Immune Cell Markers Research Articles

M2 macrophages, B cells, and PD-L1 immune checkpoint protein exhibit sexual dimorphism in the outcomes of non-muscle invasive bladder cancer

Abstract Non-muscle-invasive bladder cancer (NMIBC) is more than three times as common in men as it is in women. However, female patients with NMIBC do not respond as well to immunotherapeutic treatments and experience worse clinical outcomes than their male counterparts. The underlying causes of these discrepancies have yet to be fully elucidated. We hypothesized that sexual dimorphism in the tumor immune microenvironment (TIME) may contribute to the inferior clinical outcomes observed in female patients with NMIBC. To test this hypothesis, we investigated immune-associated gene expression in tumors from male (n=357) and female (n=103) patients. High-grade tumors from female patients exhibited significantly increased expression of B cell-associated genes CD40 and CXCL13, and the immune checkpoint genes CTLA4, PDCD1, LAG3, and ICOS. Based on these differences, we utilized multiplexed immunofluorescence to evaluate the density and spatial distribution of 12 immune cell markers (CD79a, CD3, CD8, FoxP3, Ki67, CD103, CD163, GATA3, CK5, IDO1, PD-1, and PD-L1) in tumors from an independent cohort of 332 patients with NMIBC (n=259 males and n=73 females). Tumors from female patients showed significantly higher infiltration of PD-L1+ cells and CD163+ M2-like macrophages compared to tumors from male patients. Notably, increased abundance of CD163+ macrophages and CD79a+ B cells were independently associated with decreased recurrence-free survival. This study has the potential to inform the rational utilization of immunomodulatory treatments for NMIBC based on the TIME of both male and female patients. Furthermore, these novel findings highlight the necessity of considering sexual dimorphism in the design of future immunotherapy trials.

HIV‐Associated Hypertension is TNFα‐Dependent and Involves Increased Vascular Nox1 Expression in Mice

HIV is a major global public health issue which has claimed more than 37 million lives so far. While combination antiretroviral therapies (cART) have significantly increased life expectancy, 35% of all treated HIV patients are diagnosed with hypertension. While cART treatment suppresses viral replication and further progression of the disease to AIDS, viral proteins are still present at low concentrations leading to the present clinical question of whether hypertension is the result of these viral proteins, cART regimen, or a combination of the two. We hypothesize that HIV-associated hypertension involves inflammatory response to viral proteins as well as increases in TNFα production and vascular NADPH Oxidase (Nox) 1 expression. To test this hypothesis, we utilized a transgenic mouse model (Tg26) which ubiquitously expresses seven of the nine viral proteins under the viral long terminal repeat promoter. Using quantitative RT-PCR analysis, high viral mRNA concentrations were found in lymphoid organs including the spleen, intraperitoneal immune cells, and lymph nodes but absent in the vasculature, therefore providing a mouse model mimicking clinical human pathology. Blood Pressure measurements using radio telemetry revealed that viral infection elevated mean arterial pressure in both male (WT=112.3+/-1.3 vs. Tg26=121.9+/-4.0) and female (WT=110.6+/-3.01 vs. Tg26=120.3+/-6.0mmHg) mice. Viral infection also increased systolic and diastolic blood pressure and heart rate with conserved diurnal variation. Measurement of aortic reactivity using wire myography revealed impaired endothelial relaxation (male: p<0.05/female p<0.05) to acetylcholine but intact vascular response to sodium Nitroprusside (SNP) and conserved vascular constriction to KCl and phenylephrine. Remarkably, transplant of bone marrow (BM) from Tg26 mice into WT impaired endothelial function (p<0.05) while transplant of BM from WT to Tg26 mice restored endothelial function. A multiplex cytokine panel found elevated levels of the pro-inflammatory cytokine TNFα in the plasma (p<0.05) and RT-qPCR analysis of the aorta (p<0.05) and intraperitoneal immune cells (p<0.05) also yielded increased TNFα levels. Treatment with the sTNFα inhibitor, etanercept, lowered MAP and restored endothelial function in male mice. Quantification of aortic Noxes (1,2, and 4) revealed increases in Nox1 expression (p<0.05). remarkably, selective inhibition of Nox1 via GKT771 restored endothelial dependent relaxation in both Tg26 and Tg26àWT transplanted mice. Analysis of multiple immune cell markers in the aorta via RT-qPCR found increased mRNA levels of the T-cell marker GATA3 (p<0.05). Taken together, these results support the hypothesis that HIV associated hypertension at levels of low viremia and independent of cART involves bone-derived immune cells, is TNFα dependent and involves increased vascular Nox1 expression.

