SNAT5 amino acid transporter: from pathophysiology to pro-cancer roles in the tumor microenvironment.

SLC38A5/SNAT5 is a system N transporter that can mediate net inward or outward transmembrane fluxes of neutral amino acids coupled with Na+ (symport) and H+ (antiport). Its preferential substrates are not only amino acids with side chains containing amide (glutamine and asparagine) or imidazole (histidine) groups, but also serine, glycine, and alanine are transported by the carrier. Expressed in the pancreas, intestinal tract, brain, liver, bone marrow, and placenta, it is regulated at mRNA and protein levels by mTORC1 and WNT/β-catenin pathways, and it is sensitive to pH, nutritional stress, inflammation, and hypoxia. SNAT5 expression has been found to be altered in pathological conditions such as chronic inflammatory diseases, gestational complications, chronic metabolic acidosis, and malnutrition. Growing experimental evidence shows that SNAT5 is overexpressed in several types of cancer cells. Moreover, recently published results indicate that SNAT5 expression in stromal cells can support the metabolic exchanges occurring in the tumor microenvironment of asparagine-auxotroph tumors. We review the functional role of the SNAT5 transporter in pathophysiology and propose that, due to its peculiar operational and regulatory features, SNAT5 may play important pro-cancer roles when expressed either in neoplastic or in stromal cells of glutamine-auxotroph tumors.NEW & NOTEWORTHY The transporter SLC38A5/SNAT5 provides net influx or efflux of glutamine, asparagine, and serine. These amino acids are of particular metabolic relevance in several conditions. Changes in transporter expression or activity have been described in selected types of human cancers, where SNAT5 can mediate amino acid exchanges between tumor and stromal cells, thus providing a potential therapeutic target. This is the first review that recapitulates the characteristics and roles of the transporter in physiology and pathology.

Silencing of CDC42 inhibits contraction and growth-related functions in prostate stromal cells, which is mimicked by ML141

BackgroundProstate smooth muscle contraction and stromal growth may contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia, but are incompletely understood. A role of the monomeric GTPase CDC42 for smooth muscle contraction and proliferation appears possible, but is unknown for the prostate. Here, we silenced CDC42 expression in prostate stromal cells (WPMY-1), and examined contractility, growth-related functions and responses to the presumed CDC42 inhibitor, ML141. MethodsWPMY-1 cells were transfected with scrambled or CDC42-specific siRNA, and characterized for GTPase activities, contraction, proliferation, colony formation, apoptosis, cell death and viability. Effects of ML141 were examined in cells with and without silencing. ResultsCDC42 silencing was confirmed by reduced mRNA and protein expression, and reduced CDC42 activity. Silencing impaired contraction (23–47 %), actin organization (25 %), proliferation (17–63 %), colony formation and viability (64–89 %), and increased the percentage of dead cells (2.6-fold). ML141 mimicked the phenotype of silencing in scrambled siRNA-transfected controls, and in non-transfected WPMY-1 cells, including inhibition of contraction, proliferation, colony formation and viability, breakdown of actin organization and increased cell death. In CDC42-silenced cells, ML141 still affected phalloiding organization, proliferation and cell death, with effect sizes resembling controls without silencing. ML141 inhibited RhoA activity in CDC42-silenced cells, but not in cells without silencing. ConclusionsCDC42 promotes contraction of prostate stromal cells, and drives stromal growth by CDC42-mediated proliferation and suppression of apoptosis-independent cell death. ML141 mimicks all effects of CDC42 silencing, but its specificity may be limited and depends on GTPase phenotypes of cells.

Expression of Amine Oxidase Proteins in Adrenal Cortical Neoplasm and Pheochromocytoma.

We delved into the expression of amine oxidase family proteins and their potential significance in adrenal gland neoplasm. Tissue microarrays were prepared for 132 cases of adrenal cortical neoplasm (ACN) consisting of 115 cases of adrenal cortical adenoma (ACA), 17 cases of adrenal cortical carcinoma (ACC), and 163 cases of pheochromocytoma (PCC). Immunohistochemical stainings for MAOA, MAOB, LOX, and AOC3 were performed to evaluate the H-scores and compare them with clinicopathological parameters. The H-scores of MAOA (T; p = 0.005) and MAOB (T; p = 0.006) in tumor cells (T) were high in ACN, whereas LOX (T, S; p < 0.001) in tumor and stromal cells (S) and AOC3 (T; p < 0.001) were higher in PCC. In stromal cells, MAOA (S; p < 0.001) and AOC3 (S; p = 0.010) were more expressed in ACA than in ACC. MAOB (S) in PCC showed higher H-scores when the grading of adrenal pheochromocytoma and paraganglioma (GAPP) score was 3 or higher (p = 0.027). In the univariate analysis, low MAOA expression in stromal cells of ACN was associated with shorter overall survival (p = 0.008). In conclusion, monoamine oxidase proteins revealed differences in expression between ACN and PCC and also between benign and malignant cells.

