The Role And Mechanism Of M2 Type Tumor Associated Macrophages Mediated By IL6/JAK3 Signaling Axis Participating In Glioma Chemotherapy Resistance

Objective:Glioblastoma(GBM)is the most common malignant tumor of the central nervous system,with high incidence and poor prognosis.Despite continuous improvements in the clinical diagnosis and treatment,the median survival rate of GBM is only 14.6months.More and more studies have shown that the tumor microenvironment is a key factor involved in multiple stages of cancer development,especially local drug resistance,immune escape and distant metastasis.The process of glioma development is the process of interactive transformation between GBM cells and their microenvironment,and the two together form a complex “community of destiny”.Research on the mechanism of chemotherapy resistance in GBM has always been a hot spot in the field of GBM.For example,the accelerated efflux of chemotherapy drugs,the feedback enhancement of DNA damage repair enzyme activity,the existence of tumor stem cells can affect the chemotherapy of tumor cells.In recent years,with the concept of tumor “purity”,people have begun to pay more attention to the mutual modification between the tumor microenvironment and tumor cells.This modification mediates the malignant progression of tumors and the occurrence of chemoradiotherapy resistance.Our previous research found that the proportion of tumor cells(tumor purity)in the most malignant glioblastoma tissue was only 74.1%.Immune cells are selectively recruited to tumor tissue and held hostage by tumor cells,jointly constructing a local immunosuppressive microenvironment.Among the many immune cells,macrophages are extremely abundant in glioma tissues.Most of them are polarized into the cancerpromoting M2 type,participating in the formation of an immunosuppressive microenvironment,accelerating GBM proliferation and angiogenesis,and triggering radiotherapy,chemotherapy and immunotherapy resistance.Therefore,studying the interaction mechanism between GBM cells and macrophages will help improve the theory of GBM microenvironmental regulation and propose new microenvironmental intervention targets.Through weighted gene co-expression network analysis(WGCNA),we identified the purity-related core gene JAK3,which encodes a receptor tyrosine kinase,a member of the Janus kinase family.JAK/STAT mediated by this family plays an indispensable role in tumor proliferation and invasion,stemness maintenance,angiogenesis and immune microenvironment regulation.However,there are few reports on the expression pattern,function and related mechanism of JAK3 in GBM and its microenvironment.In-depth exploration of the clinical value of JAK3 in GBM and its regulatory mechanism in the immune microenvironment will provide new ideas for the clinical treatment of chemotherapy-insensitive GBM subgroups.Methods:1.Database and clinical samples: In this study,the expression profiles and relevant clinical information of GBM patients were mainly obtained from three public database platforms,CGGA,TCGA and GEO.The CGGA database was used as the internal discovery set,and the TCGA database and GEO database were used as the external validation set.Tissue samples from GBM patients were obtained from neurosurgery in the First Affiliated Hospital of China Medical University with the informed consent of the patients and their families.The research has been approved by the hospital ethics committee.2.Bioinformatics analysis: The immune score,the stromal score and tumor purity score of GBM patients were calculated according to the ESTIMATE algorithm.The hub genes related to tumor purity were identified through WGCNA.Univariate difference analysis was performed by limma.|Log FC|>2 and FDR<0.05 were selected as the screening criteria for differential genes.The enrichment analysis of differential genes was performed using Clue Go of Cytoscape software.The proportion of immune cells in glioma patients was calculated based on the CIBERSORT and algorithms.3.Molecular biology experiments: Tissue immunofluorescence was used to determine the co-localization of the target molecule JAK3 and GBM cells.q PCR,Western and immunohistochemistry was performed to detect the expression levels of different molecules.MTS,Transwell and apoptosis assays were used to detect the functional changes of cells under different treatment conditions in vitro.Orthotopic tumor transplantation experiments in mice were used to further verify the conclusions.4.Statistical analysis: R 4.0.2 and Graph Pad Prism 8 were used for statistics analysis.Differences between groups were tested by two-tailed t-test or one-way analysis of variance.Kaplan-Meier survival analysis was used to evaluate the prognostic value of different indicators.Univariate cox regression analysis was used to verify independent prognostic factors.P<0.05 was considered to be statistically different.Results:Part Ⅰ:Description of GBM microenvironment purity factors affecting patients’ chemotherapy sensitivity and identification of purity associated hub gene JAK3Based on the public CGGA and TCGA GBM databases,we conducted bioinformatics analysis at the transcriptional level,and to some extent explored the influence of GBM microenvironment heterogeneity on chemosensitivity.We first calculated the purity score of each GBM patient through the ESTIMATE algorithm,and divided the patients into high and low groups according to the purity score.We found that the prognosis of patients with high tumor purity was significantly improved after receiving chemotherapy drugs,while chemotherapy had no significant impact on survival in the low-purity patient group.Therefore,we speculated that the