Clinical Significance And Mechanism Of PD-L1 Expression In Glioma

Background and purposeGlioma is the most common malignant tumor of the central nervous system,and its incidence accounts for 40%-50%of primary brain tumors.Although the current standard therapies for glioma are continuously developing and improving,including surgical treatment,adjuvant chemotherapy,radiation therapy and targeted therapy,glioblastoma is still the most malignant tumor（Glioblastoma,GBM,WHO grade IV）with the median survival time about 14 months and the 5-year survival rate less than 10%.Considering the characteristics of gliomas,especially glioblastomas that with poor prognosis and high recurrence rates,it is necessary to find new therapeutic methods to improve clinical treatment.Programmed death-ligand 1,PD-L1,also called cluster of differentiation 274（CD274）or B7 homolog 1,B7-H1),a classical co-suppressor molecule of the B7family,binds primarily to programmed cell death 1（PD-1）on the surface of T cells to inhibit T cell function and promote immune escape.Blocking PD-L1/PD-1pathway can restore T cell function,inhibiting immune escape and tumor progression.PD-L1/PD-1 blocking antibodies have been successfully used clinically,which have altered the treatment of advanced non-small cell lung cancer.Consistently,PD-L1,as a star molecule of immunotherapy,provides more possibilities for improving the therapeutic status of gliomas.Therefore,it is extremely important to investigate the expression and clinical significance of PD-L1in gliomas.In the tumor microenvironment of glioma,macrophage plays a vital role.For example,in some patients,macrophages may account for more than half of the cells in tumor microenviroment.Current evidence has shown that PD-L1 is expressed on the surface of both tumor cells and tumor-associated macrophages（TAMs）in the glioma microenvironment and synergistically plays an immunosuppressive role in microenvironment.Therefore,exploring the mechanism of up-regulation of PD-L1in tumor cells and macrophages may provide new opportunities of immunotherapy for gliomas.Meanwhile,further investigating the mechanism of PD-L1,as an important immunosuppressive checkpoint,to promote immune escape can better optimize PD-L1-associated immunotherapy.Thus,apart from suppressing T cell function,whether PD-L1 has other mechanisms to promote immune escape and tumor progression needs further exploration.In summary,the purpose of this study was to investigate the expression and clinical significance of PD-L1 in gliomas and to find a common mechanism for the simultaneous up-regulation of PD-L1 in tumor cells and macrophages in the glioma microenvironment,which can play an important foundation for further exploring the unrevealed mechanism of gliomas to promote the immune escape and tumor progression.Part One The Expression and Clinical Significance of PD-L1 in GliomaMethod1.qRT-PCR was used to detect the expression of PD-L1 in gliomas and adjacent normal tissues;2.Immunohistochemistry was used to detect the expression of PD-L1 in gliomas and adjacent normal tissues;3.Using flow cytometry,we analyzed the PD-L1 expression in glioma infiltrating macrophages and peripheral blood mononuclear cells;we also detected the PD-L1 expression in peripheral blood mononuclear cells from healthy donors;4.Using CGGA（Chinese Glioma Genome Atlas）,TCGA（The Cancer Genome Atlas）database and tissue specimens,we analyzed the relationship between PD-L1expression in samples and WHO grading;5.We analyzed the relationship between PD-L1 expression（flow cytometry data）and WHO classification;6.We analyzed the relationship between PD-L1 and isocitrate dehydrogenase（IDH）mutations in CGGA and TCGA database and the diagnostic value for IDH wild-type（IDH WT）in gliomas;7.We analyzed the relationship between PD-L1 and glioma molecular subtypes in CGGA and TCGA database and the diagnostic value for mesenchymal subtype;8.We analyzed the correlation between PD-L1 and other immune checkpoints in CGGA and TCGA database;9.Using CGGA and TCGA,we analyzed the relationship between PD-L1expression in samples and clinical data of glioma patients;10.Using CGGA TCGA and tissue specimens,we analyzed the relationship between PD-L1 expression and the prognosis of glioma patients.Results1.Compared with adjacent normal tissues,tumor samples had high expression of PD-L1 at mRNA level;2.The results of immunohistochemistry showed that the expression of PD-L1was significantly higher in glioma tissues than in adjacent normal samples;3.According to flow cytometry data,PD-L1 expression in TAMs of glioma tissue was significantly higher compared with peripheral blood