Co-Stimualtory Molecules SLAM Family Members Promote Malignant Progression And Immunosuppression Of Glioma

1.IntroductionGliomas are the most common primary intracranial tumors in the central nervous system,accounting for more than 50%.They are highly malignant,aggressive and poor prognostic.Despite the advances in the treatment of glioma and the use of STTUP regimen for standardized treatment,the therapeutic effect is not satisfactory.The median survival time of the most aggressive glioblastoma(GBM)after radiotherapy and chemotherapy is only 14.6 months.How to effectively treat glioma has become one of the difficult problems in the field of cancer treatment.Nowdays immunotherapy has brought new hope to cancer treatment.Checkpoint blockade therapy is a hotspot in current cancer immunotherapy.Immune checkpoints include co-stimulatory and co-suppressive molecules.At present,checkpoint blockade therapy mainly focuses on co-suppressor molecules,such as CTLA-4,PD-1 and PD-L1.In many malignant tumors,breakthroughs have been made in immune checkpoint blockade therapy,such as melanoma,non-small cell lung cancer,breast cancer,and glioma.However,due to the unique microenvironment of brain glioma and the characteristics of brain tissue itself,the effect of immune checkpoints in glioma therapy is suboptimal,only a small number of patients benefiting from it.Immune checkpoint blockade therapy is often accompanied by immune inflammation-related side effects,such as dermatitis,colitis,inflammatory endocrine disease,and even fatal brain edema.Because of the weak regeneration ability of the central nervous system,inflammation damage to the brain parenchyma is often more serious.Therefore,the most important thing is to find effective targets of immune checkpoint blockade therapy for glioma and balance the immune activation and inflammation suppression.We found that signal transduction lymphocyte activating molecule family(SLAMF)molecules play an important role in the immune response,and can regulate the development and function of many immune cells.Signal transduction lymphocyte activating molecule family 8(SLAMF8,CD353)can activate macrophages during inflammation.Overexpression of SLAMF8 in autoimmune or inflammatory diseases,suggests that SLAMF8 plays an important role in immune-related inflammation.Signal transduction lymphocyte activation molecule family 2(SLAMF2,CD48)is an adhesion costimulatory molecule expressed on most hematopoietic cells,especially in antigen presenting cells(APC).By binding to its receptor CD2,CD48 participates in a variety of innate and adaptive immune responses.It has been found that CD48 interacts with its high affinity receptor 2B4(CD244)in hepatocellular carcinoma,which can lead to NK cell dysfunction induced by monocytes/macrophages.However,there is no study on SLAMF8 and CD48 in glioma.Here,we use the DNA sequencing(RNA-seq)data of 946 gliomas from China Glioma Genome Atlas(CGGA)and The Cancer Genome Atlas(TCGA)to explore the clinical features and functions of SLAMF8 and CD48 in gliomas,in order to provide new referrence for the immune checkpoint therapy of gliomas.2.Materials and Methods 2.1 MaterialsWe analyzed 946 glioma RNA-sequencing data with detailed clinical information,310 from the CGGA database and 636 from the TCGA database.TCGA data is downloaded from a public database.Overall survival(OS)is the time from diagnosis to death or the end of final follow-up.Clinical specimens of glioma were collected from the Department of Neurosurgery,First Affiliated Hospital of China Medical University.Informed consent of patients and their families was obtained.2.2 Bioinformatics Analysis Method 2.2.1 Glioma purity and microenvironment cell populationsWe used R language to calculate glioma stromal scores,glioma immune scores,glioma purity and microenvironment cell populations.2.2.2 Go AnalysisUsing Pearson correlation analysis,we obtained genes with obvious correlation with the target gene and used them for Go analysis.The Go analysis was conducted mainly through DAVID.2.2.3 Gene Set Enrichment Analysis(GSEA)Gene set enrichment analysis(GSEA)software was used to analyze the difference of gene set enrichment between the low and high groups,in order to explore whether the difference has statistical significance.2.2.4 Principal Component Analysis(PCA)and Gene Set Variation Analysis(GSVA)Principal Component Analysis(PCA)and Gene Set Variation Analysis(GSVA)were performed in R language to distinguish the differences of transcriptome and inflammation-related functions among different components.2.2.5 Western BlottingExtraction of tissue proteins from glioma specimens: frozen specimens were taken out from the freezer at-80 ℃ and added with appropriate amount of prepared protein lysis.After crushing by homogenizer,the specimens were crushed on ice for 30 minutes,then centrifuged at 4 ℃ and 12 000 rpm for 10 minutes,and the supernatant was taken for protein quantification to prepare 4ug/ul WB sample.Western blot: Gel electrophoresis,transmembrane,blocked with nonfat milk,incubation with primary and secondary antibodies and finally exposure with ECL light to detect the expression of CD48 2.2.6 Immunohistochemistry Paraffin sections were baked at 60 degrees Celsius,then dewaxed,rehydrated with alcohol gradient,and hydrated.Then antigen was repaired,endogenous peroxidase was blocked,and the first antibody stayed overnight at 4 degrees Celsius.On the second day,PBS was washed and dripped with reactive enhancer,followed by enhanced enzyme-labeled goat anti-rabbit IgG polymer,followed by DAB coloration.Finally,it was dyed,dehydrated,transparent and sealed.2.3 Statistical analysis methodsStatistical