The Effect And Mechanism Of IKZF1-CD84-PTPN11 Pathway On The Malignant Phenotype Of Glioma By Promoting The Polarization Of Tumor-associated Macrophages

Background and objectiveGlioblastoma（GBM）is a common and fatal primary brain tumor of the central nervous system.The glioma immune microenvironment is mainly composed of glioma cells,infiltrating lymphocytes and myeloid inflammatory cells.Myeloid inflammatory cells mainly include monocytes,macrophages,microglia,antigen presenting cells,neutrophils and myeloid immunosuppressive cells.The immunosuppressive microenvironment of glioma is the key reason for the malignant progression of GBM.However,the underlying mechanism is still unclear.Macrophages can be polarized into a wide range of subtypes,including M1-and M2-like phenotypes.The main markers of M1 macrophages are CD80,secreting cytokines IL-1B and TNF-α.The markers of M2 macrophages are CD163,which mainly secrete cytokines such as IL-10 and TGF-βcells.In addition,tumor-associated macrophages（TAMs）originate from monocytes and are characterized by M2-like phenotypic macrophages.TAM-M2 has a strong ability of immunosuppression,inducing tumor malignant phenotype and promoting angiogenesis.Therefore,targeting TAM-M2 is a promising treatment strategy for GBM.IKZF1 gene encodes a transcription factor,which belongs to the zinc finger DNA binding protein family associated with chromatin remodeling.The expression of this protein is limited to the hemolymphatic system of fetuses and adults,and it acts as a regulator of lymphocyte differentiation.However,it is not clear whether IKZF1 plays a role in monocyte-macrophage differentiation.This study focused on the effects of IKZF1on glioma immune microenvironment at cellular and animal level,especially on glioma-associated macrophages,and the effects of glioma-associated macrophages on glioma stem cells,including the effects of dryness,proliferation and invasion of glioma stem cells,and explored the downstream targets of IKZF1 and its mechanism of regulating glioma-associated macrophages at cellular and molecular levels.Finally,this mechanism is used to explore new glioma therapy by targeting glioma-associated macrophages.Methods1.Ethical:This study was approved by the Institution Evaluation Committee of the first Hospital of China Medical University（approval No.KT2022173）.All animal experiments are supervised by the Animal Ethics Committee of China Medical University（approval No.2022098）.2.Cell culture.The cells used in the study were THPI cells,human primary glioma stem cells G1-GSC and G2-GSC.The freshly resected clinical glioma tissue was isolated into single cells and cultured in an incubator of 5%,CO2,37 degrees Celsius with stem cell culture medium to culture glioma stem cells.3.Transfection.Plasmid,lentivirus vector IKZF1,CD84,PTPN11 control and si RNA were used for silencing.The c DNA of plasmid,lentivirus vector IKZF1 and CD84 was used for overexpression.The effects of gene silencing and overexpression were detected by WB.4.Immunoblotting analysis and immunoprecipitation test.Western bolt was performed by protein extraction,agarose agglutination antibody incubation,protein quantification,electrophoresis,membrane transfer,milk blocking,first antibody,second antibody,luminescence and so on.IKZF1,PTPN1,P-PTPN11,SOX2,CD84,CD80 and C163 antibodies were used to detect the expression of corresponding proteins.It was quantified by Image J software.5.Immunofluorescence.The cells were fixed by paraformaldehyde,permeated by Triton XMel 100,blocked by 3%BSA,the first antibody 4o C was overnight,and the fluorescence second antibody was observed under fluorescence microscope after 1 hour.CD80 and CD163 antibodies were used to detect the expression of corresponding proteins in immunofluorescence assay.6.Elisa.The secretion of TNF-α,TGF-βand IL-1B,IL-10 in the supernatant was detected by Elisa kit.The concentration of cytokines can be detected by coating,sample addition,washing,adding enzyme labeled antibody,substrate coloration,termination reaction,result measurement,data analysis and so on.TNF-α,TGF-βand IL-1B,IL-10 antibodies were used to detect the secretion of corresponding proteins in Elisa assay.7.Limit dilution neuroball formation test:The self-renewal ability of neural stem cells was evaluated by a neurosphere formation experiment,which was dissociated and implanted into a 96-well plate at a rate of 50,100,500 or 1000 cells per well.After 7 days,the size of the sphere was observed and the spheres with a diameter of more than 50μm were counted.8.Cell viability assay.The cell survival rate was detected by 96-well plate according to the cell proliferation analysis kit,and the cell viability was measured and the curve was drawn every days.9.Transwell.The GSC cell spheres were made into single cell suspension and put into the upper chamber,and 10%serum was put into the lower chamber to invade the filter membrane coated with Matrigel for 20 hours.The invasive cells were photographed by microscope and counted by Image J software.10.Detection of cell proliferation by Edu.Edu proliferation assay kit