mononuclear cells both from patients and healthy donors;4.In both TCGA/CGGA databases and tissue specimens,the expression of PD-L1 in GBM was significantly accumulated compared with WHO grade II and III gliomas at mRNA level;5.According to flow cytometry data,the expression of PD-L1 in TAMs from GBM was significantly higher than that in WHO grade II and III gliomas;6.Compared with IDH mutant-type（IDH MT）gliomas,PD-L1 was highly specific in IDH WT gliomas;7.Compared with other molecular subtypes of glioma,PD-L1 is highly expressed in mesenchymal subtype;8.PD-L1 was mainly associated with WHO classification,IDH mutation,mesenchymal subtype and age in gliomas,while had no relations with gender;9.PD-L1 expression in gliomas was positively correlated with CD80,CD86,PD-L2,ICOSLG and B7-H3 in the B7 family,as well as ICOS,PD-1,TIM3 and IDO1;10.The median survival of PD-L1 high expression patients was significantly lower than that of PD-L1 low expression patients both in gliomas and GBM.Summary1.PD-L1 was highly expressed in gliomas and closely related to the malignant phenotype of gliomas;2.PD-L1 may have synergistic effects with multiple immune checkpoints to create a immunsuppressive microenvironment;3.PD-L1 may serve as an index of prognosis for gliomas and GBM.Part Ⅱ Down-regulation of miR-106a promotes progression of Glioma via Interaction with PD-L1 through direct and indirect wayMethods1.The relationship between PD-L1 expression and monocyte function was analyzed by flow cytometry and magnetic bead sorting and T cell co-incubation;2.Through GEO database and TargetScan,we analyzed the potential miRNAs that may participate in PD-L1 regulation in glioma TAMs;3.qRT-PCR was used to verify the differential expression of miRNAs in peripheral blood mononuclear cells and paired TAMs from glioma patients;4.Flow cytometry and q RT-PCR were used to validate the relationship between miRNAs and PD-L1 expression by using THP-1-derived macrophages;5.miR-106a was transfected into THP-1-derived macrophage,and then we detected the PD-L1-induced function changes;6.Dual luciferase reporter assay was used to explore the direct targeting relationship between miR-106a and HIF-1α,STAT3,and PD-L1;7.qRT-PCR was used to detecte the expressions of HIF-1αand STAT3 after transfected THP-1-derived macrophages by miR-106a;8.qRT-PCR was used to detect the differential expression of miR-106a in glioma cell lines and the immortalized cell lines;9.miR-106a mimics and inhibitors were transfected into U87 and U251 cells and then we detected the expression levels of HIF-1α,STAT3 and PD-L1;10.Constructing the PD-L1 overexpressing glioma cell line（U251）,then we detected the effect of miR-106a on PD-L1 expression;11.We investigated the effect of miR-106a on tumorigenicity and PD-L1expression of U251 PD-L1 overexpressing cell lines in vivo;12.We analyzed the differential expression of miR-106a in CGGA,TCGA database and tissue specimens,as well as the relationship with WHO grade;13.We analyzed the correlations of miR-106a with HIF-1α,STAT3,and PD-L1expression in tissue specimens;14.We analyzed the relationships between miR-106a and the prognosis of glioma and GBM patients in CGGA,TCGA databases and detected clinical specimens.Results1.According to flow cytometry data,we found that monocytes with high expression of PD-L1 secreted more immunosuppressive factors.Co-incubation PD-L1 high-expressing monocytes with T cells after magnetic bead sorting,we found that PD-L1 high-expressing monocytes had stronger ability to induce apoptosis in T cells;2.There were 9 miRNAs that were significantly down-regulated in glioma TAMs and may directly regulate PD-L1 expression,including miR-15a,miR-16,miR-636,miR-20b,miR-106a,miR-20a,miR-93,miR-17 and miR-425;3.According to the data from tissue specimens,we cnfirmed 8 miRNAs,including miR-15a,miR-16,miR-636,miR-20b,miR-106a,miR-20a,miR-93 and miR-425,were significantly down-regulated in TAMs;4.Transfection of miRNAs in macrophages revealed that only miR-106a can significantly reduce PD-L1 expression;5.miR-106a significantly inhibited PD-L1-mediated macrophage function;6.Double luciferase reporter assay showed that miR-106a can directly target HIF-1α,STAT3 and PD-L1;7.miR-106a significantly inhibited the expression of HIF-1αand STAT3 in macrophages;8.Compared with the immortalized cell line HBE,miR-106a expression was significantly down-regulated in glioma cell lines;9.The expressions of HIF-1α,STAT3 and PD-L1 were down-regulated in U87and U251 cell lines when ransfected with mi R-106a mimics.The expressions of HIF-1α,STAT3 and PD-L1 were significantly up-regulated in U87 and U251 cell lines