转移性结直肠癌免疫微环境及预后相关的免疫相关差异表达基因分析

Analysis of immune microenvironment and immune-related differentially expressed genes related to prognosis in metastatic colorectal cancer

目的通过生物信息学方法, 分析转移性结直肠癌原发肿瘤组织及转移灶组织的免疫微环境差异, 筛选与预后相关的转移性结直肠癌特异性免疫相关差异表达基因。方法从基因表达综合(GEO)数据库中下载结直肠癌及转移灶基因芯片数据集GSE131418, 包括莫菲特癌症中心MCC队列的样本数据517例和Consortium队列样本数据618例;从免疫学数据库和分析门户IMMPORT中下载免疫相关基因集, 包含免疫相关的基因2 483个。从癌症基因组图谱(TCGA)数据库中下载结直肠癌组织及癌旁组织的RNA测序数据共695例, 临床信息627例。采用ESTIMATE算法计算转移灶组织和原发肿瘤组织的基质细胞评分、免疫细胞评分、基质免疫总评分, 并用CIBERSORT反卷积算法对结直肠癌原发肿瘤组织及转移灶组织的22种免疫细胞浸润情况进行比较分析。筛选免疫相关差异表达基因并进行京都基因和基因组百科全书(KEGG)信号通路富集分析。根据各免疫相关差异表达基因表达水平的中位数将患者分为高、低表达组, 分别使用Kaplan-Meier法和Cox比例风险模型对免疫相关差异表达基因进行分析, 筛选两种方法结果中与预后显著相关的基因(均P<0.01), 用Cox回归进行多因素分析。比较TCGA数据库和GEO数据库GSE131418数据集中各基因在肿瘤组织和癌旁组织、原发肿瘤组织和转移灶组织中的表达差异, 并进行生存分析。结果转移性结直肠癌转移灶组织基质细胞评分、免疫细胞评分、基质免疫总评分均低于原发肿瘤组织(均P<0.001)。与原发肿瘤组织相比, 转移灶组织中活化的自然杀伤(NK)细胞、单核细胞、CD8+ T细胞、T细胞、活化的树突细胞比例增高, 而未活化的肥大细胞、未活化的树突细胞、未活化的NK细胞、活化的记忆CD4+ T细胞、M1型巨噬细胞以及中性粒细胞比例降低。转移性结直肠癌转移灶组织与原发肿瘤组织免疫相关差异表达基因289个, 其中上调基因101个, 下调基因188个。KEGG信号通路富集结果显示, 在转移性结直肠癌转移灶组织的免疫微环境中, 发生了血管内皮生长因子(VEGF)信号通路、程序性死亡受体配体1(PD-L1)表达与程序性死亡受体1(PD-1)检查点通路、辅助性T细胞(Th)1和Th2细胞分化、Th17细胞分化、NF-κB信号通路、白细胞介素17(IL-17)信号通路、趋化因子信号通路、T细胞受体信号通路、MAPK信号通路、自然杀伤细胞介导的细胞毒性等信号通路的富集。筛选出7个预后相关的免疫相关差异表达基因, 分别为ANGPTL5、FPR1、HSPA8、NR2E3、PSMD2、PSMD8、SBDS。Cox回归多因素分析结果显示, 转移性结直肠癌免疫相关差异表达基因ANGPTL5(HR=2.69, 95%CI 1.22~5.92, P<0.05)、HSPA8(HR=0.57, 95%CI 0.33~0.97, P<0.05)、SBDS(HR=2.23, 95%CI 1.18~4.21, P<0.05)是转移性结直肠癌独立的预后影响因素。ANGPTL5在肿瘤组织中表达低于正常组织, 在转移灶组织中的表达高于原发肿瘤组织, 而肿瘤组织高表达ANGPTL5的患者预后更差。HSPA8在肿瘤组织中表达高于正常组织, 在转移灶组织中的表达低于原发肿瘤组织, 而肿瘤组织高表达HSPA8的患者预后更好。SBDS在肿瘤组织中表达低于正常组织, 在转移灶组织中的表达低于原发肿瘤组织, 而肿瘤组织高表达SBDS的患者预后更差。结论转移性结直肠癌的免疫微环境与原发肿瘤相比存在较大差异, 其免疫细胞浸润程度降低, 整体处于免疫抑制状态, 筛选出预后相关的转移性结直肠癌特异性免疫相关差异表达基因可能是新的转移性结直肠癌治疗靶点。