was used to detect the DNA absorption and proliferation of cells.First,cells were incubated with Edu,fixed with paraformaldehyde,penetrated by Triton X-ray 100,blocked by light-avoiding 3%H₂O₂,incubated with Edu solution at 37℃for 90 minutes,and added 488ram 555 fluorescent dye at the same time.DAPI was used to dye the nucleus.The samples were observed under fluorescence microscope and the proliferation ratio was calculated.11.Detection of apoptosis by flow cytometry.Apoptosis was detected by flow cytometry and AnnexinⅤfluorescent antibody.The single cell suspension was taken and washed with PBS and incubated with blinding buffer and AnnexinⅤ.Flow cytometry and Flowjo software were used to analyze the results.12.Luciferase activity analysis.Jaspar website bioinformatics predicts the IKZF1 binding site upstream of the transcriptional initiation site of the CD84 promoter region.The CD84 promoter region with wild type and mutant binding sites was cloned into luciferase vector fragment.The luciferase activity of the transfected plasmid was detected by Promega double luciferase reporter kit.13.Orthotopic transplantation of intracranial glioma.GSCs and TAM were co-injected into anesthetized female BALB/c nude mice with stereotactic apparatus,and IKZF1 inhibitors were given at the same time.The symptoms and survival time of mice were observed,and the tumor growth was evaluated.The brains of mice were collected for analysis and HE staining was used to evaluate the size of tumor.14.Immunohistochemistry（IHC）.The paraffin-embedded sections of gliomas were stained with anti-Ki67,CD163 and CD44 antibodies.Observe and take pictures under an optical microscope.15.Bioinformatics analysis and statistical analysis.All experiments were repeated at least three times,and the data were expressed as mean±standard deviation.Chi-square test,t-test or analysis of variance were used for comparison between groups.Access and process TCGA glioma database and CGGA glioma database through Glio Vis and GEPIA2 online platform.TISH database was used to analyze the glioma single cell sequencing database.Survival differences were analyzed by logrank test and Kaplan-Meier analysis.The relationship between IKZF1 and CD84expression was analyzed by Pearson correlation.Double-tail P<0.05 is considered to be statistically significant.SPSSv19.0 software was used for statistical analysis.Results1.IKZF1 and CD84 are highly expressed in glioma microenvironment and lead to poor prognosis.The high expression of IKZF1 and CD84 is significantly correlated with the adverse clinical features of glioma patients,including high expression in glioma,high pathological grade,mes type pathological classification,glioma recurrence,IDH wild type,1p19q deletion and so on.Single cell sequencing analysis showed that IKZF1 and CD84 were significantly expressed in monocytes and macrophages in glioma immune microenvironment,which led to poor prognosis of glioma patients by promoting M2polarization of TAM.2.IKZF1 promotes TAM-M2 polarization by up-regulating the expression of CD84.Silencing IKZF1 in TAM significantly inhibited the expression of M2 marker CD163,significantly increased the expression of M1 marker CD80,inhibited the secretion of M2cytokines TGF-βand IL-10,and promoted the secretion of M1 cytokines TNFαand IL-1B.IKZF1 upregulates the expression of CD84 at the transcriptional level and stimulates the M2 polarization of TAM by up-regulating the expression of CD84.3.CD84 promotes the polarization of TAM-M2 by binding and activating PTPN11.IP confirmed that CD84 and PTPN11 have protein interaction.Overexpression of CD84significantly increased the activation level of PTPN11 and promoted the polarization of TAM-M2 by stimulating activated PTPN.4.IKZF1 promotes the supporting effect of TAM on the malignant phenotype of GSC by up-regulating CD84.Under the condition of GSC co-culture,silencing the expression of IKZF1 in TAM significantly inhibited the supporting effect of TAM on the malignant phenotype of GSC,including cell proliferation,invasion,anti-apoptosis ability,stem cell renewal ability,stem cell marker expression and so on.Restoring the expression of CD84 in TAM with silenced IKZF1 significantly restored the ability to promote the malignant phenotype of GSC,so IKZF1 in TAM promoted the malignant phenotype of GSC by up-regulating CD84.5.Targeted inhibition of IKZF1 in vivo to treat mouse glioma model.Silence of IKZF1 in TAM or administration of IKZF1 inhibitor in vivo could significantly reduce the tumor volume and improve the prognosis of glioma model,and significantly reduce the proliferation rate of tumor tissue,tumor dryness and M2 TAM infiltration in vivo.ConclusionIn the process of TAM polarization,IKZF1 can up-regulate CD84 to activate PTPN11,promote the M2 polarization of TAM,secrete M2 cytokines IL-10 and TGF-β,promote the malignant phenotype of GSC,and improve the tumorigenicity and TAM infiltration in vivo.Targeted inhibition of IKZF1 in TAM can significantly treat mouse glioma animal model,reduce tumor tissue,weaken tumor dryness,weaken TAM infiltration and improve prognosis.