| Background: Glioma is the most common primary tumor of the central nervous system,which is invasive growth.It is difficult to completely resect it.It's clinical progress is fast and the prognosis is poor.It has the characteristics of high invasiveness and destruction,high recurrence rate,and resistance to radiotherapy and chemotherapy.The mortality rate of advanced gliomas,especially Glioblastoma Multiforme(GBM),is very high,and the 5 year survival rate is not more than 5%.An important challenge for the currently treatment of glioma is the ability of tumor cells to induce local and systemic immunosuppression,which limits the effect of innate immunity on tumor defense and adaptive immunotherapy.Glioma has many ways to evade the body's immune surveillance,this immune escape process is affected by the tumor cells and the tumor microenvironment.Studies have shown that Glioma Stem Cells(GSCs)as "seeds" and tumor microenvironment as "soil" play an important role in immune escape of glioma.At present,most of researches on the cell surface molecules related to immune escape are the B7 family of co-stimulatory molecules,of which Programmed Death Ligand 1(PD-L1)is one of the current hot topics.In glioma tissue,GSCs,Tumor-associated macrophages(TAMs),which are the main cells in tumor microenvironment,and the negative co-stimulatory molecule PD-L1 exist at the same time,previous reports have only studied the expression of negative co-stimulatory molecule PD-L1 in each of the two kinds of cells,but the study of the three in the immune escape mechanism of glioma are rare reported at home and abroad.Objective: This study will explore the expression of GSCs,TAMs and PD-L1 in different pathological grades of glioma at mRNA and protein levels and the correlation between them,and use the negative co-stimulatory molecule PD-L1 as a bridge to explore the relationship between GSCs and TAMs in the immune escape behavior of glioma and the possible therapeutic targets.Methods: Taking CD133 as a marker of glioma stem cells and CD68 as a marker of tumor-associated macrophages.The co-expression of CD68 and PD-L1 protein in different pathological grades of glioma was detected by double immunofluorescence staining method.Immunohistochemistry was used to detect the expression of CD133,CD68 and PD-L1 protein and the correlation between them in different pathological grades of glioma at protein level.Quantitative real-time fluorescent PCR(qRT-PCR)was used to detect the expression of CD133,CD68 and PD-L1 genes and the correlation between them in different pathological grades of glioma at mRNA level.Results:1.Double immunofluorescence staining results showed that all glioma tissues have the expression of CD68 and PD-L1 proteins,which are mainly located in cell membrane and cytoplasm;double staining results showed that most of the PD-L1 molecules are expressed on TAMs,the majority of PD-L1 positive cells were tumor-associated macrophages;with the increase of pathological grade of glioma,the co-expression of CD68 and PD-L1 protein increased.2.The results of immunohistochemistry showed that all glioma tissues had the expression of CD133,CD68,and PD-L1 proteins,which were mainly expressed in the cytoplasm,and were slightly expressed on the cell membrane,mostly yellow or brown-yellow particles;with the increase of glioma pathological grade,the expression levels of CD133,CD68,and PD-L1 proteins gradually increased.Spearman rank correlation analysis showed that the expression levels of CD133,CD68,and PD-L1 proteins in glioma tissues positively correlated with pathological grade(r=0.831,P<0.001;r=0.795,P<0.001;r=0.711,P<0.001);the levels of CD133,CD68 and PD-L1 proteins in high grade glioma tissues were significantly higher than those in low grade glioma tissues(t=13.854,P<0.001;t=12.838,P<0.001;t=6.794,P<0.001);spearman rank correlation analysis showed that the expression of CD133 protein was positively correlated with CD68 protein(r=0.881,P<0.001),there was a positive correlation between CD133 and CD68 in the low-grade group(r=0.425,P=0.024),and a positive correlation between CD133 and CD68 in the high-grade group(r=0.620,P<0.001);the expression of CD133 protein was positively correlated with PD-L1 protein(r=0.753,P<0.001),there was a positive correlation between CD133 and PD-L1 in the low-grade group(r=0.432,P=0.022),and a positive correlation between CD133 and PD-L1 in the high-grade group(r=0.407,P=0.031).3.The results of real-time fluorescence quantitative PCR showed that as the pathological grade of glioma increased,the expression levels of CD133,CD68,and PD-L1 mRNAs gradually increased.Spearman rank correlation analysis showed that in glioma tissues,the expression levels of CD133,CD68 and PD-L1 mRNAs was positively correlated with pathological grades(r=0.647,P<0.001;r=0.499,P<0.001;r=0.445,P=0.001);The two-sample comparison of rank sum tests showed that the expression level of CD133,CD68 and PD-L1 mRNAs in high grade glioma tissues was significantly higher than that in low-grade glioma tissues(z=6.424,P<0.001;z=3.556,P<0.001;z=2.999,P=0.003);Spearman rank correlation analysis showed that there was a positive correlation between the expression of CD133 mRNA and CD68 mRNA in glioma tissue(r=0.525,P<0.001),there was a positive correlation between CD133 and CD68 mRNAs in the low-grade group(r=0.518,P=0.005),and a positive correlation between CD133 and CD68 mRNAs in the high-grade group(r=0.500,P=0.007);The expression of CD133 mRNA was positively correlated with PD-L1 mRNA(r=0.431,P<0.001),there was a positive correlation between CD133 and PD-L1 mRNAs in the low-grade group(r=0.398,P=0.036),and a positive correlation between CD133 and PD-L1 mRNAs in the high-grade group(r=0.417,P=0.027).Conclusion: 1.The negative co-stimulatory molecule PD-L1 in glioma tissues is mainly expressed by tumor-associated macrophages in the tumor microenvironment.2.CD133,CD68 and PD-L1 are closely related to the malignant degree of glioma;GSCs are positively correlated with TAMs expressing negative costimulatory molecule PD-L1. |
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