Intrauterine hCG application increases expression of endothelial cell\u2013cell adhesion molecules in human

Endometrial receptivity is a decisive factor in human reproduction. Human chorionic gonadotropin (hCG) is one of the first embryonic signals that precedes the implantation by trophoblast invasion into the endometrium. Meta-analysis of randomized controlled trials reports a moderate-quality evidence for improved live birth rate for an intrauterine hCG dose ≥ 500 IU. Nevertheless, all hCG endometrial effects are not completely understood. We, therefore, utilized endometrial tissue from 12 patients after estradiol and progesterone treatment with or without intrauterine hCG flushing at the window of implantation (WOI) to analyze cellular composition by measuring marker proteins for stromal, endothelial, epithelial and immune cells. Flow cytometry analysis revealed that significantly more cells expressed the endothelial adhesion molecules VE-cadherin (CD144) and S-Endo-1 (CD146) after intrauterine hCG administration. In contrast, the endothelial marker CD31 and markers involved in vessel formation (VEGFR1 and VEGFR2) remained unchanged in their expression. Similarly, stroma markers (CD73, CD90 and CD105), epithelial markers (Desmocollin-2 and E-Cadherin) and immune cell markers (CD11b, CD45, CD79a and HLA-DR) displayed no alterations in their expression. This finding directs the focus on endothelial adhesion molecules as a potential mechanistically explanation of hCG conveyed increase of embryo implantation and pregnancy rates in women undergoing ART.

Suppression of neuroinflammation by an allosteric agonist and positive allosteric modulator of the \u03b17 nicotinic acetylcholine receptor GAT107

BackgroundThe α7 nicotinic acetylcholine receptor (α7 nAChR) negatively regulates the synthesis and release of pro-inflammatory cytokines by immune cells. Our previous studies showed that in encephalitogenic T cells, α7 nAChR expression is upregulated and that activation of the cholinergic system can attenuate experimental autoimmune encephalomyelitis (EAE). GAT107 is an allosteric agonist and positive allosteric modulator (ago-PAM) of α7 nAChR that can produce persistent activation of this receptor. Therefore, in the present study, we investigated the effect of GAT107 on neuroinflammation in EAE, the animal model used for the study of multiple sclerosis (MS) via α7 nAChR, and the inflammatory pathways involved.MethodsEAE was induced by administration of myelin oligodendrocyte glycoprotein (MOG35–55) in C57BL/6 mice. EAE mice were treated with the ago-PAM GAT107 or a placebo for 9 days, starting from the day of EAE induction. Clinical assessment and immunological evaluation of immune cells and cytokine production was performed.ResultsFollowing activation of the α7 nAChR by GAT107 during EAE, disease severity was significantly reduced by 70% and was correlated with a reduction in the extent of neuroinflammation in the CNS. The treatment reduced encephalitogenic T cell proliferation and the production of pro-inflammatory cytokines, as well as increased the production of the anti-inflammatory cytokine IL-10. Furthermore, the expression of immune cell markers was altered by GAT107 treatment, which induced a significant reduction in macrophages, dendritic cells, and B cells, as well as a reduction in anti-MOG35–55 antibodies. Additionally, GAT107 was found to directly activate α7 nAChR in murine macrophage RAW264.7 cells and in human PBMCs derived from MS patients and healthy donors.ConclusionsOur results show that GAT107 can be a useful molecule for harnessing the cholinergic anti-inflammatory pathway for long-lasting and wide-ranging modulation and downregulation of neuroinflammation in EAE.

Overexpressed pseudogene MT1L associated with tumor immune infiltrates and indicates a worse prognosis in BLCA

BackgroundBLCA is a common cancer worldwide, and it is both aggressive and fatal. Immunotherapy (ICT) has achieved an excellent curative effect in BLCA; however, only some BLCA patients can benefit from ICT. MT1L is a pseudogene, and a previous study suggested that MT1L can be used as an indicator of prognosis in colorectal cancer. However, the role of MT1L in BLCA has not yet been determined.MethodsData were collected from TCGA, and logistic regression, Kaplan-Meier plotter, and multivariate Cox analysis were performed to demonstrate the correlation between the pseudogene MT1L and the prognosis of BLCA. To identify the association of MT1L with tumor-infiltrating immune cells, TIMER and TISIDB were utilized. Additionally, GSEA was performed to elucidate the potential biological function.ResultsThe expression of MT1L was decreased in BLCA. Additionally, MT1L was positively correlated with immune cells, such as Tregs (ρ = 0.708) and MDSCs (ρ = 0.664). We also confirmed that MT1L is related to typical markers of immune cells, such as PD-1 and CTLA-4. In addition, a high MT1L expression level was associated with the advanced T and N and high grade in BLCA. Increased expression of MT1L was significantly associated with shorter OS times of BLCA patients (p < 0.05). Multivariate Cox analysis revealed that MT1L expression could be an independent prognostic factor in BLCA.ConclusionCollectively, our findings demonstrated that the pseudogene MT1L regulates the immune microenvironment, correlates with poor survival, and is an independent prognostic biomarker in BLCA.