Sphingosine 1-phosphate elicits a ROS-mediated proinflammatory response in human endometrial stromal cells via ERK5 activation.

Endometriosis is a chronic gynecological disease affecting ~10% women in the reproductive age characterized by the growth of endometrial glands and stroma outside the uterine cavity. The inflammatory process has a key role in the initiation and progression of the disorder. Currently, there are no available early diagnostic tests and therapy relies exclusively on symptomatic drugs, so that elucidation of the complex molecular mechanisms involved in the pathogenesis of endometriosis is an unmet need. The signaling of the bioactive sphingolipid sphingosine 1-phosphate (S1P) is deeply dysregulated in endometriosis. S1P modulates a variety of fundamental cellular processes, including inflammation, neo-angiogenesis, and immune responses acting mainly as ligand of a family of G-protein-coupled receptors named S1P receptors (S1PR), S1P1-5 . Here, we demonstrated that the mitogen-activated protein kinase ERK5, that is expressed in endometriotic lesions as determined by quantitative PCR, is activated by S1P in human endometrial stromal cells. S1P-induced ERK5 activation was shown to be triggered by S1P1/3 receptors via a SFK/MEK5-dependent axis. S1P-induced ERK5 activation was, in turn, responsible for the increase of reactive oxygen species and proinflammatory cytokine expression in human endometrial stromal cells. The present findings indicate that the S1P signaling, via ERK5 activation, supports a proinflammatory response in the endometrium and establish the rationale for the exploitation of innovative therapeutic targets for endometriosis.

#4574 ATYPICAL CHEMOKINE RECEPTOR 4 IS EXPRESSED IN GLOMERULAR PARIETAL EPITHELIAL CELLS AND MITIGATES THE SEVERITY OF EXPERIMENTAL GLOMERULONEPHRITIS

Abstract Background and Aims Cell migration contributes to the innumerable physiological and pathological processes driven and directed by chemotactic cytokines (chemokines), small structurally homologous proteins signaling via their cognate GPCRs on motile cells. In addition to GPCRs, chemokines importantly interact with atypical chemokine receptors (ACKRs) that are characterized by expression in various stromal cells and distinctive ligand specificities. ACKR4 binds CCL19, CCL20, CCL21, CCL22 and CCL25, chemokines involved primarily in adaptive immune responses. Accordingly, ACKR4 is prominently expressed in the primary and secondary lymphoid organs, thymus, bone marrow, lymph nodes and spleen where it regulates distinct cell migratory steps driven by its cognate ligands. Furthermore, ACKR4 is expressed by dissimilar cell types in multiple parenchymal organs, the heart, the liver and the gut. Currently it is not entirely clear how chemokine scavenging by ACKR4 in these organs might affect their pathophysiology. Here we investigated the expression of ACKR4 in murine kidney and assessed its contribution to nephrotoxic serum nephritis (NTSN), an experimental murine model of immune complex glomerulonephritis. The pathomechanisms in NTSN are known to rely on the contribution of multiple chemokines and their receptors and functions of Th1 cells, Th17 cells, neutrophils and macrophages exerting pathogenic effects in both lymphoid organs and the kidney. Method The expression of renal ACKR4 was evaluated in healthy ACKR4-eGFP reporter mice as well as after the induction of NTSN. To investigate the contribution of ACKR4 to NTSN, ACKR4-deficient mice and WT controls were subjected to an anti-basal membrane immunization protocol and the parameters of immunopathogenesis and the ensuing kidney disease were evaluated at 7 and 14 days after the immunization. Results Multicolour immunofluorescence confocal microscopy revealed that ACKR4 is expressed in the kidney exclusively in the glomeruli by a discrete subset of parietal epithelial cells localizing adjacently to the vascular glomerular pole. In mice with NTSN, the overall proportion of ACKR4 expressing cells in glomeruli at the vascular pole did not change as compared to healthy mice, however ACKR4+ cells were not detected in glomeruli corresponding with the increased abundance of alpha-SMA, a marker of renal fibrosis. The ACKR4-deficient mice showed delayed antibody response following immunization and reduced readouts of cellular immunity. However, despite this, disease parameters of NTSN, including albuminuria, PAS-score and crescent formation were significantly increased in ACKR4-deficient mice as compared to the WT controls. Renin, a hormone secreted by the cells adjacent to ACKR4+ cells in the kidney was significantly increased in serum of nephritic ACKR4-/- mice. Conclusion We report for the first time that ACKR4 is expressed in the kidney by a small subpopulation of parietal epithelial cells. Reduced parameters of humoral and cellular immunity in ACKR4-deficient mice contrast with a more severe manifestations of NTSN seen in these mice, suggesting a tissue-specific functional bias of ACKR4 in the kidney that limits the development of NTSN. Furthermore, the immediate proximity of ACKR4 expressing cells to the juxtaglomerular apparatus and the observed increased renin secretion in ACKR4-/- mice suggest a potential contribution of ACKR4 to the regulation of renin-driven pathways, by a yet unclear mechanism.

