The Role,Mechanism And Prognostic Analysis Of Human Cytomegalovirus,Tumor-associated Macrophages/microglia In Glioblastoma Microenvironment

Objective: To investigate the expression and potential mechanism of HCMV in GBM microenvironment.Methods: The expression of HCMVgB and gH in GBM was detected by immunofluorescence,and IDH1 R132 H mutation,PTEN deletion and p-PDGFR?,PD-L1 and CD8+T cells were detected by immunohistochemistry.The effects of these indexes on the prognosis were analyzed by multifactor analysis.Result: The positive rate of gB was 48.5%(33/68);the positive rate of gH was 42.6%(29/68),both were simultaneously expressed as 30.9%(21/68),either positive the expression was 60.3%(41/68).However,HCMV infection was not associated with clinical features(age,sex,KPS)and molecular pathology(IDH-1 mutations and PTEN deletions)in GBM patients.There was also no correlation between HCMVgB and pPDGFR?,PD-L1 and CD8+ T cells.There was no correlation between gB and gH expression and prognosis(P = 0.551;P = 0.871).Conclusion: The expression of HCMVgB and gH can be seen in the GBM microenvironment,but there is no evidence that they are involved in the regulation of GBM biological behavior.Objective: To investigate the expression of human cytomegalovirus(HCMV)glycoprotein B,H(gB,gH)in glioblastomas(GBM)and its influence on prognosis.Methods: Detect HCMVgB and gH by immunofluorescence in 68 cases of glioblastomas,including 64 cases of primary glioblastomas and 4 cases of recurrent glioblastomas.Detect IDH R132 H mutation & PTEN deletion by immunohistochemical method.And the clinical data(age,sex,KPS score,onset period,tumor location,etc.)were collected and sorted out.Finally,Implement multifactor analysis and prognosis evaluation by statistics.Results: In 68 cases of glioblastomas,the positive expression of HCMVgB was 48.5%(33/68 cases),gH positive expression was 42.6%(29/68 cases),and either positive expression was 60.3%.The mutation of IDH1 R132 H was 20.6%(14/68 case).The loss of PTEN was 16.18%(11/68).Statistical analysis showed that the expression of gB and GH in HCMV was not associated with age,gender,KPS score,IDH1 mutation,and PTEN deletion.In prognostic analysis,IDH1 mutations were associated with prognosis(P=0.006);HCMVgB,gH expression was not associated with prognosis(P=0.551;P=0.871).Conclusion: HCMVgB and gH are expressed in GBM,but have nothing to do with prognosis.Objective: To explore the correlation between the expression of HCMVgB and platelet-derived growth factor receptor ?(PDGFR?)and to evaluate whether HCMVgB promotes the proliferation of tumors by inducing the phosphorylation of PDGFR?.Methods: immunofluorescence technique was used to detect the expression of HCMVgB in 84 cases of glioblastomas tissue specimens.Meanwhile,immunohistochemical staining was used to detect the expression of phosphorylated PDGFR?.The correlation between the expression of HCMVgB and the expression of PDGFR? was analyzed by statistics and the prognosis was evaluated.Results: In 84 specimens of glioblastomas,the positive expression of HCMVgB was40/84(47.62%),and the positive expression of phosphorylated PDGFR? was47/84(55.95%).Statistical analysis did not show a correlation between HCMVgB and PDGFR?(P=0.694).The survival curve analysis showed that the expression of PDGFR? was not associated with the survival period(P =0.098).Conclusion: In vitro,it is proved that gB can bind PDGFR? and phosphorylate it,activate the phosphatidylinositol 3-kinase(PI3K)signaling pathway,and then activate Akt,promote tumor's growth and inhibit apoptosis.However,no correlation was found between HCMVgB and PDGFR? by tissue specimens detection.Objective: To explore the relationship between the expression of gB and the programmed death-ligand-1(PD-L1)and CD8+ T cells in GBM.Methods: Immunofluorescence was used to detect HCMVgB in 91 cases of GBM,including 73 cases of primary GBM and 18 cases of relapse of GBM.Immunohistochemical was used to detect the expression of PD-L1 in some of the specimens and CD8+T cells in the microenvironment,and further evaluate whether HCMV infection participate the immune regulation in GBM microenvironment by increased expression of PD-L1 and decreased number of CD8+T cells.Results: In 91 cases of GBM,the positive expression of HCMVgB was 47.25%(43/91cases),positive expression of PD-L1 was 20.99%(17/81 cases),CD8+T cells were highly expressed in 51.95%(40/77 cases).Statistical analysis showed that there was no correlation between the positive expression of HCMVgB in GBM and the positive expression of PD-L1 and the expression of CD8+T cells(P =0.522;P =0.112).Conclusion: This study found that HCMV expressed in GBM microenvironment,however it has no correlation with some immune indicators.The role of HCMV infection participating immune regulation in the tumor microenvironment need to be further studied.Objective: To investigate the phenotypic polarization and distribution characteristics of tumor-associated macrophages/microglia(TAMs)in GBM microenvironment,and to further study the immunomodulation involved in TAMs M2 polarization and effects on prognosis.Methods: 1.Using i NOS,CD206,CD68 as markers,the phenotypic polarization and distribution characteristics of TAMs in human brain glioblastoma tissue specimens were detected by immunohistochemistry.In vitro Transwell,glioma cell lines U87,U251 and THP-1 were co-cultured to simulate the interaction between tumor cells and macrophages.The polarization status of THP-1 after co-culture was detected by flow cytometry and QPCR.2.The expression of CD206,PD-L1,PD-1,CD8+ T lymphocytes,TGF-?2,STAT3 