Research On CD36 Remodeling Tumor Microenvironment By Affecting Infiltration Of Immune Cells In Glioma

BackgroudGlioma is the most common malignant tumor of the central nervous system,accounting for about 80%of malignant tumors of the brain and other central nervous system tumors.Glioma usually originate from neural stem cells,glial cells and astrocytes.The World Health Organisation(WHO)classifies adult glioma as low grade glioma(LGG)and glioblastoma(GBM).The current standard therapy for glioma is still mainly surgical resection,followed by radiotherapy and temozolomide chemotherapy.However,due to the aggressive nature and high degree of molecular heterogeneity of glioma,the presence of the blood-brain barrier and the immunosuppressive tumor microenvironment(TME),the efficacy of existing treatment for glioma is limited.Therefore,there is an urgent necessity to explore in depth the mechanisms of molecular heterogeneity in glioma and to provide a theoretical basis for the development of new therapeutic strategies.Cluster of differentiation 36(CD36)is a transmembrane glycoprotein,also known as fatty acid translocase and scavenger receptor B2,which is expressed on the membrane of various cells.CD36 can bind to different ligands and perform various biological functions,including lipid metabolism and signal transduction,etc.In the field of oncology,up-regulation of CD36 can promote the progression of gastric cancer and ovarian cancer.However,the biological functions of CD36 in glioma have not been reported.Therefore,this study intends to investigate the functions and potential mechanisms of CD36 in glioma progression and provide new measures for treatment strategies.AimsTo explore the relationship between CD36 and the prognosis of glioma patients,and to elucidate the potential mechanisms involved in glioma progression and providing a theoretical basis for treatment strategies.Methods and Results1.High expression of CD36 was associated with worse clinicopathological characteristics in glioma patientsTranscriptomic data and corresponding clinical information of glioma patients were downloaded from The Cancer Genome Atlas(TCGA)database and the Chinese Glioma Genome Atlas(CGGA)database.Using bioinformatics methods,the expression of CD36 was significantly elevated in glioma compared with normal brain tissue.In addition,the expression of CD36 was analysed between different groups of glioma patients with different clinical characteristics using the Wilcoxon rank sum test or the Kruskal-Wallis test.The results showed that patients with glioma of advanced age(>60 year)had a worse prognosis compared to patients of lower age(≤ 60 year),and that patients with WHO IV had a shorter survival compared to glioma patients with WHO Ⅱ and Ⅲ.In addition,up-regulation of CD36 expression in glioma patients was positively correlated with malignant phenotypes such as isocitrate dehydrogenase(IDH)wild-type and 1p/19q non-codel type.2.High expression of CD36 may predict poor prognosis in glioma patientsFirstly,we performed Kaplan-Meier survival analysis and glioma patients with up-regulated expression of CD36 had a worse prognosis than those with low expression of CD36.Secondly,univariate and multivariate Cox regression analyses were conducted and the results indicated that high expression of CD36 was an independent risk factor affecting the prognosis of glioma patients.Finally,a nomogram was created to predict the probability of overall survival(OS)at 1,3 and 5 years for glioma patients,and the calibration chart and the ROC curve indicated that the prognostic model was a good predictor of OS in glioma patients.3.CD36 promoted the ability of proliferation and migration of glioma cells in vitro.CD36 siRNA was transiently transfected in U87,U251 and LN229 cell lines,and the knockdown efficiency of CD36 at the mRNA level and protein level were examined using qRT-PCR and western blot assays.The knockdown of CD36 significantly inhibited the capacity of proliferation of glioma cells as demonstrated by CCK-8,colony formation and EdU assays.The results of both wound healing assay and transwell assay showed that the migratory ability of glioma cells was significantly diminished after downregulation of CD36.4.CD36 may have an important impact on the immune microenvironment of gliomaA total of 1791 differentially expressed genes were screened between glioma patients with high and low CD36 expression using the "DESeq2" R package.We performed GO and KEGG functional enrichment analyses on these differentially expressed genes.Next,the top 20 genes and proteins most associated with CD36 in the GeneMANIA database and STRING database were selected and subsequent functional enrichment analyses were performed on these genes and proteins.The results of the above enrichment analyses showed that CD36 played a significant role in the tumor immune microenvironment.5.Up-regulation of CD36 may indicate increased immune infiltration of macrophages and neutrophils in glioma TMEUsing the ESTIMATE algorithm,glioma patients with high expression of CD36 had higher immune scores and stromal scores compared to patients in the low expression group.Analyses using the CIBERSORT algorithm and the ssGSEA method revealed that glioma patients in the high expression of CD36 had significantly larger number of immune infiltrating macrophages and neutrophils.6.CD36 may promote the migration and infiltration of myeloid macrophages and neutrophils into glioma TMEInitially,spearman correlation analysis revealed that the expression of CD36 was positively correlated with the expression of monocyte chemokines(CCL2,CCL5)and neutrophil chemokines(CXCL6,CXCL8).Subsequently,qRT-PCR assay was used to demonstrate that the ability of glioma cells with knockdown of CD36 to secrete these chemokines were decreased significantly.In addition,by spearman correlation analysis,the expression of CD36 was also positively correlated with the expression of typical markers of M0-and M2-macrophages and N2-neutrophils.While we found that glioma cell lines with knockdown of CD36 secreted M2-macrophages associated polarizing factors(TGF-β1,CSF-1)and N2-neutrophils associated polarizing factors(CSF-2,CSF-3)were significantly reduced.ConclusionsCD36 may not only serve as a biomarker for determining the prognosis and tumor progression of glioma patients,but may also play an important role in remodeling the TME of glioma,and promoting the formation of immunosuppressive TME.