IL-6 Levels Correlate with Prognosis and Immunosuppressive Stromal Cells in Patients with Colorectal Cancer.

The prognosis for patients with colorectal cancer (CRC) is determined by tumor characteristics as well as the host immune response. This study investigated the relationship between an immunosuppressive state and patient prognosis by evaluating the systemic and tumor microenvironment (TME) interleukin (IL)-6 levels. Preoperative serum IL-6 levels were measured using an electrochemiluminescence assay. Expression of IL-6 in tumor and stromal cells was evaluated immunohistochemically in 209 patients with resected CRC. Single-cell analysis of tumor-infiltrating immune cells was performed using mass cytometry in 10 additional cases. Elevated serum IL-6 levels were associated with elevated stromal IL-6 levels and a poor prognosis for patients with CRC. High IL-6 expression in stromal cells was associated with low-density subsets of CD3+ and CD4+ T cells as well as FOXP3+ cells. Mass cytometry analysis showed that IL-6+ cells among tumor-infiltrating immune cells were composed primarily of myeloid cells and rarely of lymphoid cells. In the high-IL-6-expression group, the percentages of myeloid-derived suppressor cells (MDSCs) and CD4+FOXP3highCD45RA- effector regulatory T cells (eTreg) were significantly higher than in the low-IL-6-expression group. Furthermore, the proportion of IL-10+ cells in MDSCs and that of IL-10+ or CTLA-4+ cells in eTregs correlated with IL-6 levels. Elevated serum IL-6 levels were associated with stromal IL-6 levels in CRC. High IL-6 expression in tumor-infiltrating immune cells also was associated with accumulation of immunosuppressive cells in the TME.

PD-L1 expression, morphology, and molecular characteristic of a subset of aggressive uterine tumor resembling ovarian sex cord tumor and a literature review

BackgroundUterine tumors resembling ovarian sex cord tumor (UTROSCT) is a rare neoplasm of unknown etiology and has undetermined malignant potential. The emergence of recurrent UTROSCT case reports has led to its initial identification as a tumor of low malignancy potential. Owing to its low incidence, we currently lack any in-depth studies regarding the subset of UTROSCTs that may be aggressive in nature. Here, we sought to identify unique characteristics in aggressive UTROSCT.Methods19 cases of UTROSCT were collected. Their histologic and tumor immune microenvironment were evaluated by three gynecologic pathologists. The gene alteration was also detected by RNA sequencing. For later analyses regarding differences between benign and malignant tumors, we supplemented our 19 included cases with additional reports from the literature.ResultsInterestingly, we found PD-L1 expression in stromal tumor-infiltrating immune cells (stromal PD-L1) was markedly higher in aggressive UTROSCT. Patients with high stromal PD-L1 (≥ 22.5 cells/mm2) had worse prognosis. When our cases were added with previous cases identified in the literature, we discovered that aggressive UTROSCT was more likely to have significant mitotic activity and NCOA2 gene alterations than benign UTROSCT. Consistence with those results, patients with significant mitotic activity and gene alteration of NCOA2 had worse prognoses.ConclusionsCollectively, high expression of stromal PD-L1, significant mitotic activity, and gene alteration of NCOA2 may be useful markers to predict aggressive UTROSCT.

Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. Not applicable.

