Table_7_CX3CR1 Acts as a Protective Biomarker in the Tumor Microenvironment of Colorectal Cancer.xlsx

The tumor microenvironment (TME) plays an important role in the pathogenesis of many cancers. We aimed to screen the TME-related hub genes of colorectal adenoma (CRAD) and identify possible prognostic biomarkers. The gene expression profiles and clinical data of 464 CRAD patients in The Cancer Genome Atlas (TCGA) database were downloaded. The Estimation of STromal and Immune cells in MAlignant Tumours using Expression data (ESTIMATE) algorithm was performed to calculate the ImmuneScore, StromalScore, and EstimateScore. Thereafter, differentially expressed genes (DEGs) were screened. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein–protein interaction (PPI) analysis were performed to explore the roles of DEGs. Furthermore, univariate and multivariate Cox analyses were accomplished to identify independent prognostic factors of CRAD. CX3CR1 was selected as a hub gene, and the expression was confirmed in colorectal cancer (CRC) patients and cell lines. The correlations between CX3CR1 and tumor-infiltrating immune cells were estimated by Tumor IMmune Estimation Resource database (TIMER) and CIBERSORT analysis. Besides, we investigated the effects of coculture with THP-1-derived macrophages with HCT8 cells with low CX3CR1 expression on immune marker expression, cell viability, and migration. There were significant differences in the ImmuneScore and EstimateScore among different stages. Patients with low scores presented significantly lower lifetimes than those in the high-score group. Moreover, we recognized 1,578 intersection genes in ImmuneScore and StromalScore, and these genes were mainly enriched in numerous immune-related biological processes. CX3CR1 was found to be associated with immune cell infiltration levels, immune marker expression, and macrophage polarization. Simultaneous silencing of CX3CR1 and coculture with THP-1 cells further regulated macrophage polarization and promoted the cell proliferation and migration of CRC cells. CX3CR1 was decreased in CRAD tissues and cell lines and was related to T and N stages, tumor differentiation, and prognosis. Our results suggest that CX3CR1 contributes to the recruitment and regulation of immune-infiltrating cells and macrophage polarization in CRC and TAM-induced CRC progression. CX3CR1 may act as a prognostic biomarker in CRC.肿瘤微环境（tumor microenvironment, TME）在多种癌症的发病机制中发挥关键作用。本研究旨在筛选结直肠腺瘤（colorectal adenoma, CRAD）的肿瘤微环境相关核心基因，并鉴定潜在的预后生物标志物。我们从癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库中下载了464例结直肠腺瘤患者的基因表达谱与临床数据。采用基于表达数据的恶性肿瘤间质与免疫细胞估算（Estimation of STromal and Immune cells in MAlignant Tumours using Expression data, ESTIMATE）算法计算免疫评分（ImmuneScore）、间质评分（StromalScore）与估算评分（EstimateScore）。随后筛选差异表达基因（differentially expressed genes, DEGs），并通过基因本体论（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析以及蛋白质-蛋白质相互作用（protein–protein interaction, PPI）分析探究差异表达基因的功能。进一步通过单因素与多因素Cox回归分析，鉴定结直肠腺瘤的独立预后因素。选取CX3CR1作为核心基因，并在结直肠癌（colorectal cancer, CRC）患者及细胞系中验证其表达水平。通过肿瘤免疫估算资源（Tumor IMmune Estimation Resource, TIMER）数据库与CIBERSORT分析评估CX3CR1与肿瘤浸润免疫细胞的相关性。此外，本研究探究了低表达CX3CR1的HCT8细胞与THP-1源性巨噬细胞共培养对免疫标志物表达、细胞活力及迁移能力的影响。不同分期患者的免疫评分与估算评分存在显著差异，低评分组患者的生存期显著短于高评分组患者。本研究共筛选得到免疫评分与间质评分的交集基因1578个，这些基因主要富集于众多免疫相关生物学过程。研究发现CX3CR1与免疫细胞浸润水平、免疫标志物表达及巨噬细胞极化密切相关。同时沉默CX3CR1并与THP-1细胞共培养，可进一步调控巨噬细胞极化并促进结直肠癌细胞的增殖与迁移。CX3CR1在结直肠腺瘤组织与细胞系中表达下调，且与T分期、N分期、肿瘤分化程度及预后相关。本研究结果表明，CX3CR1参与了结直肠癌中免疫浸润细胞的招募与调控以及巨噬细胞极化，并介导了肿瘤相关巨噬细胞（tumor-associated macrophage, TAM）诱导的结直肠癌进展。CX3CR1可作为结直肠癌的潜在预后生物标志物。