The Mechanism Study Of Glioma Exosomes In Mediating The Formation Of An MDSCs-associated Immunosuppressive Tumor Microenvironment

BackgroundGlioma is the most common malignant tumor of the central nervous system and is resistant to conventional therapy.In recent years,tumor immunotherapies have been applied in the treatment of various tumors.However,glioma is also resistant to immunotherapies because of its complex immunosuppressive microenvironment.Myeloid-derived suppressor cells(MDSCs)play an important role in mediating the formation of the glioma immunosuppressive microenvironment.MDSCs could suppress the function of T cells by secreting immunosuppressive cytokines and expressing the ecto-enzyme CD73,which contributes to the immunotherapy resistance.Since MDSCs originate from myeloid progenitors in the bone marrow and the differentiation of MDSCs begins before their migration into glioma,there must be a long-distance interaction between glioma and MDSCs.Mesenchymal stem cells(MSCs)are a major component of the tumor microenvironment and are responsible for the malignant progression of glioma.Bone marrow-derived MSCs(BM-MSCs)could regulate the function of immune cells,such as inhibiting the maturation of dendritic cells and promoting the M2 polarization of macrophages,however,the immune regulatory effect of glioma-associated MSCs(GA-MSCs)on MDSCs and the formation mechanism of GA-MSCs remain unclear.Exosomes are small vesicles(50-150 nm in size)secreted by almost all cell types and distribute widely around multiple types of body fluids,such as cerebrospinal fluid(CSF)and plasma.Glioma cells can deliver nucleic acids and proteins through exosomes to recipient cells,such as MDSCs and MSCs,and modify their function,promoting the formation of an immunosuppressive microenvironment.The purpose of this study is to explore the formation mechanism of glioma immunosuppressive microenvironment.We found glioma exosomes could directly induce the differentiation and activation of MDSCs on the one hand,and could also modify the function of MSCs and indirectly promote the immunosuppressive function of MDSCs through modified MSCs,which together promoted the formation of an MDSCs-associated immunosuppressive tumor microenvironment and the malignant progression of glioma.Part Ⅰ:The mechanism study of glioma exosomes in promoting the formation of an MDSCs-associated immunosuppressive tumor microenvironment directlyObjectivesInvestigate the effect and underlying mechanism of glioma patient CSF-,plasma-and glioma cell-derived exosomes on the differentiation and activation of MDSCs,and identify the new immunotherapy targets for glioma.Methods1.Glioma patient CSF-,plasma-and glioma cell-derived exosomes were used to treat peripheral blood mononuclear cells(PBMCs),then the percentage and the T cell suppression ability of MDSCs were measured using flow cytometry.2.miRNA sequencing was performed in exosomes derived from glioma patient CSF and normoxia-or hypoxia-conditioned glioma cells.The highly expressed miRNAs were transfected into monocytes to find the key miRNA responsible for inducing MDSCs.3.TransmiR database was used to predict the transcription factor of the key miRNA.Chromatin immunoprecipitation(ChIP)assay was performed to validate the binding site of the transcription factor.4.The proteomic dataset from a pull-down assay of the key miRNA was analyzed to find the protein responsible for packaging the key miRNA into glioma exosomes.5.The inhibitor capable of suppressing the key exosomal miRNA expression was identified and its therapeutic effect was validated in a glioma xenograft mouse model.Results1.Glioma patient CSF-,plasma-and glioma cell-derived exosomes promoted the differentiation and T cell suppression function of MDSCs.Hypoxia-induced glioma exosomes exhibited a stronger MDSCs induction ability than normoxia-induced glioma exosomes.2.miR-1246 was highly expressed in glioma patient CSF-derived exosomes and could induce the differentiation and activation of MDSCs.miR-1246 was the most abundant miRNA in glioma cell-derived exosomes and hypoxia upregulated the expression of exosomal miR1246.3.POU5F1 was the transcription factor of miR-1246 and hypoxia increased POU5F1 expression via HIF-1α.4.hnRNPA1 selectively packaged miR-1246 into glioma exosomes and hypoxia increased hnRNPA1 expression via HIF-1α.5.2-Methoxyestradiol(2-ME2)interfered with MDSCs differentiation and immunosuppressive function by blocking HIF-lα-induced glioma exosomal miR-1246 expression,which restrained glioma progression.Conclusion1.Glioma patient CSF-and glioma cell-derived exosomal miR-1246 drives the differentiation and activation of MDSCs.2.Hypoxia increases the expression of miR-1246 in glioma-derived exosomes by promoting miR-1246 transcription and selective exosomal sorting through upregulating POU5F1 and hnRNPA1.2.2-Methoxyestradiol suppresses the differentiation and activation of MDSCs by inhibiting glioma exosomal miR-1246 expression,which in turn restrains glioma progression.Part II:The mechanism study of glioma exosomes in promoting the formation of an MDSCs-associated immunosuppressive tumor microenvironment by modifying MSCsObjectivesInvestigate the regulatory role of glioma-associated MSCs(GA-MSCs)-derived exosomes on MDSCs and the regulatory role of glioma-derived exosomes on GA-MSCs.Methods1.BM-MSCs-and GA-MSCs-derived exosomes were used to treat PBMCs,then the percentage of MDSCs and the CD73 expression on MDSCs were measured using flow cytometry.2.miRNA sequencing was performed in exosomes derived from BM-MSCs and GAMSCs.The top 10 enriched and upregulated miRNA in exosomes isolated from GA-MSCs were transfected into MDSCs to find the key miRNA responsible for inducing CD73 expression.3.Transcriptome sequencing was performed in GA-MSCs and BM-MSCs,and the upregulated genes in GA-MSCs were filtered by overlap with predicted key miRNA transcription factors(determined with the transmiR and mirTrans database).4.The transcriptome sequencing result was analyzed to compare the DNA methylation level between GA-MSCs and BM-MSCs.DNA methylation inhibitor was used to treat BM-MSCs and the key miRNA expression was measured.5.The highly expressed miRNA in glioma exosomes were transfected into BM-MSCs to find the miRNA responsible for reducing DNA methylation level in MSCs.Results1.GA-MSCs-derived exosomes had a stronger ability to promote the differentiation of MDSCs and upregulate CD73 expression on MDSCs than BM-MSCs-derived exosomes.2.GA-MSCs upregulated CD73 expression on MDSCs through exosomal miR-21.3.XBPls was the transcription factor of miR-21 and was upregulated in GA-MSCs.4.The DNA methylation level was reduced in GA-MSCs.DNA methylation inhibitor treatment increased miR-21 expression in BM-MSCs.5.Glioma exosomal miR-21 suppressed the SP1 expression in MSCs,which in turn inhibited the expression of DNA Methyltransferase 1(DNMT1)and reduced DNA methylation level.Conclusion1.GA-MSCs-derived exosomal miR-21 upregulates CD73 expression on MDSCs and enhances the immunosuppressive function of MDSCs.2.Glioma-derived exosomes deliver proteins to MSCs and upregulate XBP1s expression in MSCs.XBP1s is the transcriptional factor of miR-21 and the upregulation of XBP1s increases miR-21 expression in MSCs cells and exosomes.3.Glioma-derived exosomes transfer miR-21 to MSCs and increase miR-21 expression in MSCs cells and exosomes via miR-21/SP1/DNMT1 positive feedback loop.