Differential gene expression and protein-protein interaction networks of human periodontal ligament stromal cells under mechanical tension

Orthodontic treatment is based on complex strategies and takes up to years until a desired therapeutic outcome is accomplished, implying long periods of high costs and discomfort for the patient. Choosing the optimal settings for force intensities in the initial phase of orthodontic tooth movement is the key to successful orthodontic treatment. It is known that orthodontic tooth movement is mainly mediated by tensile and compressive forces that are communicated to the alveolar bone via the periodontal ligament. While the revelation of the complex molecular network was already approached by transcriptomic analysis of compressed periodontal ligament cells, the entity of molecular key players activated by tensile forces remains elusive. Therefore, the aim of this study was to assess the effect of mechanical tensile forces on the gene expression profile of human primary periodontal ligament stromal cells, mimicking the initial phase of orthodontic tooth movement. A transcriptomic analysis of tension-treated and untreated periodontal ligament stromal cells yielded 543 upregulated and 793 downregulated differentially expressed genes. Finally, six highly significant genes were found in the transcriptome that are related to biological processes with relevance to orthodontic tooth movement, including apelin, fibroblast growth factor receptor 2, noggin, sulfatase 1, secreted frizzled-related protein 4 and stanniocalcin 1. Additionally, differences of gene expression profiles between individual cell donors showed a high effect size. Closer understanding of the roles of the identified candidates in the initial phase of orthodontic tooth movement could help to clarify the underlying mechanisms, which will be essential for the development of personalized treatment strategies in orthodontics.

Targeting YAP1 ameliorates progesterone resistance in endometriosis

Does YAP1 inhibition alleviate progesterone resistance in endometriosis? YAP1 inhibition reduces progesterone resistance in vitro and in vivo. Progesterone resistance not only causes treatment failure for endometriosis but also inhibits eutopic endometrial cell proliferation, dysregulates decidualization, and reduces the success rates of pregnancy. Hippo/yes-associated protein 1 (YAP1) signaling pathway plays an important role in the pathogenesis of endometriosis. Paraffin-embedded tissues containing paired endometriotic and endometrial specimens (n = 42) and serum samples isolated from normal controls (n = 15) or endometriotic patients with (n = 25) or without (n = 21) prior dienogest treatment were analyzed. A mouse model of endometriosis was also used to evaluate the effects of YAP1 inhibition on progesterone resistance. Primary endometriotic and endometrial stromal cells treated with YAP1 inhibitor or miR-21 mimic/inhibitor were used for the in vitro studies including decidualization induction, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation. Tissue specimens and serum from human and mouse were used for immunohistochemistry staining, exosome isolation, and microRNA (miRNA) quantification, respectively. Herein, we report, by using ChIP-PCR and RNA-IP, that YAP1 inhibits progesterone receptor (PGR) expression through upregulation of miR-21-5p. Upregulation of miR-21-5p not only reduces PGR expression but also inhibits endometrial stromal cell decidualization. Indeed, levels of YAP1 and miR-21-5p are inversely correlated with the level of PGR in human endometrial samples. In contrast, knockdown of YAP1 or treatment with verteporfin (VP), a YAP1 inhibitor, reduces miR-21-5p expression, thus leading to an increase in PGR expression in ectopic endometriotic stromal cells. In the mouse model of endometriosis, treatment with VP increases PGR expression and enhances decidualization. More importantly, VP synergistically increases the treatment effect of progestin in causing the regression of endometriotic lesions and improves the decidualization capability of the endometrium. Interestingly, treatment with dienogest, a synthetic progestin, reduces YAP1 and miR-21-5p expression in human cells and in the mouse model of endometriosis. Patients who received dienogest treatment for 6 months show a significant decrease in serum extracellular vesicle-associated miR-21-5p level. A public dataset (GSE51981) containing a large cohort of endometriotic tissues is available from the Gene Expression Omnibus (GEO). A large cohort of clinical samples is needed to verify the current diagnostic value of miR-21-5p in future studies. The reciprocal regulation of YAP1 and PGR suggests that combined YAP1 inhibitor and progestin may be a better therapeutic approach for treating endometriosis. This study was supported by the Ministry of Science and Technology, Taiwan (MOST-111-2636-B-006-012, MOST-111-2314-B-006-075-MY3, and MOST-106-2320-B-006-072-MY3). The authors have no conflict of interest to disclose.

