Tenascin-XB plays a role in the infiltration of immune cells in tumor microenvironment.

Introduction Sarcomas, a broad family of mesenchymal malignancies, exhibit remarkable histologic diversity. Previous study has reported that the infiltration of immune and stromal cells in tumor microenvironment contribute significantly to prognosis. ESTIMATE, an algorithm to calculate immune and stromal scores, predicts the infiltration of non-cancer components. The Cancer Genome Atlas (TCGA) database is available to grasp potential correlations between gene set prolife and overall survival of malignancies, including sarcomas. To better understand the proportions of immune cells in the tumor microenvironment, We used CIBERSORT deconvolution software and ssGSEA to infer the relative proportions of several distinct leukocyte cell types in the tumors from microarray gene expression data of sarcoma patients. By taking advantage of both TCGA database of sarcomas cohorts and ESTIMATE algorithm, we extracted a list of genes that predict poor outcomes in sarcoma patients. Finally, we validated these genes in an independent sarcoma cohort from the GSE17679. Thus, we obtained a list of tumor microenvironment-related genes that predict poor outcomes in sarcoma patients. Methods Gene expression profile and clinical data for sarcoma patients was obtained from the TCGA data portal. Immune scores and stromal scores were calculated by applying the ESTIMATE algorithm to the downloaded database. Batch adjusted data was subsequently analyzed using CIBERSORT to resolve the immune composition. For validation, gene expression profiles for Ewing sarcoma patients were obtained from the Gene Expression Omnibus dataset GSE17679. Results The immune and stromal score are associated with prognosis. The type 2 macrophages in sarcoma makes up the largest composition of all immune cells in the tumor microenvironment of sarcoma (except synovial sarcoma), which predicts poor outcomes. From functional enrichment analysis of TCGA database applied by ESTIMATE algorithm-based immune scores, we extracted that NR1H3, VAMP5, GIMAP2, GBP2, HLA-E and CRIP1 are highly expressed in the immune microenvironment, predicting good outcomes in sarcoma patients. Conclusion We extracted a list of tumor microenvironment related genes. These genes were validated in an independent sarcoma cohort and that may represent promising novel signatures for the diagnosis and prognosis prediction of sarcoma. The immune composition analysis could be useful for outlining the prognosis. Citation Format: Hongmin Chen. Comprehensive bioinformatic analysis of immune composition and genes for survival prediction in sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4434.

Melanoma is the most lethal type of skin cancer. Despite the breakthroughs in the clinical treatment of melanoma using tumor immunotherapy, many patients do not benefit from these immunotherapies because of multiple immunosuppressive mechanisms. Therefore, there is an urgent need to determine the mechanisms of tumor-immune system interactions and their molecular determinants to improve cancer immunotherapy. In this study, combined analysis of microarray data and single-cell RNA sequencing data revealed the key interactions between immune cells in the melanoma microenvironment. First, differentially expressed genes (DEGs) between normal and malignant tissues were obtained using GEO2R. The DEGs were then subjected to downstream analyses, including enrichment analysis and protein–protein interaction analysis, indicating that these genes were associated with the immune response of melanoma. Then, the GEPIA and TIMER databases were used to verify the differential expression and prognostic significance of hub genes, and the relationship between the hub genes and immune infiltration. In addition, we combined single cell analysis from GSE123139 to identify immune cell types, and validated the expression of the hub genes in these immune cells. Finally, cell-to-cell communication analysis of the proteins encoded by the hub genes and their interactions was performed using CellChat. We found that the CCL5-CCR1, SELPLG-SELL, CXCL10-CXCR3, and CXCL9-CXCR3 pathways might play important roles in the communication between the immune cells in tumor microenvironment. This discovery may reveal the communication basis of immune cells in the tumor microenvironment and provide a new idea for melanoma immunotherapy.

