Microbiome-triggered reprogramming and mutation of colon epithelial cells leading to tumor stem-like cells

结直肠癌是第三大癌症原因，超过一半的病例发生在 很难治愈的晚期疾病需要新的预防战略。结肠 微生物组在结直肠癌（CRC）的病因学中起重要作用。本实验室 在炎症相关CRC模型中研究了粪肠球菌。这个人类 海藻酸钠可以刺激（或激活）巨噬细胞重新编程和转化上皮细胞 转化成癌细胞这个过程被称为“微生物组诱导的旁观者效应”。在 CRC，肿瘤干细胞样细胞被认为起源于隐窝基部的干细胞。 然而，新的数据支持在隐窝顶部的完全分化的上皮细胞作为上皮细胞。 CRC起源。这符合人类结肠腺瘤形态发生的“自上而下”模型 其中转化的干细胞样细胞向下扩展以取代正常的隐窝结构。在这 项目，我们假设微生物极化的结肠巨噬细胞引起诱变， 隐窝上部分化上皮细胞向多能性的重编程 和干性，从而推动恶性转化。使用已建立的体外和体内 微生物群触发和巨噬细胞诱导的恶性转化模型，我们将 评估分化的结肠上皮细胞在转化过程中的重编程和突变， 转化为肿瘤干细胞。关键驱动基因的突变沿着全基因组 将评估染色体、基因表达和甲基化的变化，以确定 重编程和转化过程中的事件序列。最后，恶性转化 通过类器官培养和以免疫缺陷为特征的同种异体移植来富集克隆。 小鼠益生菌（乳酸杆菌和双歧杆菌）对微生物组的预防作用- 还将评估诱导的旁观者效应。使用这些相同的体外和体内模型， 肿瘤坏死因子α作为微生物组的关键介质的信号作用- 将评估诱导的旁观者效应。我们假设阻断肿瘤受体 坏死因子α将导致功能性肿瘤干细胞样细胞标志物的下调， 双皮质素样激酶1，从而防止微生物组驱动的结肠癌发生。这些 实验将有助于确定肠上皮细胞如何刺激先天免疫细胞， 产生旁观者效应，从而诱导诱变、重编程和转化 结肠上皮细胞。最后，我们将研究益生菌是否可以抑制微生物组- 驱动的致癌作用，从而支持这作为CRC预防的方法。

Colorectal cancer is the third leading cause of cancer with more than half of cases occurring in advanced stages that are difficult to cure. New prevention strategies are needed. The colonic microbiome plays an important role in the etiology of colorectal cancer (CRC). Our laboratory has studied Enterococcus faecalis in models of inflammation-associated CRC. This human commensal can polarize (or activate) macrophages to reprogram and transform epithelial cells into cancer cells. This process is referred to as the “microbiome-induced bystander effect.” In CRC, tumor stem-like cells are believed to originate from stem cells at the base of crypts. However, newer data supports fully differentiated epithelial cells at the top of crypts as cells of origin for CRC. This fits with a “top-down” model of morphogenesis for human colon adenomas in which transformed stem-like cells expand downward to replace normal crypt structures. In this project, we hypothesize that microbiome-polarized colon macrophages cause mutagenesis and reprogramming of differentiated epithelial cells in the upper portion of crypts toward pluripotency and stemness, thereby driving malignant transformation. Using established in vitro and in vivo models for microbiome-triggered and macrophage-induced malignant transformation, we will assess differentiated colon epithelial cells for reprogramming and mutation during transfor- mation into tumor stem-like cells. Mutations in key driver genes along with genome-wide changes in chromosomes, gene expression, and methylation will be assessed to establish the sequence of events during reprogramming and transformation. Finally, transformed malignant clones will be enriched by organoid culture and allografts characterized in immunodeficient mice. The preventive effect of probiotics (Lactobacillus and Bifidobacterium) on a microbiome- induced bystander effect will also be assessed. Using these same in vitro and in vivo models, the role of signaling by tumor necrosis factor alpha as a key mediator for the microbiome- induced bystander effect will be assessed. We hypothesize that blocking a receptor for tumor necrosis factor alpha will lead to downregulation of a functional tumor stem-like cell marker, doublecortin-like kinase 1, and thereby prevent microbiome-driven colon carcinogenesis. These experiments will help determine how intestinal commensals polarize innate immune cells to produce bystander effects and thereby induce mutagenesis, reprogramming and transformation of colon epithelial cells. Finally, we will investigate whether probiotics can inhibit microbiome- driven carcinogenesis and thereby bolster this as an approach to CRC prevention.