and p-STAT3 in GBM tissue specimens was detected by immunohistochemistry.The correlation between CD206 and the above immunological parameters was statistically analyzed.In vitro co-culture experiments and CGGA transcriptome sequencing data were verified and analyzed.3.Analyze the relationship between M2 type polarization and prognosis from tissue samples and CGGA database.Results: The TAMs in the GBM microenvironment were mainly M2-type expressing CD206.M2 TAMs are mostly distributed around blood vessels and necrosis.After the glioma cell was co-cultured with THP-1,THP-1 was mainly polarized in M2 type,but it had nothing to do with the prognosis.Analysis of CGGA transcriptome sequencing data revealed that the gene encoding CD206 was associated with the genes encoding PD-L1 and PD-1(P=0.026;P=0.00).In vitro experiments are consistent with the results of the CGGA database.Conclusion: In the GBM microenvironment,TAMs is mainly M2 type and coexist with M1 type,which is involved in both immunosuppression and immune response.M2 TAMs may participate in the GBM immune microenvironment through STAT3 signaling pathway and PD-L1/PD-1.Therefore,targeting M2 TAMs may become a new direction of immunotherapy.Objective: To evaluate the phenotypic polarization and distribution characteristics of TAMs in the GBM microenvironment.Methods: Using i NOS,CD206 and CD68 as markers,the phenotypic polarization and distribution characteristics of TAMs in tissue samples were detected by immunohistochemistry.In vitro experiments using Transwell,glioma cell lines U87,U251 and THP-1 were co-cultured to simulate the interaction between tumor cells and macrophages.The polarization state of THP-1 after co-culture was detected by flow cytometry and QPCR.Results: TAMs is mainly expressed in CD206 marked M2 in tissue specimen in GBM microenvironment.M2 TAMs are mostly distributed around blood vessels and necrosis.The glioma cell lines U87,U251 were co-cultured with the human peripheral blood mononuclear cell line THP-1,and THP-1 expressed M2 polarization.Conclusion: In GBM microenvironment,TAMs is mainly M2 type,while M1 TAMs co-existing,which participate the double actions of immunosuppression and immune response.Objective: By detecting the expression of TAMs in GBM and detect immune checkpoints,CD8+ T lymphocytes,immune related signaling pathways and immunosuppressive factors,and to evaluate the immunoregulatory role and mechanism of TAMs involved in GBM microenvironment.Methods: Immunohistochemistry was used to detect CD206,PD-L1,PD-1,CD8+ T cells,TGF-?2 and signal transducer and activator of transcription factor 3(STAT3)in GBM tissue samples.The correlation between CD206 and the above immunological indexes was statistically analyzed.In vitro co-culture experiments and CGGA transcriptome sequencing data were verified and analyzed.Results: In GBM microenvironment,the positive expression of CD206 was 100%(93/93 cases),and the mean value of the count was 31.12 + 19.22(5.00-115.33).The positive expression of PD-L1 was 20.48%(17/83),and the positive expression rate of PD-1 was 0%(0/88).The positive of CD8+T cell was 100%(83/83),and the mean value was 18.59 + 16.34(1.00-79.67).The positive expression of TGF-?2 was 56.76%(21/37).The high expression of p-STAT3 was 68.35%(54/79).Statistical analysis showed that the high and low expression of CD206 in GBM had no correlation with the expression of PD-L1,CD8+T cells,TGF-?2 and p-STAT3(P=0.241,P=0.163,P=0.570,P=0.936).CGGA transcriptome sequencing data analysis showed that there was no correlation between the expression of MRC1 gene and CD8 A gene(P=0.959),MRC1 gene expression was correlated with CD274 and PDCD1 gene(P=0.026;P=0.00).In vitro,the glioma cell line was co-cultured with THP-1.After co-culture,it was found that the expression of p-STAT3 and PD-L1 was increased in U87 cells,and PD-1 was increased in M2 macrophages.Conclusion: M2 TAMs are significantly more than CD8+ T lymphocytes,further demonstrating the immunosuppressive state of the GBM microenvironment.M2 TAMs may be involved in the formation of GBM immune microenvironment through STAT3 signaling pathway and PD-L1/PD-1.Targeting M2 TAMs may become a new direction of immunotherapy.Objective: M2 TAMs plays an important role in the development of GBM,regulating the proliferation,survival,invasion and angiogenesis of tumor cells,affecting the efficacy of antiangiogenic drugs and participating in the mechanism of immune escape.The purpose of this study was to detect the expression of M2 type TAMs in GBM tumor tissue and to explore its prognostic value.Methods: Immunohistochemical method was used to detect the expression of CD206 in84 cases of glioblastomas tissue.The K-M curve was used to evaluate its effect on progression free survival(PFS)and overall survival(OS).Results: The expression of M2 type TAMs in GBM microenvironment was not related to age,sex,KPS,Ki-67 value added index(P=1.000;P=0.389;P=0.810;P=0.654).The expression of CD206 was not related to PFS(P=0.316)and OS(P=0.912)in patients with GBM.CGGA transcriptome data were used to analyze the relationship between the expression of MRC1 gene(coding CD206)and prognosis.The results showed that MRC1 gene expression was not correlated with clinical prognosis(P =0.134).Conclusion: This study analyzed the relationship between M2 TAMs polarization and prognosis basing on tissue specimens and CGGA database,the results showed no correlated with the prognosis.Obviously,although M2 type TAMs polarization plays a crucial role in the development of GBM,it is not the key factor affecting the prognosis.