Laryngeal cancer (LC) is a malignant tumor that occurs in the head and neck. Laryngeal cancer is one of the most common cancers of the neck and head, and its prognosis has always been poor. The incidence of LC increased gradually and showed an early rising trend. Laryngeal cancer is rarely studied in relation to immunity, Malignant tumors will change the state of the human body in various ways to adapt to their own survival and avoid the immune system. This study aims to explore the immune molecular mechanism of laryngeal cancer through bioinformatics analysis. The gene expression data was downloaded for 3 microarray datasets: GSE27020, GSE59102, and GSE51985. CIBERSORT algorithm was performed to evaluate immune cell infiltration in tissues between LC and healthy control (HC). Differentially expressed genes (DEGs) were screened. Functional correlation of DEGs were analyzed by Gene Ontology, Gene Set Enrichment Analysis and Kyoto encyclopedia of genes and genomes. Candidate biomarkers were identified by cytoHubba of Cytoscape. Spearman correlations between the above biomarkers and infiltrating immune cells were explored using R software analysis. The immune cell types of LC and HC were significantly different. Twenty-one DEGs were obtained by cross-screening. The function of DEGs is closely related to the number of immune cells. Five central genes (TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4) were screened. The HUB gene was demonstrated to have the ability to diagnose LC and HC with good specificity and sensitivity. The correlation between immune cells and biomarkers showed that hub gene was positively correlated with macrophages and dendritic cells, and negatively correlated with CD4 + T cell. TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4 can be used as diagnostic biomarker for LC. Macrophages, dendritic cells and CD4 + T cell may participate in the occurrence and development of LC.

Neoadjuvant chemoradiation followed by surgery is recommended for locally advanced esophageal squamous cell carcinoma (ESCC). Patients achieve pathological complete response (pCR) have better survival outcomes. Immune cells in tumor microenvironment (TME) are potential to affect chemoradiation sensitivity. Our study aimed to discover key immune-associated predictors of pCR for ESCC. The infiltrations of multiple immune cells were estimated by ImmuCellAI and cibersort based on RNA-seq data of fifty ESCC samples before treatment. The infiltrations of immune cells including Th1 cells, CD3+ T cells, CD8+ T cells, NK cells, NKT cells and DCs were evaluated by multiplex immunohistochemistry (mIHC) in 131 ESCC samples before treatment. In-vitro cell experiments were performed to discover the effects of Th1 cells on tumor cells. The analysis of RNA-seq data of ESCC revealed that the infiltration of Th1 cells was higher in pCR group. Results of mIHC demonstrated that infiltrations of Th1 cells and its relevant immune cells including CD8+ T cells, NK cells, NKT cells and DCs were positively associated with pCR. ESCC samples were divided into two subtypes based on the infiltration of these immune cells, with the high infiltration subtype was easier to achieve pCR. In-vitro cell experiments confirmed that Th1 cells were able to inhibit proliferation and improve chemoradiation sensitivity of tumor cells. Infiltrations of Th1 cells and its relevant immune cells are positively associated with pCR following neoadjuvant chemoradiation in ESCC. Th1 cells can improve the chemoradiation sensitivity of tumor cells.

Immune cells in the tumour microenvironment (TME) interact with each other and with tumour cells to promote tumour development. IL-6/STAT3 pathway induces and maintains mainly pro-tumour TME. Macrophages and lymphocytes are positive for IL-6, STAT3 and IL-17, while FoxP3 cells are mainly regulatory cells. IL-17+ and FoxP3+ immune cells have impact on gut inflammation and tumourigenesis. The aim of this study was to determine IL-6+, STAT3+, IL-17+ and FoxP3+ immune cells in colorectal cancer (CRC) TME and explore their association with clinicopathological parameters and mismatch repair (MMR) status. The investigated samples were collected from 104 CRC patients. Immunohistochemistry for the aforementioned markers and microsatellite instability (MSI) markers was performed. MSI testing was used. The investigated immune cells were significantly more in the invasive front (IF) as compared to tumour stroma (TS). IL-6+ and STAT3+ immune cells were more in the early tumour stages as compared to advanced stages. IL-17+ and IL-6+ immune cells were more in well and moderately differentiated cancers. IL-6+, STAT3+ and IL-17+ immune cells prevailed in the TME in microsatellite stable patients and only FoxP3+ cells were fewer there. Higher numbers of STAT3+ cells correlated with longer survival. These results support the suggestion that the transition of normal colonic mucosa to CRC is marked by a shift of Th programme, leading to accumulation of Th17 cells and Tregs that sustain tumour cell growth through the IL-6/STAT3 signalling pathway.