Utilisation of cytological samples for multiplex immunofluorescence assay.

Understanding the immune environment of non-small cell lung cancer (NSCLC) is important for designing effective anticancer immunotherapies. We describe the use of multiplex immunofluorescence (mIF) assays to enable characterisation of the tumour-infiltrating immune cells and their interactions, both across and within immune subtypes. Six cytological samples of NSCLC taken by transoesophageal ultrasound-guided fine needle aspiration were tested with an mIF assay designed to detect the expression of key immune cell markers such as CD3, CD8, CD20, CD11b, and CD68. Pan-cytokeratin was used to detect the NSCLC cells. Fluorescence images were acquired on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). MIF assay was able to reliably detect and quantify the myeloid cell markers CD11b, CD68, CD3+ and CD8+ T cells, and CD20+ B lymphocytes on cytological samples of NSCLC. Whole-tissue analysis and its correlation with the corresponding H&E stains allowed a better understanding of the tissue morphology and the relationship between tumour and stroma compartments. Additionally, a uniform, specific, and correct staining pattern was seen for every immune marker. The implementation of mIF assay on cytological samples taken with minimally invasive methods seems feasible and can be used to explore the immune environment of NSCLC.

Relative Predictive Value of Circulating Immune Markers in US Adults Without Cardiovascular Disease: Implications for Risk Reclassification

ObjectiveTo investigate the relative predictive value of circulating immune cell markers for cardiovascular mortality in ambulatory adults without cardiovascular disease. MethodsWe analyzed data of participants enrolled in the National Health and Nutrition Examination Survey from January 1, 1999, to December 31, 2010, with the total leukocyte count within a normal range (4000-11,000 cells/μL [to convert to cells ×109/L, multiply by 0.001]) and without cardiovascular disease. The relative predictive value of circulating immune cell markers measured at enrollment—including total leukocyte count, absolute neutrophil count, absolute lymphocyte count, absolute monocyte count, monocyte-lymphocyte ratio (MLR), neutrophil-lymphocyte ratio, and C-reactive protein—for cardiovascular mortality was evaluated. The marker with the best predictive value was added to the 10-year atherosclerotic cardiovascular disease (ASCVD) risk score to estimate net risk reclassification indices for 10-year cardiovascular mortality. ResultsAmong 21,599 participants eligible for this analysis, the median age was 47 years (interquartile range, 34-63 years); 10,651 (49.2%) participants were women, and 10,713 (49.5%) were self-reported non-Hispanic white. During a median follow-up of 9.6 years (interquartile range, 6.8-13.1 years), there were 627 cardiovascular deaths. MLR had the best predictive value for cardiovascular mortality. The addition of elevated MLR (≥0.3) to the 10-year ASCVD risk score improved the classification by 2.7%±1.4% (P=.04). Elevated MLR had better predictive value than C-reactive protein and several components of the 10-year ASCVD risk score. ConclusionAmong ambulatory US adults without preexisting cardiovascular disease, we found that MLR had the best predictive value for cardiovascular mortality among circulating immune markers. The addition of MLR to the 10-year risk score significantly improved the risk classification of participants.

Hepcidin Upregulation in Lung Cancer: A Potential TherapeuticTarget Associated With Immune Infiltration.

Lung cancer has the highest death rate among cancers globally. Hepcidin is a fascinating regulator of iron metabolism; however, the prognostic value of hepcidin and its correlation with immune cell infiltration in lung cancer remain unclear. Here, we comprehensively clarified the prognostic value and potential function of hepcidin in lung cancer. Hepcidin expression was significantly increased in lung cancer. High hepcidin expression was associated with sex, age, metastasis, and pathological stage and significantly predicted an unfavorable prognosis in lung cancer patients. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) results suggested that hepcidin is involved in the immune response. Furthermore, hepcidin expression was positively correlated with the infiltration levels of immune cells and the expression of diverse immune cell marker sets. Importantly, hepcidin may affect prognosis partially by regulating immune infiltration in lung cancer patients. Hepcidin may serve as a candidate prognostic biomarker for determining prognosis associated with immune infiltration in lung cancer.

Identification of cluster of differentiation molecule-associated microRNAs as potential therapeutic targets for gastrointestinal cancer immunotherapy.