Objective To analyze the difference in immune microenvironment between primary tumor tissues and metastatic tumor tissues of metastatic colorectal cancer,and to screen specific immune-related differentially expressed genes(DEG)related to prognosis of metastatic colorectal cancer via bioinformatics methods.Methods The GSE131418 microarray dataset of colorectal cancer and metastases was downloaded from gene expression omnibus(GEO)database,including 517 samples from the MCC cohort and 618 samples from the Consortium cohort in Moffitt Cancer Center.Immune-related gene sets were downloaded from immunology database and analysis portal IMMPORT,including 2483 immune-related genes.A total of 695 cases of RNA sequencing data and 627 cases of clinical information of colorectal cancer tumors and adjacent tissues were downloaded from Cancer Genome Atlas(TCGA)data.The stroma cell score,immune cell score and stromal immune total score of metastatic tumor tissues and primary tumor tissues were calculated by using ESTIMATE algorithm,and 22 kinds of immune cell infiltration in primary tumor and metastatic tumor tissues of colorectal cancer were compared and analyzed by using CIBERSORT deconvolution algorithm.Immune-related DEG were screened to make Kyoto Encyclopedia of Genes and Gnomes(KEGG)signaling pathway enrichment analysis.The patients were divided into high and low expression groups according to the median expression levels of immune-related DEG.The Kaplan-Meier method and Cox regression risk model were used to analyze immune-related DEG,and the genes significantly related to prognosis in the results of the two methods were screened(all P<0.01),and multivariate analysis was performed by using Cox regression method.The expression differences of each gene in tumor tissues,adjacent tissues,primary tumor tissues and metastatic tissues in GSE131418 data sets of TCGA database and GEO database were compared,and survival analysis was also performed.Results The stroma cell score,immune cell score and stromal immune total score of colorectal cancer metastatic tissues were lower than those of primary tumor tissues(all P<0.001).Compared with primary tumor tissues,the proportion of activated natural killer(NK)cells,monocytes,CD8+T cells,T cells,activated dendritic cells in metastatic colorectal cancer tissues was increased,while the proportion of inactive mast cells,inactive dendritic cells,inactive NK cells,activated memory CD4+T cells,M1 macrophages,and neutrophils was decreased.There were 289 immune-related DEG in metastatic tissues and primary tumor tissues of metastatic colorectal cancer,including 101 up-regulated genes and 188 down-regulated genes.KEGG signaling pathway enrichment analysis showed that in the immune microenvironment of metastatic tissues in metastatic colorectal cancer,vascular endothelial growth factor(VEGF)signaling pathway,programmed death ligand 1(PD-L1)expression and programmed death 1(PD-1)checkpoint pathway,T helper cell(Th)1,Th2 and Th17 cell differentiation,NF-kappa B signaling pathway,interleukin 17(IL-17)signaling pathway,chemokine signaling pathway,T cell receptor signaling pathway,MAPK signaling pathway,and NK cell-mediated cytotoxicity pathways enrichment were detected.Immune-related DEG related to prognosis including ANGPTL5,FPR1,HSPA8,NR2E3,PSMD2,PSMD8 and SBDS were screened out.Cox regression multivariate analysis showed that immune-related DEG ANGPTL5(HR=2.69,95%CI 1.22-5.92,P<0.05),HSPA8(HR=0.57,95%CI 0.33-0.97,P<0.05),and SBDS(HR=2.23,95%CI 1.18-4.21,P<0.05)were independent prognostic factors for metastatic colorectal cancer.The expression of ANGPTL5 in tumor tissues was lower than that in normal tissues,and the expression of ANGPTL5 in metastatic tissues was higher than that in primary tumor tissues.Patients with high expression of ANGPTL5 in tumor tissues had worse prognosis.The expression of HSPA8 in tumor tissues was higher than that in normal tissues,and the expression of HSPA8 in metastatic tissues was lower than that in primary tumor tissues.Patients with high expression of HSPA8 in tumor tissues had a better prognosis.The expression of SBDS in tumor tissues was lower than that in normal tissues,and the expression of SBDS in metastatic tissues was lower than that in primary tumor tissues.Patients with high expression of SBDS in tumor tissues had worse prognosis.Conclusions Immune microenvironment of metastatic colorectal cancer is quite different from that of primary tumor.The degree of immune cell infiltration is reduced and the whole is immunosuppressed.The specific immune-related DEG related to prognosis of metastatic colorectal cancer may be new therapeutic targets of metastatic colorectal cancer.