Abstract 2505: PYCR1 inhibition in multiple myeloma-associated stroma limits tumor growth

Abstract Introduction Multiple myeloma (MM) is a hematological cancer, characterized by the accumulation of monoclonal plasma cells in the bone marrow. It remains an incurable cancer due to drug resistance, wherein the bone marrow microenvironment plays a crucial role. We have previously shown that glutamine-to-proline conversion is upregulated in MM cells upon hypoxic culture. Moreover, inhibition of proline production by blocking its converting enzyme PYCR1 successfully reduced proliferation and viability in vitro. Importantly, PYCR1 inhibition combined with standard-of-care agent bortezomib decreased tumor load in vivo. As PYCR1 is also highly expressed in stromal cells and proline an important component of extracellular matrix proteins, we investigated whether PYCR1 targeting in stromal cells affects MM viability and its structural microenvironment. Material and methods PYCR1 expression was investigated by the use of microarray data from the Heidelberg/Montpellier cohort. For in vitro experiments, the human MM cell line OPM-2 and human stromal cell line HS-5 were used. CD138+ and CD138- fractions from primary patient samples were separated by MACS. To obtain primary stromal cells, the CD138- fraction was plated out in fresh medium. After 48h, the medium was refreshed and all adherent cells were further cultured and used as primary stromal cells. PYCR1 expression in HS-5 cells was reduced through siRNA. Hypoxic culture (&amp;lt;1% O2) was established through chambers. RNA expression was measured by RT-qPCR, while protein expression was measured by western blot. Viability was assessed by CellTiterGlo assay. Results Gene expression analysis shows high RNA expression of PYCR1 in healthy bone marrow plasma cells (BMPCs), plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and MM patients, but also in bone marrow stromal cells (BMSCs). PYCR1 expression was low or absent in other cell types, including T cells, osteoclasts, monocytic and granulocytic cells. On protein level, we also confirmed PYCR1 expression in MM cells (CD138+), CD138- fraction (including stromal cells) and cultured primary stromal cells. Moreover, PYCR1 expression increased upon hypoxic culture in primary stromal cells and stromal cell line HS-5. PYCR1 knockdown in stromal cell line HS-5 did not affect viability of the stromal cells, but its conditioned medium did reduce its protective effects when OPM-2 MM cells were treated with standard-of-care agent bortezomib. Further investigation revealed that knockdown of PYCR1 in HS-5 reduces RNA expression of structural proteins col1a1, col1a2, col3a1, ctgf and acta2 in HS-5. Conclusion PYCR1 is highly expressed in myeloma cells and stromal cells. PYCR1 inhibition in stromal cells reduces its proliferative effect on myeloma cells when combined with bortezomib. Preliminary data indicates a link between PYCR expression in stromal cells and rearrangement of extracellular matrix proteins. Citation Format: Inge Oudaert, Catharina Muylaert, Hatice Satilmis, Sylvia Faict, Kim De Veirman, Elke De Bruyne, Karin Vanderkerken, Eline Menu. PYCR1 inhibition in multiple myeloma-associated stroma limits tumor growth [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 2505.

375 Vorinostat for Missense Mutated Von Hippel Lindau Disease Associated Hemangioblastomas: A Pilot Study

INTRODUCTION: Missense mutated VHL protein (pVHL) maintains intrinsic function but undergoes proteasomal degradation, loss of function and tumor initiation/progression of VHL-associated tumors. Histone deacetylase inhibitors (HDACi) can rescue missense mutated pVHL and arrest tumor growth in pre-clinical models. METHODS: Seven subjects with missense VHL received 400mg/day of vorinostat (7-days) and surgical resection of hemangioblastomas. We analyzed vorinostat-treated HBs (and paired untreated HBs from the same patients) for gene and protein expression in the pVHL-HIF-VEGF pathway, and protein-protein interactions. We explored the transcriptional landscape of these tumors with single nucleus gene expression analysis. We recapitulated patient specific missense mutations and validated the clinical findings using lentiviral expression recombinant constructs in VHL-/- 786-O cells. RESULTS: Short-term oral vorinostat was well tolerated by all patients (age 46.0 ± 14.5 years; 25-61 years). In the 6 evaluable tumors, pVHL expression in neoplastic stromal cells of vorinostat treated tumors was significantly elevated compared to untreated tumors (p = 0.01). We found transcriptional suppression of downstream effectors including VEGF-A and GLUT1 in human tumor samples and validated the effects in patient-specific missense mutations expressed in VHL 786-O cells. Mechanistically, we found that vorinostat prevented Hsp90 recruitment to missense mutated pVHL. The vorinostat effects on pVHL rescue, transcriptional repression and Hsp90-pVHL interaction was independent of the location of the mutation on VHL. Lastly, we found a HB stromal cell specific effect in suppression of pro-tumorigenic pathways with single nucleus gene expression analysis. CONCLUSIONS: Short-term treatment with vorinostat increased pVHL levels and suppressed tumor progression signaling in VHL-associated hemangioblastomas. These results indicate that proteostasis modulation can rescue tumor pVHL in germline missense mutated VHL patients and such treatment could arrest tumor growth.