purity in the microenvironment might have a key regulatory effect on the chemosensitivity of glioma.To further identify the core targets that may be involved in causing changes in the purity of the GBM microenvironment,we applied the method of weighted correlation network analysis(WGCNA)and screened out the hub gene Janus kinase 3(JAK3),which was most correlated with purity and had stable prognostic value.We further identified co-localization of JAK3 and GBM cell marker GFAP by tissue immunofluorescence,confirming the important role of JAK3 in GBM cells.In order to determine the targeting value of JAK3 in GBM cells,we knocked down JAK3 on tumor cells when treated with temozolomide,and found that the proliferation ability of glioma cells was significantly down-regulated,and the apoptosis ability was significantly up-regulated.The intracranial xenograft tumor model also suggested that targeting JAK3 combined with temozolomide therapy could significantly prolong the survival time of tumor-bearing mice,confirming that the expression of endogenous JAK3 in GBM can affect the therapeutic response of GBM cells themselves to temozolomide.Part Ⅱ : Mechanism of JAK3-mediated activation of STAT3/PARP1 dual pathway in GBM cells to promote nuclear translocation of DNA damage repair key enzyme MGMTTo further explore the molecular mechanism of JAK3 regulation of chemotherapy resistance in GBM cells,we first performed RNA sequencing on the GBM cells that knocked down JAK3.Through differential gene enrichment analysis,we found that DNA damage repair signaling pathway,JAK-STAT signaling pathway and PARP signaling pathway were mainly enriched in the tumor cells of the control group compared with the tumor cells of the knockdown JAK3 group.Considering the changes in chemosensitivity caused by DNA damage repair,we detected the protein level of the classic DNA damage repair enzyme MGMT in tumor cells.We found that while tumor cells were treated with temozolomide,the nuclear protein levels of MGMT were significantly downregulated after knocking down JAK3 in tumor cells.Through mass spectrometry results combined with enrichment analysis,we identified that the activation of JAK3 can simultaneously activate the downstream STAT3 and PARP1 signaling pathways,both of which promote the nuclear translocation of MGMT to achieve DNA damage repair in tumor cells.After overexpressing JAK3 and targeting STAT3 and PARP1 at the same time,the up-regulated MGMT level in the nucleus will be restored,confirming the key role of STAT3/PARP1 dual pathway in glioma cells,and suggesting that their common upstream JAK3 played an important role as a key regulatory target of GBM chemotherapy resistance.Part Ⅲ: Experimental exploration of the interaction between GBM cells and macrophages mediated by JAK3Through the enrichment analysis of sequencing data,we also found that with the knockdown of JAK3 gene,the ability of tumor cells to recruit immune cells was also significantly down-regulated.Since JAK3 is a hub gene related to tumor purity,we performed CIBERSORT and MCP algorithms based on CGGA and TCGA two databases.We found that macrophages accounted for the largest proportion in the glioma microenvironment,and were significantly enriched in patients with low purity.Immunohistochemistry suggested that the expression level of JAK3 was highly positively correlated with the content of M2 macrophages.We also found that the conditioned medium of GBM cells pretreated with JAK3 inhibitors also significantly weakened the chemotactic ability of macrophages,and at the same time caused the ability of macrophages to polarize to the M2 type to be significantly weakened,further confirming that JAK3 may be involved in the regulation of M2 macrophages in the microenvironment.Considering the regulation of JAK3-mediated paracrine effects on M2 macrophages,we found that the protein level of CCL2 was significantly downregulated after JAK3 inhibitor treatment through the secretory factor Array and PCR,which may be the role of JAK3 in affecting macrophage chemotaxis and M2 macrophages polarization potential mechanism.After exploring the key role of tumor cells on macrophages,we further verified the key regulatory role of macrophages on tumor cell chemotherapy resistance.We induced THP1 monocytes into M0/M1/M2 macrophages,and then collected conditioned medium to stimulate GBM cells.We found that the JAK3 signaling pathway in GBM cells treated with the conditioned medium of M2 macrophages was activated,and their sensitivity to temozolomide was significantly weakened,and this drug resistance was identified by M2 macrophages that secrete IL-6 to achieve it;We also orthotopically implanted tumor cells and macrophages in equal proportions into the brains of mice,and the tumor-bearing mice that were originally sensitive to temozolomide became resistant.The downregulated chemosensitivity was restored after targeting JAK3,suggesting a key role for JAK3-mediated chemoresistance in macrophage-promoting GBM cells.Conclusions:Part Ⅰ: Purity factors in the GBM microenvironment exert a key regulatory effect on the chemosensitivity of GBM cells,and JAK3,as a stable purity-related hub gene,mediates the chemoresistance of GBM cells.Part Ⅱ: JAK3 promotes the nuclear translocation of DNA damage repair key enzyme MGMT by regulating the STAT3/PARP1 dual pathway in GBM cells to achieve chemotherapy resistance of GBM.Part Ⅲ: JAK3 in GBM cells promotes macrophages chemotaxis and polarization to M2 phenotype by secreting CCL2;Macrophages after chemotaxis and polarization further activate JAK3 signal pathway in glioma cells through IL-6 secretion factors,forming a positive feedback loop for microenvironment interaction.