Cluster of differentiation molecules are markers of immune cells that have been identified as a potential immunotherapeutic target for cancer treatment. MicroRNAs are small non-coding RNAs that act as tumor suppressors or oncogenes whose importance in diagnosis, prognosis, and treatment of gastric and colorectal cancers has been widely reported. However, their association with cluster of differentiation molecules in gastrointestinal cancers has not been well studied. Therefore, our study aimed to analyze the relationship between microRNAs and cluster of differentiation molecules in gastrointestinal cancers, and to identify cluster of differentiation molecule-associated microRNAs as prognostic biomarkers for gastrointestinal cancer patients. Targetscan, Starbase, DIANA microT, and miRDB were used to investigate microRNA profiles that might be correlated with cluster of differentiation molecules in gastrointestinal cancers. Moreover, The Cancer Genome Atlas data analysis was used to investigate the association between cluster of differentiation molecules and microRNA expression in patients with gastric, colon, rectal, pancreatic, and esophageal cancers. The Kaplan-Meier plotter was used to identify the association between overall survival and cluster of differentiation molecule-associated microRNA expression in gastrointestinal cancer patients. miR-200a, miR-559, and miR-1236 were negatively associated with CD86, CD81, and CD160, respectively, in almost all types of gastrointestinal cancers, which were further verified in the in vitro studies by transfecting microRNA mimics in gastric cancer, colon cancer, pancreatic, and esophageal cell lines. Our study showed that miR-200a, miR-1236, and miR-559 are identified as cluster of differentiation-associated microRNAs in gastrointestinal cancers, providing a novel perspective to identify new therapeutic targets for cancer immunotherapy in gastrointestinal cancer patients.

Characterization of innate and adaptive immune cells involved in the foreign body reaction to polypropylene meshes in the human abdomen

BackgroundPolypropylene (PP) mesh is widely used to reinforce tissues. The foreign body reaction (FBR) to the implant is dominated by innate immune cells, especially macrophages. However, considerable numbers of adaptive immune cells, namely T cells, have also been regularly observed, which appear to play a crucial role in the long-term host response.MethodsThis study investigated the FBR to seven human PP meshes, which were removed from the abdomen for recurrence after a median of one year. Using immunofluorescence microscopy, the FBR was examined for various innate (CD11b+ myeloid, CD68+ macrophages, CD56+ NK) and adaptive immune cells (CD3+ T, CD4+ T-helper, CD8+ cytotoxic, FoxP3+ T-regulatory, CD20+ B) as well as “conventional” immune cells (defined as cells expressing their specific immune cell marker without co-expressing CD68).ResultsT-helper cells (19%) and regulatory T-cells (25%) were present at comparable rates to macrophages, and clustered significantly toward the mesh fibers. For all cell types the lowest proportions of “conventional” cells (< 60%) were observed at the mesh–tissue interface, but increased considerably at about 50–100 µm, indicating reduced stimulation with rising distance to the mesh fibers.ConclusionBoth innate and adaptive immune cells participate in the chronic FBR to PP meshes with T cells and macrophages being the predominant cell types, respectively. In concordance with the previous data, many cells presented a “hybrid” pattern near the mesh fibers. The complexity of the immune reaction seen within the foreign body granuloma may explain why approaches focusing on specific cell types have not been very successful in reducing the chronic FBR.

Systematic review on the effects of physical exercise on cellular immunosenescence-related markers - An update.

Immunosenescence is a remodeling of the immune system occurring with aging that leads to an increased susceptibility to auto-immunity, infections and reduced vaccination response. A growing consensus supports the view that physical exercise may counteract immunosenescence and improve the immune response. Unfortunately, evidence regarding the effects of exercise on markers of cellular immunosenescence lacked uniformity at the time of an extensive literature review in 2016. Moreover, exercise-induced effects in older adults were underrepresented compared to young adults or completely lacking, such as for senescent T-cells and apoptosis of T-lymphocytes. The aim of this systematic literature study was to collect and appraise newly available data regarding exercise-induced changes on immunosenescence-related markers of immune cells and compare this against data that was already available in 2016. Systematic reviewing of newly available data in the field of exercise immunology provides additional evidence for the effect of exercise on immunosenescence-related cellular markers. Importantly, this review provides evidence for the effect of long-term exercise on senescent T-lymphocytes in older adults. Additionally, newly retrieved evidence shows an acute exercise-induced mobilization of naïve and memory cells in older adults. In general, data regarding long-term exercise-induced effects in older adults remain scarce. Noteworthy was the high number of articles describing exercise-induced effects on regulatory T-cells. However exercise-induced effects on this cell type are still inconclusive as some articles reported an exercise-induced up- or downregulation, while others reported no effects at all. Numerous studies on Natural Killer cell counts did not provide uniformity among data that was already available. Recent data regarding dendritic cells mostly described an increase after exercise. Overall, our literature update highlights the major influence of the type and intensity of exercise on immunosenescence-related markers, especially in older adults.

