The Mechanism Study Of Auophagy And M2-like Polarization Of Tumor Associated Macrophages Promoted By Hypoxia Induced Glioma-derived Exosomes

BackgroundGliomas are most commonly encountered in cerebrospinal tumors.Even if patients with glioma receive active treatment,they will still relapse in the short term.As a new strategy,immunotherapy for glioma has made considerable progress,but few clinical trials have achieved favorable results,mainly due to the existence of an immunosuppressive glioma microenvironment.TAMs,as the principal immune cells infiltrated in the glioma microenvironment(accounting for more than 30%),are closely related to the malignant degree of glioma.There were two diverse polarization types of activated macrophages in tumor microenvironment:M1-like type and M2-like type.Among them,interleukin(IL)-4,IL-6,IL-10 and other Th2-type cytokines can induce the production of M2-type TAMs,and also secrete numberous factors which can fight inflammation and promote tumor.Therefore,M2-like type TAMs related positively with the establishment of the inhibitory immune microenvironment of glioma.The microenvironment of solid tumor also has the features of hypoxia,which promotes tumor to create blood vessels,invade,migrate etc.,and also funcions in the establishment of tumor suppressive immune microenvironment.We have reported that there exist extensive hypoxic regions in glioma,and massive TAMs(especially in hypoxic regions)are infiltrated in the microenvironment.The increasing number of M2-like subgroup means more severe hypoxic degree and malignant degree of glioma.Through in-depth study,we found for the first time that glioma cells in hypoxic microenvironment can also induce TAMs to M2-like type macrophage polarization by massively secreting cytokines such as POSTN.Studies have shown that up-regulating the autophagy level of TAMs can also promote its M2-like polarization.In consequence,autophagy pathway may correlate with the M2-like polarization of TAMs.Autophagy is a biological process to meet the survival needs of cells by wrapping non-essential cell contents such as damaged or aging organelles to form autophagy bodies and decompose them into the basic components required for cells.Therefore,autophagy usually occurs when cells are in a state of starvation,hypoxia,immune injury and radiotherapy and chemotherapy.However,due to the extremely vigorous proliferation of tumor cells and severe ischemia and hypoxia in the core area,autophagy has also become an important means for hypoxic cells to overcome various adverse survival pressure in tumor microenvironment.It was found that autophagy played a crucial role in the malignant progression of tumors.Our previous studies confirmed that hypoxia up-regulated the autophagy level of glioma through IL-6 autocrine and paracrine,and its downstream molecule miR-155-3p played a crucial role.Moreover,glioma cells mainly release IL-6 and other cytokines into the microenvironment through vesicle secretion.Exosomes,as a significant regulatory exocrine vesicle,can carry abundant IL-6 and other cytokines,targeting immune cells in the microenvironment.Therefore,hypoxic glioma cells may be involved in TAMs autophagy and M2-like type macrophage polarization by secreting exosomes,and IL-6 related pathways may play a key role.In the present study,we investigated the relationship among hypoxic glioma-derived exosomes(H-GDEs),TAMs autophagy and macrophage M2-like polarization.We first demonstrated that M2-like macrophage polarization could be induced by H-GDEs through autophagy activation.According to our previous studies,hypoxia induces the upregulation of IL-6 and miR-155-3p in GBM cells.Then,we detected and compared the content of IL-6 and miR-155-3p between H-GDEs and normoxic glioma-derived exosomes(N-GDEs)and found that IL-6 and miR-155-3p were expressed higher in H-GDEs than in N-GDEs.Furthermore,we investigated the function of STAT3 pathway activation in the IL-6-autophagy-M2-like macrophage polarization process using S3I-201,a STAT3 inhibitor.Finally,we showed that IL-6 and miR-155-3p,which were delivered by exosomes,enhanced autophagy induction and M2-like macrophage polarization.In conclusion,this study provides a new basis to explore the glioma development mechanism on glioma cell immune inhibitory micro-environment,autophagy and glioma malignant biological behavior research.IL-6 and miR-155-3p may be potential biomarkers of treatment of glioma.Treatment of targeting autophagy and the STAT3 pathway could reverse immunosuppression in the tumor microenvironment,which played an anti-tumor role.1.Objective(1)To study the effect of glioma-derived exosomes on M2-like polarization and the transformation of autophagy.(2)To confirm the enrichment of IL-6 and miR-155-3p in GDEs and macrophages.(3)To explore the effects of IL-6 and miR-155-3p on autophagy and on M2-like polarization of macrophages.2.Method(1)Isolation and identification of GDEsCentrifugalization etc.