| Background and purpose:Immune checkpoint inhibitor therapy（ICT）for Glioblastoma（GBM）often difficult to achieve effective benefits,which is attributed to the central inhibitory tumor microenvironment（TIME）in GBM,so in-depth research on the molecular mechanism of the formation of GBM immunosuppressive microenvironment is helpful to provide a theoretical basis for ICT therapy of GBM.Extracellular vesicles（EVs）or exosomes（Exos）have attracted much attention in the diagnosis and treatment of GBM,but limited by the conditions of brain biopsy,EVs/Exos in Cerebrospinal Fluid（CSF）has great potential for immunodynamic monitoring and therapeutic strategies making for GBM.Therefore,in this study,cerebrospinal fluid of GBM patients was obtained.EVs/Exos proteomics in CSF from GBM was further studied to screen new immune monitoring or therapeutic targets,and hope our results provide new research ideas for ICT treatment of GBM patients.Methods:1.For the clinical study,patients with GBM and GⅡ-Ⅲ glioma（WHO Grade Ⅱ and Ⅲ,GⅡ-Ⅲ）were included,and a healthy control group was set.CSF of all subjects were obtained,lymphoid and myeloid immune cell ratios and immunophenotypes were determined by flow cytometry in GBM cerebrospinal fluid.EVs/Exos in CSF of GBM and GⅡ-Ⅲ patients were extracted and purified by ultrafiltration,characterized by Nanoparticle Tracking Analysis（NTA）and biological transmission electron microscopy.Western blotting was used to identify the surface markers of EVs/Exos.Liquid chromatography-mass spectrometry（LC-MS）was used for proteomic analysis of EVs/Exos to screen out EVs/Exos cargo protein LGALS9,which was related to GBM immunosuppressive phenotype.2.In vitro study,the expression of LGALS9,Rab27a and n SMase2in human malignant glioma cells U87-MG was deleted using CRISPR/Cas9 technology to inhibit the release of EVs/Exos-LGALs9.WT,n SMase2-/-,Rab27a-/-or LGALS9-/-U87-MG were co-cultured with human Dendritic Cells（DCs）and T Cells in vitro.DCs antigen presentation related immunophenotypes and T cell activation levels were determined by flow cytometry and enzyme-linked immunosorbent assay（ELISA）.Furthermore,the antigen presentation inhibition effect of GBM-derived EVs/Exos-LGALS9 on DC cells was determined by knockout of TIM3 receptor gene in DC cells to disable the recognition function of LGALS9 li gand.3.In vivo study,the constructed mouse glioblastoma GL-261 WT,n SMase2-/-,Rab27a-/-and LGALS9-/-cells were transplanted subcutaneously or in the brain of C57BL/6J mice and immunodeficient NOD/SCID mice to construct tumor-bearing animals.The tumor volume and Overall Survival（OS）of mice were observed,and the immunophenotype of related immune cells in mice CSF was analyzed by flow cytometry to determine the effect of EVs/Exos-LGALS9 on TIME formation in CSF of mice.Results:1.Compared with the healthy control group（n=4）,the proportion of granulocytes and CD8+T cells in the CSF of GBM group（n=7）was down-regulated（P<0.05）,and the proportion of monocytes/macrophages and dendritic cells in the GBM and GⅡ-Ⅲ（n=7）was up-regulated（P<0.05）.There was no significant difference in the proportion of CD4+T cells in CSF between GBM and GⅡ-Ⅲ group（P>0.05）.Immunophenotypic analysis showed that CD8+T cells in GBM-CSF were immunosuppressed,including low clonal activity and Type Ⅱ IFN response inactivation.The DCs activity of GBM patients was significantly lower than that of GⅡ-Ⅲ patients,and even lower than that of healthy subjects without tumor antigen stimulation,including decreased expression of antigen-recognized HLA-A,costimulatory molecules CD40 and CD86,and antigen-processing protein TAP1（P<0.05）.Patients in the GBM and GⅡ-Ⅲ tumor groups showed higher concentrations of EVs and smaller nanosized particles in CSF than healthy controls（P<0.05）.Small extracellular vesicles obtained by ultrafiltration（SEVs;≦0.1μM）with Exos surface markers（TSG101,CD63）and morphology.GBM-SEVs/Exos can