Superovulation with gonadotropin-releasing hormone agonist or chorionic gonadotropin for ovulation trigger differentially affects leukocyte populations in the peri-implantation mouse uterus

To investigate the effect of superovulation with human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone agonist (GnRHa) trigger on leukocyte density and expression of leukocyte-specific genes in the peri-implantation period in the mouse uterus. Laboratory research. University laboratory facility. Female mice were mated to fertile male mice in one of three protocols: (1) natural mating or mating following injection with pregnant mare serum gonadotropin followed by trigger with (2) GnRHa or (3) hCG. Female mice were killed prior to implantation, 3 days after ovulation (E3.5), and the ovaries and uterine tissue were collected. Total RNA was isolated and assayed using quantitative reverse transcription polymerase chain reaction, and the uterine tissue was stained for histologic analysis of immune cell markers. Endometrial leukocyte (CD45) and vessel density (CD31) by immunohistochemical staining; expression of leukocyte markers CD11b, CD335, and CD22, by quantitative reverse transcription polymerase chain reaction in the whole uterine tissue. Superovulation decreased (compared with controls) the endometrial leukocyte density, based on the number of cells staining for CD45, and endometrial vessel density, based on the number of cells staining for CD31. Leukocyte density was additionally decreased in the GnRHa trigger group compared with that in the hCG trigger group. Superovulation with hCG and GnRHa triggers decreased the uterine expression of the B-cell marker CD22 compared with controls. The expression of the natural killer cell marker CD11b was decreased by the hCG trigger but not by the GnRHa. Abundance of mRNA encoding the CD335 natural killer cell marker was not affected by superovulation or trigger agent. In mice, superovulation with the GnRHa trigger compared with that with the hCG trigger differentially alters key immunologic factors in the uterine peri-implantation. These altered immunologic factors have roles in angiogenesis that may assist in elucidating the effects of assisted reproductive technologies on implantation efficiency and fetal growth and development.

Prognostic biomarkers related to tumoral microenvironment in pancreatic ductal adenocarcinoma: a systematic review.

Over the past decades, pancreatic ductal adenocarcinoma (PDAC) has been coming into view due to increased mortality, the 5-year survival rate being the lowest of all cancers (around 6%). In PDAC, microenvironmental components possess prognostic relevance. The aim of this article is to perform a review of studies evaluating the composition of the tumor microenvironment to identify tumor microenvironment-related prognostic biomarkers in patients with PDAC. A literature search has been performed in three major databases PubMed®, Embase®, Web of Science® using the search terms: pancreatic adenocarcinoma in combination with one of the following: alpha-smooth muscle actin (α-SMA), collagen I, cluster of differentiation (CD)31, CD105, CD3–CD4–CD8, CD68 and CD206. Total number of articles identified through database searching was 1185. After title and abstract review, we have selected 92 articles in which the markers sought were studied. Tumor microenvironment-related biomarkers appear to also possess role in monitoring the response to treatment. Thus, CD105 angiogenetic immunomarker, stromal immunomarkers such as α-SMA and collagen I, immune cells markers represented by CD4/CD8 ratio, CD206 and CD68 were correlated with negative prognosis, while CD3+, CD8+ immune cells markers and CD31 angiogenetic immunomarker proved to be correlated with good prognosis. Furthermore, most studies were performed on resected specimens and culture cells, while only a few studies used specimens obtained through endoscopic ultrasound-guided fine-needle biopsy (EUS–FNB). To increase the therapeutic response and reduce toxicity, prognostic targets should be determined on a large scale, not only based on resected specimens. EUS–FNB represents a feasible method to provide sufficient tissue for diagnosis and additional immunohistochemistry analysis.

Biological Heterogeneity of Chondrosarcoma: From (Epi) Genetics through Stemness and Deregulated Signaling to Immunophenotype.

Simple SummaryChondrosarcoma (ChS) is the second most frequently diagnosed malignant bone tumor of cartilaginous origin and is generally resistant to standard treatment options. In this paper, we aim to review the current state of the knowledge regarding ChS. We discuss the genetic, epigenetic, and molecular abnormalities underlying its substantial biological and clinical heterogeneity. This review summarizes the critical genetic and molecular drivers of ChS development and progression, contributing to its radio- and chemotherapy resistance. We describe genomic aberrations and point mutations, as well as epigenetic modifications and deregulated signal transduction pathways. We provide an insight into the stem-like characteristics and immunophenotype of ChS. The paper also outlines potential diagnostic and prognostic biomarkers of ChS and recently identified novel targets for future pharmacological interventions in patients.Chondrosarcoma (ChS) is a primary malignant bone tumor. Due to its heterogeneity in clinical outcomes and resistance to chemo- and radiotherapies, there is a need to develop new potential therapies and molecular targets of drugs. Many genes and pathways are involved in in ChS progression. The most frequently mutated genes are isocitrate dehydrogenase ½ (IDH1/2), collagen type II alpha 1 chain (COL2A1), and TP53. Besides the point mutations in ChS, chromosomal aberrations, such as 12q13 (MDM2) amplification, the loss of 9p21 (CDKN21/p16/INK4A and INK4A-p14ARF), and several gene fusions, commonly occurring in sarcomas, have been found. ChS involves the hypermethylation of histone H3 and the decreased methylation of some transcription factors. In ChS progression, changes in the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K–AKT–mTOR) and hedgehog pathways are known to play a role in tumor growth and chondrocyte proliferation. Due to recent discoveries regarding the potential of immunotherapy in many cancers, in this review we summarize the current state of knowledge concerning cellular markers of ChS and tumor-associated immune cells. This review compares the latest discoveries in ChS biology from gene alterations to specific cellular markers, including advanced molecular pathways and tumor microenvironment, which can help in discovering new potential checkpoints in inhibitory therapy.