(2)Autophagy assays in vitroBoth MCherry-GFP-LC3 fluorescence results and western blot results indicated that hypoxic glioma exosome treatment could upregulate the autophagy level of macrophages.3-MA was used as an autophagy blocker.S3I-201 was used as a STAT3 inhibitor.(3)Exploration of the variations of IL-6 and miR-155-3p in GDEsWestern blot etc.(4)Cell transfection and construction of monocyte cell line stably expressing IL-6 and miR-155-3pThe constructed plasmids were transfected with Lipo3000 to express the related molecule.Furthermore,the binding of miR-155-3p to the 3’UTR of CREBRF was investigated.Different lentivirus were used to transfect the U937and THP-1 cell lines to construct miR-155-3p-expressing U937 and THP-1 cell lines.(5)Evaluation of M2-like macrophage polarizationConfirmation of M2-like polarization:qRT-PCR and ELISA.S3I-201 was used as a STAT3 inhibitor and its effect on M2-like macrophage polarization was valuated.(6)Determination of the glioma proliferationEdU assay was employed in related experiments.(7)Determination of the glioma migrationTranswell migration was employed in different related experiments..(8)Vivo experiments in nude mice modelsIn situ tumorigenesis model etc.3.Results(1)Identification of GDEs and indication of GDEs being uptaken by macrophagesTEM images showed that the GDEs and exosomes expressed typical exosomal markers.Using macrophages being cocultured with PKH67-labeled GDEs,PKH67 signals were detected in the macrophages by confocal microscopy,indicating efficient uptake of GDEs by macrophages.(2)Hypoxic glioma-derived exosomes(H-GDEs)enhance autophagy in macrophagesBoth the fluorescence results of mCherry-GFP-LC3 and Western blot indicated that H-GDEs significantly increased the puncta to cell ratio in macrophages.In addition,the puncta to cell ratio decreased in macrophages treated with 3-MA,an inhibitor of autophagy.(3)H-GDEs promote M2-like macrophage polarization and can be inhibited by 3-MABy performing qRT-PCR,flow cytometry and ELISA,we revealed that H-GDEs significantly induced M2-like macrophage polarization,and it could be blocked by 3-MA.(4)H-GDEs-treated macrophages promote glioma malignant progressionIn vitro results showed that macrophages co-cultured with hypoxic glioma exosomes promoted the proliferation and migration of gliomas.The results of in vivo experiments showed that the tumors of mice in the co-injection group of glioma cells and macrophages treated with hypoxic glioma exosomes were more malignant.(5)Hypoxic glioma exosomes are enriched for IL-6 and miR-155-3p and can be delivered to macrophagesWestern blot and QRT-PCR results revealed hypoxic glioma exosomes are enriched for IL-6 and miR-155-3p,and miR-155-3p is delivered to macrophages via exosomes.(6)IL-6 enhances autophagy induction via STAT3 activation in macrophagesBoth the fluorescence results of mCherry-GFP-LC3 and Western blot indicated that exogenous IL-6 significantly increased the puncta to cell ratio in macrophages.In addition,the puncta to cell ratio decreased in macrophages treated with S3I-201,a STAT3 inhibitor.(7)MiR-155-3p is involved in IL-6-induced autophagy by directly targeting CREBRF in macrophagesQRT-PCR results confirmed in experiment.S3I-201 eliminated the promoting effect of the IL-6-p-STAT3 pathway on miR-155-3p expression.In addition,the puncta to cell ratio decreased in macrophages treated with 3-MA.However,the puncta to cell ratio did not decreased in macrophages treated with S3I-201.The target gene of miR-155-3p was predicted to be CREBRF by searching some databases,and was confirmed finally.(8)IL-6-and miR-155-3p-induced autophagy promotes M2-like macrophage polarization via STAT3 activationBy performing qRT-PCR,flow cytometry and ELISA,we revealed that IL-6 or miR-155-3p overexpression significantly induced M2-like macrophage polarization,and it could be blocked by S3I-201.However,3-MA was used to inhibit autophagy induction.We observed that the effect of miR-155-3p was blocked completely;however,the effect of IL-6 was attenuated incompletely.(9)Overexpression of IL-6 and miR-155-3p in macrophages promotes glioma tumorigenesis in vitro and in vivoMacrophages overexpressing IL-6 or miR-155-3p both promoted glioma proliferation and migration,and could be inhibited.The results in mice indicated that the tumors in the co-injection group of glioma cells and macrophages overexpressing IL-6 or miR-155-3p were more malignant.4.Conclusions(1)H-GDE-derived IL-6 and miR-155-3p can induce M2-like macrophage polarization via the IL-6-pSTAT3-miR-155-3p-autophagy-pSTAT3 positive feedback loop,which promotes glioma progression.(2)IL-6 and miR-155-3p may be potential biomarkers for treating glioma and that treatment targeting autophagy and the STAT3 pathway may impair the immunosuppressive tumor microenvironment and participate in antitumor immunotherapy.