P-0035 - Immunosuppressive Protein Expression on Tumor and the Presence of Immune Cells Within Tumor Microenvironment in Curatively Resected Gastric Cancer

Background: The tumor microenvironment (TME) of breast cancer (BRCA) is a complex and dynamic micro-ecosystem that influences BRCA occurrence, progression, and prognosis through its cellular and molecular components. However, as the tumor progresses, the dynamic changes of stromal and immune cells in TME become unclear. Objective: The aim of this study was to identify differentially co-expressed genes (DCGs) associated with the proportion of stromal cells in TME of BRCA, to explore the patterns of cell proportion changes, and ultimately, their impact on prognosis. Methods: A new heuristic feature selection strategy (CorDelSFS) was combined with differential co-expression analysis to identify TME-key DCGs. The expression pattern and co-expression network of TME-key DCGs were analyzed across different TMEs. A prognostic model was constructed using six TME-key DCGs, and the correlation between the risk score and the proportion of stromal cells and immune cells in TME was evaluated. Results: TME-key DCGs mimicked the dynamic trend of BRCA TME and formed cell type-specific subnetworks. The IG gene-related subnetwork, plasmablast-specific expression, played a vital role in the BRCA TME through its adaptive immune function and tumor progression inhibition. The prognostic model showed that the risk score was significantly correlated with the proportion of stromal cells and immune cells in TME, and low-risk patients had stronger adaptive immune function. IGKV1D-39 was identified as a novel BRCA prognostic marker specifically expressed in plasmablasts and involved in adaptive immune responses. Conclusions: This study explores the role of proportionate-related genes in the tumor microenvironment using a machine learning approach and provides new insights for discovering the key biological processes in tumor progression and clinical prognosis.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world with an immunosuppressive Tumor microenvironment (TME). Ferroptosis plays an essential role in tumor proliferation, invasion, and metastasis. However, the relationship between ferroptosis and TME of HCC has remained elusive. Differentially expressed ferroptosis-related genes (DE FRGs) between normal liver tissues and HCC tissues were obtained from The Cancer Genome Atlas (TCGA). On this basis, we identified the molecular subtypes mediated by DE FRGs and TME cell infiltration. Next, a predictive signature was established to quantity the ferroptosis-related characteristics by performing the least absolute shrinkage and selection operator Cox regression analyses. Univariate and multivariate COX analyses determined the independent prognostic factors. Finally, the expression stability of 3 ferroptosis-related signature genes was verified in cancer and paracancerous normal tissues of HCC. We identified three different molecular subtypes and found that the subtype with the better prognosis was associated with high enrichment of immune- and metabolic-related hallmark signaling pathways and high infiltration of immune cells in TME. The signature was considered to be an independent prognostic factor. We also found that the signature can reflect the infiltration characteristics of different immune cells in TME. Immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), regulatory T cells, and type 17 T helper cells were significantly enriched in the high-risk group. The analysis data of immune checkpoints and tumor mutation load indicated that the signature had great potential in predicting Immunotherapy response and chemotherapeutic sensitivity. In addition, the overexpression of 3 ferroptosis-related signature genes was confirmed in HCC tissues and HCC cell lines. Ferroptosis inducer RSL3 inhibited the proliferation of HCC cells and was a potential cancer immunotherapy agent. These findings enhanced our understanding of the regulatory mechanism of ferroptosis in HCC and provided new insights into evaluating prognosis and developing more effective Immunotherapy and chemotherapy strategies.