transfected with miR-106a inhibitor;10.miR-106a significantly decreased PD-L1 expression in U251 PD-L1overexpressing cell line;11.In nude mice tumorigenesis experiments,mi R-106a inhibited the growth of PD-L1 over-expressing U251 cells and reduced the expression of PD-L1;12.The expression of miR-106a in glioma specimens was significantly lower than that in adjacent normal tissues,and was negatively correlated with WHO grade;13.According to the data from clinical specimen detection,we found a significant negative correlation between the expression of miR-106a and the expressions of HIF-1α,STAT3 or PD-L1;14.In both gliomas and GBM,the mean survival of patients with high expression of miR-106a was significantly longer than that of patients with low expression of miR-106a.Summary1.mi R-106a was down-regulated both in tumor cells and TAMs in glioma samples,and regulated PD-L1 expression through direct and indirect targeting;2.To a certain extent,miR-106a can reverse PD-L1-induced immunosup-pression;3.The low expression of miR-106a was closely related to the progression of glioma,and may be a prognostic indicator of glioma and GBM.Part Ⅲ PD-L1 promotes progression of Glioma through up-regulating the expression of CCL2Methods1.Using the TCGA database,we analyzed differentially expressed genes in patients with high and low expression of PD-L1,and performed GO and KEGG analysis on highly expressed genes;2.Using Wayne and correlation analysis,we screened the downstream of PD-L1;3.Using cytokine microsphere detection technology,we screened the chemokines that regulated by PD-L1;4.Using PCR and Elisa assays,we validated the effect of PD-L1 on CCL2secretion;5.miR-106a mimics were transfected to verify the regulatory relationship of mi R-106a-PD-L1-CCL2;6.Using bioinformatics analysis,we analyzed the possible involvement biological process of CCL2 in glioma;7.We analyzed the relationship between CCL2 expression and chemotaxis and function of glioma macrophages;8.We analyzed the relationship between CCL2 expression and angiogenesis;9.We analyzed the correlation of PD-L1 and CCL2 expression in CGGA,TCGA database and tissue specimens;10.We analyzed the relationship between CCL2 expression in CGGA and TCGA databases and WHO grading,IDH mutation and molecular typing;11.We analyzed the prognostic impact of CCL2 on glioma and GBM patients in CGGA,TCGA database and tissue specimens.Results1.Highly expressed genes in PD-L1^hi glioma group were mainly enriched in immune response related biological processes and cytokine-cytokine receptor interactions related pathway,especially chemokine-related pathway;2.Bioinformatics suggested that PD-L1 may be involved in the regulation of CXCL10,CCL2,CXCL11,CCL20,CXCL9,CCL8,CXCL14,CCL5,CCL13 and CCL27;3.Cytokine microsphere detection technology results showed that anti-PD-L1significantly reduced the secretion of CCL2;4.CCL2 expressin was regulated by PD-L1 according to PCR and elisa results;5.miR-106a significantly reduced PD-L1-mediated CCL2 expression;6.Bioinformatics analysis suggested that CCL2 may be involved in immune response,chemotaxis and angiogenesis in gliomas;7.Bioinformatics analysis suggested that the expression of CCL2 in glioma was closely related to the chemotaxis,differentiation and function of monocytes;8.Bioinformatics analysis suggested that CCL2 expression was closely related to angiogenesis in gliomas;9.There was a significant positive correlation between PD-L1 and CCL2expression both in glioma and GBM;10.The expression of CCL2 in gliomas was enriched in GBM,IDH-WT gliomas and mesenchymal subtype;11.In both glioma and GBM,the average survival of patients with high expression of CCL2 was significantly lower than that of patients with low expression of CCL2.Summary1.PD-L1 can up-regulate the expression of CCL2,which in turn promote the recruitment,differentiation and function of mononcytes and macrophages,further create a microenvironment for immunosuppression,and mediate angiogenesis through CCL2,which furthur promote tumor progression.2.The high expression of CCL2 was closely related to the progression of gliomas,and may act as an indicator of prognosis of glioma and GBM.Conclusion1.Up-regulation of PD-L1 expression in gliomas was closely related to the malignant phenotype and poor prognosis of gliomas.2.miR-106a was down-regulated in both glioma tumor cells and macrophages,and promoted progression of glioma via interaction with PD-L1 through direct and indirect way;3.PD-L1 was involved in the regulation of CCL2 expression,and induced immunosuppression and tumor infiltration.It may be a new mechanism for PD-L1 to promote the progression of glioma.