be ingested by DCs and significantly reduced the antigen presentation activity of DCs.Proteomic analysis showed that 2958 proteins were identified in all CSF-EVs,and the levels of 487 protein cargo in GBM-SEVs/Exos group were higher than those in GⅡ-Ⅲ-SEVs/Exos group（P<0.05）.GO and Pathway analysis showed that 18 protein molecules in GBM-SEVs/Exos were involved in antigen presentation signaling pathway of DCs,among which LGALS9 has attracted our attention.2.Compared with primary astrocytes（HA）,the m RNA transcription level of LGALS9 in U87 MG was significantly up-regulated（P<0.05）.However,there was no significant change in LGALS9 protein translation level（P>0.05）,and LGALS9 protein carrying amount in U87MG-derived Exos was 100 times higher than that in HA-derived Exos（P<0.05）.Compared with U87 WT,the secretion of U87 MG n SMase2-/-Exos and U87 MG Rab27a-/-Exos was significantly reduced.DCs uptake of m Cherry labeled tumor antigen was significantly increased in U87 MG n SMase2-/-and U87 MG Rab27a-/-co-culture systems.The expression of CD40,HLA-A and TAP1 in DCs activated by U87 MG n SMase2-/-and U87 MG Rab27a-/-was higher than that in DCs activated by U87 MG n SMase2-/-and U87 MG Rab27a-/-（P<0.05）.The content of exosomal LGALS9 was lower than U87 MG WT（P<0.05）.Activated DCs promoted clonal proliferation of co-cultured T cells（CSFE incorporation）and Type Ⅱ IFN Response（IFN-γand Granzyme B release）.This process was also significantly enhanced in the co-culture systems of U87 MG n SMase2-/-and U87 MG Rab27a-/-（P<0.05）.3.The effect of LGALS9 knockout on exosomes of mouse GL-261cells was similar to that of human U87 MG cells.n SMase2,Rab27a and LGALS9 knockout GL-261 cells showed no significant in the inhibition of cell proliferation activity.Tumor growth and OS up to 32 days were observed in mice with in-situ brain implantation of GL-261wt cells at about 20 days.In contrast,GL-261 n SMase2-/-,Rab27a-/-and LGALS9-/-tumor-bearing mice had significantly reduced the tumor growth rate and achieved longer OS（P<0.05）.Under NOD/SCID immunodeficiency background,there was no significant difference in tumor growth rate and OS between GL-261 n SMase2-/-,Rab27a-/-and LGALS9-/-mice and GL-261wt mice（P>0.05）.Almost no expression of LGALS9 was detected in the CSF of GL-261 LGALS9-/-tumor-bearing mice.In GL-261 WT tumor-bearing mice,more than 60%of DCs were LGALS9 highly expressed(LGALS9hiDCs),while LGALS9hiDCs accounted for less than 5%of the total DCs in GL-261LGALS9-/-.Importantly,The expressions of MHC I,CD40 and TAP1 in the CSF of GL-261 LGALS9-/-tumor-bearing mice were significantly higher than those of GL-261 WT tumor-bearing mice（P<0.05）.Compared with GL-261 WT tumor-bearing mice,CD8+T cells in the CSF of GL-261 LGALS9-/-tumor-bearing mice exhibited enhanced anti-tumor immune activity,including increased Ki67 positive cells and INF-γ/Gzm B expression.In addition,mice exposed to GL-261LGALS9-/-for the first time will developed resistance to re-inoculated GL-261 WT cells,as demonstrated by inhibition of tumor growth and improved survival.Conclusions:1.The antigen presentation function of DC cells in cerebrospinal fluid from GBM is inactivation,which may be the reason that GBM is more malignant than GⅡ-Ⅲ.2.LGALS9 is unique in GBM CSF SEVs/Exos and may be related to DCs antigen presentation inactivation.3.GBM-exos-derived LGALS9 promotes tumor growth by inhibiting DC cell antigen presentation.4.LGALS9 in GBM-Exos inhibits the antigen presentation process of DCs through TIM3 receptor pathway.5.Reduction of GBM-derived exosomal LGALS9 restores the anti-tumor immune activity in the GBM immune microenvironment.6.DCs that exposed to tumor-associated antigen in the microenvironment that absence of exosomal LGALS9 can successful complete the antigen presentation and resist to the immunosuppressive effect of exosomal LGALS9... |
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