The invasion of immune cells in the tumor microenvironment (TME) is closely related to cancer development. Studies have demonstrated that N6-methyladenosine (m6A) can affect the invasion of immune cells in TME as well as cancer development. We comprehensively analyzed the RNA-seq data of 16 different cancer types based on 20 m6A regulators and identified two distinct m6A modification patterns, which were closely associated with TME cell infiltration and overall patient survival. Then, we used principal component analysis (PCA) to construct m6Ascore based on the expression of m6A-related prognostic genes, which can successfully predict patient survival. The low-m6Ascore subtype is characterized by more immune cell infiltration, good prognosis and lower TNM stages, while the high-m6Ascore subtype is characterized by low immune infiltration, stromal activation, and poor prognosis. m6Ascore was also closely associated with immunotherapy response and was significantly higher in complete response/partial response (CR/PR) patients than in stable disease/progressive disease (SD/PD) patients in both immunotherapy cohorts. Therefore, our study indicates that m6A modification plays an important role in the prognosis of pan-cancer and the formation of complex TME in pan-cancer. Our research helps to improve the cognition of m6A modifications at pan-cancer levels and identify more effective strategies for immunotherapy.

Extracellular adenosine (eAdo) bridges tumor metabolism and immune regulation. CD39-CD73-eAdo/A2aR axis regulates tumor microenvironment (TME) and immunotherapy response. In the era of immunotherapy, exploring the impact of the CD39-CD73-eAdo/A2aR axis on TME and developing targeted therapeutic drugs to enhance the efficacy of immunotherapy are the current research hotspots. This study summarizes and explores the research trends and hotspots of the adenosine axis in the field of TME to provide ideas for further in-depth research. Literature information was obtained from the Web of Science core collection database. The VOS viewer and the bibliometric tool based on R were used to quantify and identify cooperation information and individual influence by analyzing the detailed information of the global annual publication volume, country/region and institution distribution, article authors and co-cited authors, and journal distribution of these articles. At the same time, the distribution of author keywords and the co-occurrence of author keywords, highly cited articles, and highly co-cited references of CD39-CD73-eAdo/A2aR in the field of TME were analyzed to determine research hotspots and trends. 1,721 articles published in the past ten years were included in this study. Through bibliometric analysis, we found that (1) 69 countries and regions explored the effect of the CD39-CD73-eAdo/A2aR on TME, and the research was generally on the rise. Researchers in the United States dominated research in this area, with the highest total citation rate. China had the most significant number of publications. (2) Harvard University has published the most articles in this field. (3) 12,065 authors contributed to the publication of papers in this field, of which 23 published at least eight papers. STAGG J had significant academic influence, with 24 published articles and 2,776 citations. Co-cited authors can be clustered into three categories. Stagg J, Allard B, Ohta A, and Antonioli, L occupied a central position in the network. (4) 579 scholarly journals have published articles in this field. The journal FRONTIERS IN IMMUNOLOGY published the most significant number of papers, with 97 articles and a total of 2,317 citations, and the number of publications increased year by year. (5) "The ectonucleotidases CD39 and CD73: Novel checkpoint inhibitor targets" was the most frequently local cited article (163 times). The "A2A adenosine receptor protects tumors from antitumor T cells" was the most co-cited reference (224 times). (6) Through the analysis of author keywords, we found that the relationship between adenosine and immunotherapy was a core concept for many researchers in this field. Breast cancer, melanoma, colorectal cancer, ovarian cancer, glioblastoma, pancreatic cancer, hepatocellular carcinoma, and lung cancer were the most frequent cancer types in adenosine-related tumor studies. Immunotherapy, immunosuppression, immune checkpoint, and immune checkpoint inhibitors were the hot keywords in the research, reflecting the importance of the adenosine metabolic pathway in tumor immunotherapy. The keywords such as Immunogenic cell death, T cells, Sting, regulatory T cells, innate immunity, and immune infiltration demonstrated the pathways by which adenosine affected the TME. The famous author keywords in recent years have been immunotherapy, immunogenic cell death, inflammation, lung cancer, and gastric cancer. The effect of CD39-CD73-eAdo/A2aR on the infiltration and function of various immune cells in TME, tumor immunotherapy response, and patient prognosis has attracted the attention of researchers from many countries/regions. American scholars still dominate the research in this field, but Chinese scholars produce the most research results. The journal FRONTIERS IN IMMUNOLOGY has published the wealthiest research in the field. Stagg J was a highly influential researcher in this field. Further exploration of targeted inhibition of CD39-CD73-eAdo/A2aR alone or in combination with other immunotherapy, radiotherapy, and chemotherapy in treating various cancer types and developing effective clinical therapeutic drugs are continuous research hotspots in this field.