ITGA5 is a prognostic biomarker and correlated with immune infiltration in gastrointestinal tumors

BackgroundIntegrin Subunit Alpha 5 (ITGA5), belongs to the integrin alpha chain family, is vital for promoting cancer cell invasion, metastasis. However, the correlation between ITGA5 expression and immune infiltration in gastrointestinal tumors remain unclear.MethodsThe expression level of ITGA5 was detected by Oncomine and Tumor Immune Estimation Resource (TIMER). The association between ITGA5 and prognosis of patients was identified by Kaplan–Meier plotter, Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and PrognoScan. We evaluated the correlation between ITGA5 expression and immune infiltrating level via TIMER. Besides, TIMER, immunohistochemistry (IHC) staining and western blot were used to explore correlations between ITGA5 expression and markers of immune infiltrates cells. Furthermore, we constructed protein-protein interaction (PPI) network and performed functional enrichment by GeneMANIA and Metascape.ResultsITGA5 was generally overexpressed and correlated with worse prognosis in multiple types of gastrointestinal tumors. In addition, ITGA5 expression level was significantly associated with tumor purity and immune infiltration levels of different immune cells in gastrointestinal tumors. Interestingly, immune markers for monocytes, tumor - associated macrophages (TAMs), macrophages 2 (M2) cells and T-helper 2 (Th2) cells were found to be significantly and positively correlated with ITGA5 expression levels in colon and gastric cancer. Results from IHC staining and western blot further proved that markers of Th2 and M2 cell were significantly increased in gastric cancer patients with high ITGA5 expression levels. Lastly, interaction network and function enrichment analysis revealed ITGA5 was mainly involved in “integrin mediated signaling pathway”, “leukocyte migration”, “cell-substrate adhesion”.ConclutionsOur study demonstrated that ITGA5 may act as an essential regulator of tumor immune cell infiltration and a valuable prognostic biomarker in gastrointestinal tumors. Additional work is needed to fully elucidate the underlying mechanisms behind these observations.

FUT7 Promotes the Epithelial-Mesenchymal Transition and Immune Infiltration in Bladder Urothelial Carcinoma.

BackgroundBladder urothelial carcinoma (BLCA) is one of the most frequently appearing, lethal and aggressive malignancies of the genitourinary system with growing morbidity and mortality, which affects human health seriously. Protein glycosylation, catalyzed by specific glycosyltransferase, has been found to be abnormal in several diseases, especially cancer. Fucosyltransferase VII (FUT7), one of the fucosyltransferases, was observed abnormally expressed in various cancers, however, the role of FUT7 in BLCA, and the association between its expression and clinical outcomes or immune infiltration remains unclear.MethodologyFUT7 expression in BLCA was analyzed in Oncomine database, which was further confirmed with immunohistochemistry and ELISA. The prognostic value of FUT7 for BLCA was evaluated with PrognoScan database, and its genetic alteration was examined in cBioPortal database. The proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) changes of bladder cancer cells after FUT7 siRNA or cDNA transfection were determined by CCK8, colony formation, transwell and Western blot, respectively. The correlation between FUT7 expression and immune infiltration levels was analyzed in TIMER and TISIDB databases, and the methylation level of FUT7 was detected in UALCAN database.ResultsThe results showed that the expression of FUT7 was increased in BLCA, and patients with high FUT7 level were predicted to have lower overall survival and disease-specific survival rates, which were not influenced by FUT7 genetic alterations. Downregulation FUT7 inhibited the proliferation, migration, invasion and EMT of bladder cancer cells, whereas upregulation of FUT7 showed the opposite effects. We found that FUT7 was positively correlated with immune cell infiltration levels (CD8+ T cells, CD4+T cells, macrophage, neutrophil and dendritic cells), and also the expression of gene markers of immune cells. The negative correlation between FUT7 expression and FUT7 methylation level was observed, among which FUT7 expression was positively correlated with the abundance of 28 kinds of tumor-infiltrating lymphocytes (TILs), while FUT7 methylation level was negatively correlated with TILs.ConclusionAltogether, these findings suggested that FUT7 possessed the potential to serve as a detection biomarker or immunotherapeutic target for BLCA.

