Genetic mutations and chronic inflammation of the colon contribute to the development of colorectal cancer (CRC). Using a murine model of inflammation-induced colon tumorigenesis, we determined how genetic mutations alter colon tumor cell differentiation. Inflammation induced by enterotoxigenicBacteroides fragilis(ETBF) colonization of multiple intestinal neoplasia (MinApcΔ716/+) mice triggers loss of heterozygosity ofApccausing colon tumor formation. Here, we report that the addition ofBRAFV600Emutation (BRAFF-V600ELgr5tm1(Cre/ERT2)CleMinApcΔ716/+, BLM) or knocking outMsh2(Msh2LoxP/LoxPVil1-creMinApcΔ716/+, MSH2KO) in the Min model altered colon tumor differentiation. Using single-cell RNA sequencing, we uncovered the differences between BLM, Min, and MSH2KO tumors at a single-cell resolution. BLM tumors showed an increase in differentiated tumor epithelial cell lineages and a reduction in the tumor stem cell population. Interestingly, the tumor stem cell population of BLM tumors had revival colon stem cell characteristics with low WNT signaling and an increase in RevCSC marker gene expression. In contrast, MSH2KO tumors were characterized by an increased tumor stem cell population that had higher WNT signaling activity compared to Min tumors. Furthermore, overall BLM tumors had higher expression of transcription factors that drive differentiation, such asCdx2, than Min tumors. Using RNA velocity, we identified additional potential regulators of BLM tumor differentiation such as NDRG1. The role of CDX2 and NDRG1 as putative regulators for BLM tumor cell differentiation was verified using organoids derived from BLM tumors. Our results demonstrate the critical connections between genetic mutations and cell differentiation in inflammation-induced colon tumorigenesis. Understanding such roles will deepen our understanding of inflammation-associated colon cancer.
遗传突变与结肠慢性炎症共同促进结直肠癌的发生发展。本研究利用炎症诱导结肠肿瘤发生的小鼠模型，探究了遗传突变如何改变结肠肿瘤细胞的分化状态。产肠毒素脆弱拟杆菌定植于多发性肠腺瘤小鼠（MinApcΔ716/+）引发的炎症反应，通过诱导Apc基因杂合性缺失触发结肠肿瘤形成。本研究发现，在Min模型中引入BRAFV600E突变（BRAFF-V600ELgr5tm1(Cre/ERT2)CleMinApcΔ716/+，简称BLM）或敲除Msh2基因（Msh2LoxP/LoxPVil1-creMinApcΔ716/+，简称MSH2KO）均会改变结肠肿瘤分化特征。通过单细胞RNA测序技术，我们在单细胞分辨率水平揭示了BLM、Min和MSH2KO肿瘤的差异：BLM肿瘤表现为分化型肿瘤上皮细胞谱系增多而肿瘤干细胞群减少，其肿瘤干细胞群呈现复苏性结肠干细胞特征——WNT信号通路活性降低且复苏性结肠干细胞标志基因表达升高；相比之下，MSH2KO肿瘤以肿瘤干细胞群扩增为特征，其WNT信号通路活性较Min肿瘤显著增强。进一步分析发现，BLM肿瘤整体表达更高水平的分化驱动转录因子（如Cdx2）。通过RNA速率分析，我们鉴定出NDRG1等可能调控BLM肿瘤分化的潜在因子。利用BLM肿瘤来源的类器官模型，验证了CDX2和NDRG1作为BLM肿瘤细胞分化调控因子的功能。本研究结果揭示了炎症诱导结肠肿瘤发生过程中遗传突变与细胞分化间的关键联系，这一发现将深化对炎症相关性结肠癌发病机制的理解。
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