N6-methyladenosine (m6A) is the most common type of RNA methylation and is considered to participate in various biological and pathological processes, specifically in the regulation of tumorigenesis and metastasis. However, the exact prognostic role of m6A methylation regulators in early-stage clear cell renal cell carcinoma (ccRCC) is currently unknown. In the present study, a prognostic model consisting of m6A RNA methylation regulators in early stage ccRCC was constructed and the reliability of the signature was assessed by proteomics and immunohistochemistry. Additionally, the relationship between the prognostic model and tumor infiltrating immune cells within the tumor microenvironment was investigated. Gene mutation and RNA sequencing data of 19 m6A methylation regulators for early-stage ccRCC patients were extracted from The Cancer Genome Atlas (TCGA) database with the corresponding clinical information. Univariate and multivariate Cox regression analysis were applied to construct a prognostic model and the proteomic data as well as immunohistochemistry were used to validate the result. The correlations between the prognostic model and tumor infiltrating immune cells were assessed using Spearman's rank correlation analysis. A total of 192 early stage ccRCC gene mutation data as well as 261 RNA sequencing data with relative clinical data were extracted from the TCGA. The overall mutation frequency of the 19 m6A RNA methylation regulators was relatively low with 4.69%. The transcriptome data revealed that 11 genes were differentially expressed between cancer tissues and relatively normal tissues. Survival analysis highlighted four specific genes as having a significant influence on overall survival. An established model with four genes demonstrated the best predictability for early-stage ccRCC. After integrating clinical characteristics into the multivariate analysis, the model remained effective at predicting ccRCC prognosis. Spearman's rank analysis suggested several tumor infiltrating immune cells such as dendric cells, CD4+ cells, CD8+ T cells and macrophages were significantly correlated with the model. Proteomic data analysis as well as immunohistochemistry from the Human Protein Atlas showed that all the genes used to construct the model were differentially expressed between ccRCC and normal tissues. In conclusion, a novel m6A methylation regulators-based prognostic signature was established and validated with proteomics and immunohistochemistry. In addition, the model was significantly correlated with multiple infiltrating immune cells in tumor microenvironment.

e16500 Background: Bladder cancer (BC) is the most common malignant tumor of the urinary system, with various heterogeneity. Immunotherapy has become one of the hot areas of cancer research. Tumor immune microenvironment is closely related to prognosis and immunotherapy effect. Therefore, Immune cell infiltration analysis is necessary for the prognosis and treatment of BC. Methods: In this study, we analyzed IMvigor210 dataset, which included complete expression data and detailed clinical information from a cohort associated with Bladder Urothelial Carcinoma (BLCA) immunotherapy. The dataset was divided into two groups based on tumor remission after immunotherapy, including 68 remission samples and 230 stable samples. The CIBERSORT program package was used for immune invasion analysis to compute the relative proportion of immune cells in the tumor tissue from tissue gene expression profiles. Results: Significant results of differentially immune cells proportion were obtained in 126 BC patients by CIBERSORT analysis ( p< 0.05). Compared with the stable group, the number of resting memory CD4+ T cells decreased and the number of CD8+T cells increased in the remission group. The relative proportion of immune cells in tumor microenvironment has certain influence on the occurrence and development of tumor. Conclusions: Memory CD4+ T cells in the resting state did not promote the activation and proliferation of CD8+ T cells, whereas resting memory CD4+ T cells were associated with poor outcome. The increased level of CD8+ T cells in tumor microenvironment was associated with the anti-tumor effect and improved prognosis of various cancers. The results indicate that resting memory CD4+ T cells and CD8+ T cells may be the target of immunotherapy.