Clinical and molecular immune characterization of ERBB2 in glioma

ERBB2 is a well-studied oncogene that promotes progression of multiple cancers, especially in breast cancer. However, the expression status of ERBB2, the values of ERBB2 on prognosis, and its molecular characterization in glioma have not been well examined. We explored the expression of ERBB2 and its clinical and molecular immune characterization in human glioma samples using the extracted genetic and clinical data from the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) databases. Immunohistochemistry (IHC) performed on the tissue microarray slide was used to validate the expression and clinical and prognostic values of ERBB2 in glioma. Higher ERBB2 expression was found in patients with higher grades gliomas than in patients with lower grades gliomas. Besides, patients with higher ERBB2 expression showed poor prognosis. The IHC and clinical data next validated the expression pattern and prognostic value of ERBB2 in glioma. Further analysis showed that there was a strong positive correlation between ERBB2 and common immune checkpoints as well as immune markers of various immune cells in both TCGA and CGGA databases, and the IHC data further validated the positive correlation between ERBB2 and PD-L1 expression. Besides, analysis of ERBB2-related immune genes and signatures showed the significant role of ERBB2 in mediating tumor immune response in glioma. To sum up, our findings summarize the expression pattern and clinical characteristics of ERBB2 in glioma, which may be useful for expanding our understanding of the critical role of ERBB2 in antitumor therapy in glioma.

Increased Tumor Immune Microenvironment CD3+ and CD20+ Lymphocytes Predict a Better Prognosis in Oral Tongue Squamous Cell Carcinoma.

Background: Oral tongue squamous cell carcinoma (OTSCC) causes over 350,000 cases annually and particularly impacts populations in developing countries. Smoking and alcohol consumption are major risk factors. Determining the role of the tumor immune microenvironment (TIME) in OTSCC outcomes can elucidate immune mechanisms behind disease progression, and can potentially identify prognostic biomarkers.Methods: We performed a retrospective study of 48 OTSCC surgical specimens from patients with tobacco and alcohol exposures. A panel of immunoregulatory cell subpopulations including T (CD3, CD4, CD8) and B (CD20) lymphocytes, dendritic cells (CD1a, CD83), macrophages (CD68), and immune checkpoint molecules programmed cell death protein 1 (PD-1) and ligand 1 (PD-L1) were analyzed using immunohistochemistry. The levels of immune effector cell subpopulations and markers were analyzed in relation to overall survival.Results: Pathological characteristics of the tumor microenvironment included inflammatory infiltrates (83.3%), desmoplasia (41.6%), and perineural invasion (50.0%). The TIME contained high levels of T cells (CD3+, CD4+, and CD8+) and B cells (CD20+), as well as immature (CD1a) and mature (CD83) dendritic cells, PD-1, and PD-L1. Higher numbers of TIME infiltrating CD3+ T cells and CD20+ B cells were predictive of better survival, while higher levels of CD83+ mature dendritic cells predicted better survival. CD3+ T cells were identified as an independent prognostic marker for OTSCC. Lastly, CD3+ T cells were strongly correlated with the number of CD8+ cells and PD-L1 expression.Conclusion: Our findings provide evidence that the TIME profile of OTSSC impacted prognosis. The high expression of CD3+ T cells and B cells are predictive of better overall survival and indicative of an immunologically active, inflammatory TIME in patients with better survival. The number of CD3+ T cells was an independent prognostic marker.

Abstract PS18-01: Glucocorticoid receptor expression in early-stage triple-negative breast cancer is associated with a high level of tumor immune cell infiltrates

