Mechanism Of Hypoxia Glioblastoma Cell-Derived Exosomes Regulating Polarization Of M2 Microglia

Background: Microglia are immune cells that reside in the brain and play a dual role in promoting inflammation and anti-inflammation in the central nervous system.Glioblastoma as a primary malignant tumor in the brain together with microglia,astrocytes,neurons,blood vessels and extracellular matrix components constitute a dynamic hypoxia immunosuppressive tumor microenvironment.In the tumor microenvironment,tumor cells can recruit microglia to accumulate in hypoxia zones,while glioblastoma cell-derived exosomes,as carriers of intercellular communication connections,can deliver components such as active substances or proteins carried into microglia,driving microglia to polarize toward the M2 phenotype,prompting glioblastoma to evade immune surveillance,and facilitating the formation of an immunosuppressive tumor microenvironment to promote tumor proliferation and invasion.To explore the mechanism of phenotypic polarization of glioblastoma cells to microglia,it is important to understand the role of microglial cell activation in glioblastoma.Objective: Firstly,to determine the effect of hypoxia glioblastoma cell-derived exosomes on the M2 phenotype of microglia,and then combined with bioinformatics analysis to screen the differential proteins carried within the exosomes of hypoxia glioblastoma cells expressed by glioblastoma and enrich the signaling pathways associated with the differential proteins.To explore the possible involvement of proteins carried within the exosomes of hypoxia glioblastoma cells in the signaling pathways regulating the M2 phenotype polarization of microglia,and to provide a reference for clinical research on the treatment of glioblastoma.Methods: Exosomes were extracted from glioblastoma C6 and U87 cells under normoxia and hypoxia using differential centrifugation and intervened in BV2 microglia and primary microglia,respectively.The migration ability of microglia was observed by scratch assay,and the expression levels of M2 microglia marker proteins CD206 and Arg-1 were detected by Western Blot.Meanwhile,an in vivo glioblastoma model was established,and tumor tissue proteins were extracted for Western Blot to detect microglia phenotypes in tumor tissues.In addition,the primary microglia culture medium after the intervention of C6 cell-derived exosomes under normoxia and hypoxia conditions was collected and co-cultured with C6 cells.The migration and proliferation of C6 cells were detected by scratch assay and CCK-8.Combined with bioinformatics analysis to screen for differential proteins expressed by glioblastoma carried within hypoxia glioblastoma cell-derived exosomes,predict the possible involvement of differential proteins in signaling pathways regulating the activation of M2-type microglia,and analyze the effect of tumor-associated microglia on glioblastoma proliferation and migration.Results: In vitro experiments,it was observed that hypoxia glioblastoma cellderived exosomes enhanced the migration of microglia and that hypoxia glioblastoma cell-derived exosomes significantly upregulated the expression of M2-type markers CD206 and Arg-1 in microglia.In vivo experiments observed that glioblastoma tissues were predominantly surrounded by M2-type microglia.It was found that the microglial culture medium induced by hypoxia glioblastoma cell-derived exosomes was more significant in promoting tumor cell proliferation and migration.Meanwhile,combined with bioinformatics analysis to screen the differential proteins expressed in glioblastoma carried by the exosome of hypoxia glioblastoma cells,GO-enriched functional annotation of the differential proteins revealed that they were mainly present in the cellular exosome fraction,and KEGG analysis enriched them mainly in the PI3K/AKT signaling pathway,and the proteins in the exosome fraction were compared with those involved in the PI3K/AKT signaling pathway Venn analysis was performed.Differential proteins carried by exosomes involved in the PI3K/AKT signaling pathway were obtained,and immuno-infiltration analysis revealed that there were FN1,COL6A3,ITGB1,and YWHAQ proteins proportional to the abundance of M2 tumorassociated macrophage infiltration.Bioinformatics analysis revealed that tumorassociated microglia were associated with poorer prognosis in patients with glioblastoma and that tumor-associated microglia could be involved in cell proliferation and migration in the tumor microenvironment.Based on experimental and bioinformatic analyses,it was predicted that FN1,COL6A3,ITGB1,and YWHAQ proteins carried in hypoxia exosomes may co-regulate M2 microglia activation through PI3K/AKT signaling pathway.Conclusion: Hypoxia glioblastoma cell-derived exosomes induce polarization of microglia toward the M2 phenotype,and M2-type microglia promote proliferation and migration of glioblastoma cells.Bioinformatics analysis of hypoxia glioblastoma cellderived exosomes carrying highly expressed FN1,COL6A3,ITGB1,and YWHAQ proteins associated with M2 macrophage immune infiltration may cascade with microglia through PI3K/AKT signaling pathway and jointly participate in the regulation of microglia M2 phenotype.