Single-cell and spatial transcriptomics reveal 5-methylcytosine RNA methylation regulators immunologically reprograms tumor microenvironment characterizations, immunotherapy response and precision treatment of clear cell renal cell carcinoma

BackgroundDespite the huge success of immunotherapies targeting T cells, a substantial proportion of patients experience resistance or relapse due to the immunosuppressive nature of tumor microenvironment. Increasing number of studies...

Background: The tumor microenvironment (TME) is composed of not only cancer cells but also an extracellular matrix and many types of non-cancerous cells, including fibroblasts and immune cells. In recent years, infiltrating immune cells in TME are known as a predictor of chemotherapy and immunotherapy effectiveness. The purpose of this study is to evaluate infiltrations of lymphocytes and macrophages in biliary tract cancers (BTCs). Methods: Clinical data and tissues were obtained from 130 patients who underwent surgical treatment for BTCs (intrahepatic, perihilar, and distal bile duct cancer, gallbladder cancer, and ampullary cancer) at our institution between 2001 and 2017.The immunohistochemical evaluation was performed to evaluate the number of CD8+ T cells, CD4+ T cells and FOXP3+ T cells and CD68+ Macrophages around the tumor cells. We defined Immunoscore based on the status of infiltrating immune cells. With CD8-high, CD4-high, and FOXP3-high TILs and CD68-low TAMs as one factor, the number of factors was counted in each case, and Immunoscore was assigned a score of 5 stages, from 0 to 4. It was high for the score of 3-4 and low for the score of 0-2. Also, to evaluate the correlation with TME and systemic inflammatory reaction, we examined blood inflammatory biomarker from peripheral blood samples. Results: A total of 59 cases had high Immunoscore and 71 cases had low Immunoscore. Patients with high Immunoscore showed significantly superior overall survival (OS) and recurrence free survival (RFS) than those with low Immunoscore (median OS 60.8 vs. 26.4 months, p = 0.001; median RFS not reached vs. 17.2 months, p < 0.001). For OS, low Immunoscore, positive lymph node metastasis, presence of distant metastasis, and high serum CA19-9 level were independent poor prognostic factors (hazards ratio 2.05, 3.48, 1.7, and 2.05; p = 0.01, p = 0.005, p = 0.05, and p = 0.01, respectively). For RFS, low Immunoscore, T classification of pT3-4, and presence of distant metastasis were independent poor prognostic factors with hazards ratio of 2.41 (p = 0.001), 2.16 (p = 0.005), and 3.58 (p = 0.005), respectively. Patients with high preoperative Neutrophil-to-lymphocyte ratio (NLR) had a significantly lower average number of CD8+ T cells as compared to patients with low NLR (p = 0.02). Preoperative NLR was associated with infiltrating CD8+ T cells in TME. Conclusions: High Immunoscore group had significantly longer OS and RFS and was an independent good prognostic factor. Also, infiltrating CD8+ T cells in TME correlated preoperative NLR. Our findings indicated that in biliary tract cancer, the evaluation of infiltrating immune cells in TME was useful to predict patient prognosis, and preoperative NLR may predict the tumor infiltrating CD8+ T cells. Citation Format: Kousuke Hatta, Ryota Tanaka, Kenjiro Kimura, Shinpei Eguchi, Go Ohira, Hiroji Shinkawa, Kohei Nishio, Masahiko Kinoshita, Shigeaki Kurihara, Shuhei Kushiyama, Takahito Kawaguchi, Naoki Tani, Koichi Nakanishi, Mizuki Yoshida, Takeaki Ishizawa. Evaluating tumor-infiltrating immune cells helps predicting prognosis in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6884.