analysis was carried out using software of SPSS,GraphPad Prism 7 and R 3.3.3(https://www.r-project.org/).Student’s t-test was applied to evaluate differences in expression,and Pearson correlation was analyzed to calculate correlations.Survival distribution was assessed via Kaplan-Meier survival analysis,and the log-rank test was used to estimate significance between stratified groups.Receive operating characteristic(ROC)curve were made using Medcalc software.Other statistical computations and figures were built with R(ggplot2,corrplot,pheatmap).Statistical significance was defined as a 2-tailed p-value < results of clinical controlled trial.3.Results Part 1: SLAMF8 is highly expressed in malignant phenotypes of glioma and glioblastoma,and is a good predictor of mesenchymal subtypes of glioma.The expression level of SLAMF8 is closely related to the poor prognosis of glioma,and it is an independent prognostic factor of glioma.High expression of SLAMF8 is an important marker of the poor prognosis of glioma.High expression of SLAMF8 glioma has obvious chemotherapeutic resistance.SLAMF8 can affect the immune microenvironment of glioma by altering the recruitment of monocytes and dendritic cells.Its function is closely related to the immune response.The high expression SLAMF8 group and the low expression group showed different response phenotypes.The malignant high SLAMF8 glioma showed strong immune response phenotype,but the effective anti-tumor immunity was inhibited.SLAMF8 was strongly positively correlated with multiple immunosuppressive regulatory points,but had very low correlation with PD-L1.Finally,we found that SLAMF8 glioma with high expression was associated with strong immune inflammation,both chronic and acute inflammation.Part 2: CD48 is highly expressed in glioblastoma,IDH wild type and mesenchymal subtype with malignant phenotype.As an independent risk factor of glioma,the prognosis of patients with high expression of CD48 is obviously poor.CD48 can affect the purity of glioma and local immune cell subsets.The expression level of CD48 is positively correlated with the enrichment of bone marrow dendritic cells(r = 0.470),B cell lines(r = 0.397),CD8 T cells(r = 0.343)and monocyte lines(r = 0.236).CD48 is closely related to interferon-gamma-mediated signaling pathway,T-cell co-stimulation and antigen processing presentation through MHC-I in glioma.Glioma patients with high expression of CD48 have strong innate immune response and T cell mediated immune response phenotype.At the same time,the high expression of CD48 glioma is in the state of immunosuppression.CD48 is closely related to a variety of immunosuppressive molecules and immunosuppressive regulatory points,but not to CTLA-4 and PD-L1.In addition,we also found that similar to SLAMF8,CD48 expression level is closely related to immune inflammation.Part 3: The immune checkpoint signature of CD48,SLAMF8 and PD-L1 were established by co-stimulus analysis and co-suppressor.We found that the risk score of glioma with malignant phenotype was significantly higher,such as glioblastoma,IDH wild type and mesenchymal subtype.Compared with CD48 and SLAMF8,the immune regulatory point risk score can predict mesenchymal subtype glioma more accurately.Immune checkpoint risk score was an independent prognostic factor for glioma,and high risk score had a significantly poor prognosis.Considering age and molecular subtypes,we found that patients with low risk and high risk score had similar prognosis when they were younger than 40 years old.However,in patients over 40 years of age,the prognosis of patients with high and low risk score is significantly different.The prognosis of immune score evaluation is influenced by age factors.Similarly,the immune risk score has prognostic value in IDH wild-type GBM and MGMT promoter methylated GBM,but not in IDH mutant GBM and MGMT promoter non-methylated GBM.4.Conclusion Part 1: Through multidimensional systematic analysis of large samples of glioma cases,we found that SLAMF8 was related to malignant progression,poor prognosis and chemotherapeutic resistance.SLAMF8 was closely related to immunosuppression and inflammation of glioma.It is suggested that SLAMF8 plays an important role in the immune response of glioma.We will further study the co-stimulatory molecule SLAMF8 in glioma in order to further understand its immune regulation and provide reference for effective immunotherapy of glioma.Part 2: CD48 is highly expressed in glioblastoma,IDH wild type and mesenchymal subtype with malignant phenotype.As an independent risk factor of glioma,the prognosis of patients with high expression of CD48 was obviously poor.CD48 could affect the purity and local immune cell subsets of glioma and was closely related to immune function.Glioma patients with high expression of CD48 had a strong immune response phenotype,but at the same time they were in a state of immunosuppression accompanied by a strong immune inflammatory response.Part 3: The immune checkpoint signature constructed by CD48,SLAMF8 and PD-L1 was closely related to the malignant progression of glioma,which can more accurately predict the mesenchymal subtype glioma with higher malignant phenotype.As an independent prognostic factor,a higher checkpoint risk score indicated a poor prognosis of glioma.Age,IDH status and MGMT promoter status of glioma patients will affect the prognostic value of regulatory points.CD48,SLAMF8 and PD-L1 were used to evaluate the decisive role of immune regulatory points in glioma.IDH wild-type glioma patients older than 40 years with MGMT promoter methylation may be more suitable for immunoregulatory point blocking therapy.