Abstract Background: Triple-negative breast cancer (TNBC) with high glucocorticoid receptor activity (GR-high) is associated with a high risk of relapse and chemotherapy resistance. Glucocorticoids (GC) are essential for the regulation of immune and inflammatory responses. Multiple mechanisms of GC-induced immunosuppression have been proposed, including suppression of T cell proliferation and dendritic cell antigen presentation and function. The importance of intact immune surveillance in controlling neoplastic transformation is well-established, and accumulating evidence demonstates a correlation between tumor-infiltrating lymphocytes (TILs) in cancer tissue and favorable prognosis. In particular, the presence of CD8+ T-cells and the ratio of CD8+ effector T-cells / Foxp3+ regulatory T-cells (Tregs) appears to correlate with improved prognosis and long-term survival. We hypothesized that tumor GR activity would modulate the tumor immune microenvironment (TME), thereby suppressing anti-tumor immunity in TNBC. Methods: We identified 46 patients with TNBC who received neoadjuvant chemotherapy at The University of Chicago between 2002-2014, and had pretreatment tissue available for study. Tumors were stained for GR expression using the anti-GR rabbit monoclonal XP antibody (Cell Signaling, 1:200 dilution). GR expression was determined independently by two pathologists. Using multiplex immunohistochemistry, six phenotypes for immune cell markers (BATF3+ dendritic cells, CD8+ T-cells, Foxp3+ Tregs, PD-L1+tumor, PD-L1-tumor, PD-L1+immune cells) were identified in the tumor and stroma of the pretreatment biopsy specimens. Absolute counts of each type of immune cell as well as ratios between immune cell types were compared in GR-low, GR-moderate, and GR-high expressing TNBC specimens using the Kruskal-Wallis test. Results: There were 8 GR-low, 23 GR-moderate, and 15 GR-high expressing tumors in our cohort. When comparing GR-low to GR-moderate or GR-moderate to GR-high expressing tumors, there were no significant difference observed in immune cell types. However, when comparing GR-high to GR-low expressing TNBCs, there were significantly higher levels of CD8+ T-cells, Foxp3+ Treg cells, and BATF3+ dendritic cells in GR-high vs GR-low TNBC tumors (p=0.0004, p=0.011, and p=0.0072, respectively). When comparing the ratio of CD8+ T-cells to Foxp3+ Treg cells, PD-L1+ tumor or immune cells, no significant differences were observed by level of GR expression (p=ns for all comparisons). Conclusions: We report for the first time that GR-high, treatment-naïve, primary TNBCs have significantly higher CD8+ T cells, Foxp3+ T cells, and BATF3+ dendritic cells compared to GR-low TNBCs, consistent with an inflamed TME. While historically a robust immune infiltrate in the TME in early TNBC has been associated with a better response to chemotherapy and a more favorable long-term outcome, high GR activity in early TNBC has been associated with a worse outcome. Our observation that GR-high tumors have an inflamed TME, suggests that checkpoint blockade to either suppress Foxp3+ Tregs or activate CD8+ T cells may be able to restore anti-tumor immunity. Citation Format: Deniz N Dolcen, Marie Dreyer, Aaron Miller, Anna Biernacka, Ken Hatogai, Randy Sweis, Rita Nanda, Suzanne D Conzen. Glucocorticoid receptor expression in early-stage triple-negative breast cancer is associated with a high level of tumor immune cell infiltrates [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-01.

Molecular analyses of triple-negative breast cancer in the young and elderly

BackgroundBreast cancer in young adults has been implicated with a worse outcome. Analyses of genomic traits associated with age have been heterogenous, likely because of an incomplete accounting for underlying molecular subtypes. We aimed to resolve whether triple-negative breast cancer (TNBC) in younger versus older patients represent similar or different molecular diseases in the context of genetic and transcriptional subtypes and immune cell infiltration.Patients and methodsIn total, 237 patients from a reported population-based south Swedish TNBC cohort profiled by RNA sequencing and whole-genome sequencing (WGS) were included. Patients were binned in 10-year intervals. Complimentary PD-L1 and CD20 immunohistochemistry and estimation of tumor-infiltrating lymphocytes (TILs) were performed. Cases were analyzed for differences in patient outcome, genomic, transcriptional, and immune landscape features versus age at diagnosis. Additionally, 560 public WGS breast cancer profiles were used for validation.ResultsMedian age at diagnosis was 62 years (range 26–91). Age was not associated with invasive disease-free survival or overall survival after adjuvant chemotherapy. Among the BRCA1-deficient cases (82/237), 90% were diagnosed before the age of 70 and were predominantly of the basal-like subtype. In the full TNBC cohort, reported associations of patient age with changes in Ki67 expression, PIK3CA mutations, and a luminal androgen receptor subtype were confirmed. Within DNA repair deficiency or gene expression defined molecular subgroups, age-related alterations in, e.g., overall gene expression, immune cell marker gene expression, genetic mutational and rearrangement signatures, amount of copy number alterations, and tumor mutational burden did, however, not appear distinct. Similar non-significant associations for genetic alterations with age were obtained for other breast cancer subgroups in public WGS data. Consistent with age-related immunosenescence, TIL counts decreased linearly with patient age across different genetic TNBC subtypes.ConclusionsAge-related alterations in TNBC, as well as breast cancer in general, need to be viewed in the context of underlying genomic phenotypes. Based on this notion, age at diagnosis alone does not appear to provide an additional layer of biological complexity above that of proposed genetic and transcriptional phenotypes of TNBC. Consequently, treatment decisions should be less influenced by age and more driven by tumor biology.