PTX3 Mediates The Infiltration,migration,and M2-polarization Of Macrophage In Glioblastoma

Objective: Pentraxin 3(PTX3)is a soluble inflammatory factor in the tumor microenvironment,a candidate marker of inflammation and an innate immune modulator associated with immune escape,PTX3 has the functions of activating complement,neutralizing pathogens,affecting apoptotic cells and regulating function of inflammation.Macrophages,an important component of the tumor microenvironment,also significantly influence the development of glioma.However,the immune profile and clinical significance of PTX3 in gliomas have not been fully elucidated.To comprehensively define the role of PTX3 in the glioma tumor microenvironment,bioinformatics analysis and in vitro experiments were performed.This is the first extensive study of the molecular and immunerelated features of PTX3 in gliomas,with a particular focus on the interrelationship between PTX3 and macrophages,and will help advance the clinical management of gliomas.Methods: RNA-sequencing data from low-grade glioma(LGG)and glioblastoma(GBM)samples were collected from the TCGA and CGGA datasets.The molecular and clinical features of PTX3 were explored.Somatic mutations and copy number variations associated with PTX3 expression were determined using GISTIC 2.0 analysis.Immune-related functional annotation of PTX3 was performed.Single-cell sequencing data from primary GBM samples were collected to explore the cell-to-cell interactions associated with PTX3 expression in the tumor microenvironment of GBM.Potential transcription factors for PTX3 in GBM were further predicted.Tumor tissues from patients undergoing neurosurgical glioma resection at Xiangya Hospital of Central South University were used for immunohistochemical staining for PTX3,HAVCR2,PD-1,PD-L1,and CD276.GBM samples from the Xiangya cohort and pan-cancer samples from tissue arrays were used for multiplex immunofluorescence staining for PTX3,CD68,and CD163.The microglia cell line HMC3 was co-cultured with the si-PTX3-transfected GBM cell line U251 and U87.Transwell assays were performed on co-cultured HMC3 cells to determine migratory capacity,and flow cytometry assays were performed on co-cultured HMC3 cells to determine polarization capacity.Peripheral blood-derived monocyte THP-1 cell line was induced to obtain M2 macrophages,and M2 macrophages were co-cultured with GBM cell line U251 transfected with si-PTX3.Transwell assay was performed on co-cultured M2 macrophages to determine the migration ability.CCK-8 assay determined the optimal concentration of PI3K/Akt/m TOR pathway inhibitor NVP-BEZ235.The protein expressions of PI3K/Akt/m TOR pathway molecules and PTX3 were detected after adding NVP-BEZ235 to U251 cells and transfecting U251 cells with si-PTX3.The protein expression of PI3K/Akt/m TOR pathway molecules with PTX3 was detected after the addition of r PTX3 recombinant protein to M2 macrophages.Results: PTX3 expression was significantly increased in GBM compared to LGG in TCGA and CGGA.PTX3 can be used as a predictive marker for IDH status,1p19 q status,MGMT status and molecular subtypes.PTX3 expression levels were associated with different genomic alterations,in which IDH1,TP53,ATRX were more frequently mutated in the PTX3 low expression group,while EGFR,PTEN,TTN were more frequently mutated in the PTX3 high expression group.PTX3 is also associated with multiple immune-infiltrating cells and inflammatory activity in gliomas.High PTX3 expression predicted poorer survival in the Xiangya cohort based on the H-score calculated in the IHC staining results.In addition,based on IHC staining results,PTX3 was closely related to the expression of PD-1,PD-L1,CD276,and HAVCR2 in the glioma tumor microenvironment.Furthermore,at the single-cell sequencing level,PTX3 is involved in tumorigenic and immunogenic processes.PTX3 was found to be involved in cellular communication between GBM cells and immune cells,especially macrophages,through several canonical signaling pathways.In the results of multiplex immunofluorescence staining,PTX3 was mainly co-expressed with CD68 and CD163 in GBM samples and pancancer samples.In addition,PTX3 mediates macrophage migration and M2 polarization in in vitro experiments,and PTX3 can also directly affect M2 macrophages migration.PI3K/Akt/m TOR has been shown to be an important regulatory axis of PTX3 in GBM,and PTX3 inhibition in GBM cells activates the PI3K/Akt/m TOR pathway negatively.PTX3 also activates the PI3K/Akt/m TOR pathway in M2 macrophagesConclusion: Taken together,this study sheds light on the characteristics of PTX3 in glioma development and the tumor immune microenvironment.The immunosuppressive properties of PTX3 make it a potential new therapeutic target and prognostic marker for the treatment of glioma.PTX3 mediates microglial infiltration,migration and M2 polarization in GBM.PTX3 mediates M2 macrophage migration in GBM.This study is expected to provide a theoretical basis for further in-depth study of the PTX3-related immune microenvironment in glioma.