Based On Bioinformatics To Explore Clinical Value Of CTLA4 And GREM1 As An Immunogenicity Indicators In Tumor Microenvironment Of Gastric Cancer

Object:In our study,we tried to screen of prognostic genes as indicators for predicting immunogenicity in tumor microenvironment(TME)of gastric cancer(GC),and investigate immuno-molecular subtypes classification of GC that were sensitive to precise immunotherapy.Methods:(1)A total of 111 clinical cases of postoperative GC were collected,and paraffin tissue specimens were also obtained.The total 817 GC transcriptome RNA-Seq and clinical dataset were obtained from the the Cancer Genome Atlas Program(TCGA)and Gene Expression Omnibus(GEO)databases.A total of 47 immune checkpoint-related genes were obtained through the Molecular signatures database module on the Gene Set Enrichment Analysis(GSEA)online website.(2)R package’’limma’’was performed to selected differentially expressed genes(DEGs).The infiltration status of 22 immune cells and Immune/Stormal scores were calculated using the CIBERSORT algorithm and ESTIMATE.The“Consensu Cluster Plus”R-package was used to identify the three immune cell infiltration clusters for molecular classifications.CTLA4 and GREM1 expression-associated signal pathways were investigated by GSEA enrichment analysis.GEPIA database was used to verify the expression difference of target genes in408 GC tissues and 211 normal tissues.Correlations of GREM1 with immune signature molecules and drug susceptibility were investigated by TISIDB and Cell Miner database.(3)The protein expressions of CTLA4 and GERM1 in tumor and paracancerous tissues were detected by immunohistochemistry and western blot.The m RNA Expression of CTLA4,GREM1,CD8,CD68,INOS and CD163 were detected by quantificational real time-PCR(q RT-PCR).(4)Univariate and multivariate Cox regression analysis were used to determine the prognostic factors of DEGs.ROC curve was displayed to explore the clinical value of CTLA4 and GREM1 in GC patients after surgery.Results:(1)CTLA4 was regarded as the only survival-related immune checkpoint-DEGs of total 47 genes by Cox analysis.The higher Immune scores(HR=0.71,P=0.027)and Stormal scores(HR=1.51,P=0.008)were both in significant correlation with GC survival.Besides,A total of 40 shared TME-related DEGs were selected in the high and low groups of Immune score and Stromal score.And Four survival-related immuno-DEGs were obtained by Cox analysis,which were GREM1,SFRP2,CYP1B1 and MGP.GEPIA databases revealed that GREM1 and SFRP2 expression in GC was significantly higher in comparison with that in normal tissues.Compared with SFRP2,GREM1 expression was more powerfully correlated with Immune score and Stromal score.(2)It was found that the expression of CTLA4 was negatively correlated with the N and Grade,and was positively associated with the survival time of GC patients(HR=0.78,P=0.02).While the expression of GREM1 was positively correlated with Tand Grade,and was negatively associated with clinical outcomes of GC patients(HR=1.52).GREM1 was also positively associated with TGF-β1(r=0.50),ENTPD1(r=0.67),CCL14(r=0.56)as well as CCR2(r=0.38,P<0.001).Moreover,GC patients with high expression of GREM1 might more sensitive to drug Vismodegib therapy(r=0.37,P=0.004).(3)GSEA results showed that both CTLA4 and GREM1 high-expression group were mainly enriched in the immune-related active genomes.Moreover,CTLA4 expression was positively associated with CD8~+Tcell and M1 macrophages,and was negatively correlated to M2 macrophages.However,GREM1 expression was positively correlated to M2 macrophages,and was negatively CD8~+T cell and M1macrophages.(4)Three immune cell infiltration clusters for molecular classifications of GC patients of public databases were identified.Meanwhile,the results showed that patients of the immune cell infiltration clusters A with a satisfactory prognosis might have potent immunogenicity.Interestingly,the higher expression of CTLA4 and lower expression of GREM1 was found in cluster A.(5)The higher m RNA expression of CTLA4 and GREM1 of GC tissues detected by q RT-PCR,the further Western Blot and IHC showed that the expression of CTLA4 and GREM1 protein were also higher in GC tissues than in normal tissues.The m RNA/protein expression of CTLA4 were both negatively associated with T,N and Stage,but was positively correlated to a improved outcome.However,higher m RNA/protein expression of GREM1of GC patients indicated a poor prognosis,and were positively related to T,N and Stage.GC patients with a higher m RAN expression of CD8(HR=0.29,P=0.001),CD68(HR=0.30,P=0.001)and INOS(HR=0.24,P<0.001)were found to have better prognosis,while CD163(HR=5.31,P<0.001)was associated to the low survival of GC patients.CTLA4 m RNA expression was positively correlated with CD8(r=0.48,P<0.001),CD68(r=0.49,P<0.001)and INOS(r=0.43,P<0.001),and was negatively correlated with CD163(r=-0.58,P<0.001).However,GREM1 m RNA expression was negatively associated to CD8(r=-0.67,P<0.001),CD68(r=-0.66,P<0.001)and INOS(r=-0.47,P<0.001),and was positively associated with CD163(r=0.61,P<0.001).The combined indicator of CTLA4 and GREM1 has the powerful predictive potent for postoperative survival of GC patients(AUC=0.858).Conclusion:Thus,we concluded that CTLA4 and GREM1 could regard as an immunogenicity clinical indicator in TME of GC.Besides,the expression levels of CTLA4 and GREM1 in GC can be used as indicator for immune molecular classification.Moreover,patients(immuno-cluster A)with high expression of CTLA4 and low expression of GREM1 could have favorable clinical outcomes and potent.