The high incidence of colorectal cancer (CRC) is closely associated with environmental pollutant exposure. To identify potential intestinal carcinogens, we developed a cell transformation assay (CTA) using mouse adult stem cell-derived intestinal organoids (mASC-IOs) and assessed the transformation potential on 14 representative chemicals, including Cd, iPb, Cr-VI, iAs-III, Zn, Cu, PFOS, BPA, MEHP, AOM, DMH, MNNG, aspirin, and metformin. We optimized the experimental protocol based on cytotoxicity, amplification, and colony formation of chemical-treated mASC-IOs. In addition, we assessed the accuracy of in vitro study and the human tumor relevance through characterizing interdependence between cell–cell and cell–matrix adhesions, tumorigenicity, pathological feature of subcutaneous tumors, and CRC-related molecular signatures. Remarkably, the results of cell transformation in 14 chemicals showed a strong concordance with epidemiological findings (8/10) and in vivo mouse studies (12/14). In addition, we found that the increase in anchorage-independent growth was positively correlated with the tumorigenicity of tested chemicals. Through analyzing the dose–response relationship of anchorage-independent growth by benchmark dose (BMD) modeling, the potent intestinal carcinogens were identified, with their carcinogenic potency ranked from high to low as AOM, Cd, MEHP, Cr-VI, iAs-III, and DMH. Importantly, the activity of chemical-transformed mASC-IOs was associated with the degree of cellular differentiation of subcutaneous tumors, altered transcription of oncogenic genes, and activated pathways related to CRC development, including Apc, Trp53, Kras, Pik3ca, Smad4 genes, as well as WNT and BMP signaling pathways. Taken together, we successfully developed a mASC-IO-based CTA, which might serve as a potential alternative for intestinal carcinogenicity screening of chemicals.

结直肠癌（CRC）的高发病率与环境污染物暴露密切相关。为了识别潜在的肠道致癌物，我们使用小鼠成体干细胞来源的肠道类器官 (mASC-IO) 开发了一种细胞转化测定 (CTA)，并评估了 14 种代表性化学物质的转化潜力，包括 Cd、iPb、Cr-VI、iAs- III、Zn、Cu、PFOS、BPA、MEHP、AOM、DMH、MNNG、阿司匹林和二甲双胍。我们根据化学处理的 mASC-IO 的细胞毒性、扩增和集落形成优化了实验方案。此外，我们通过表征细胞-细胞和细胞-基质粘附之间的相互依赖性、致瘤性、皮下肿瘤的病理特征和CRC相关分子特征来评估体外研究的准确性和人类肿瘤的相关性。值得注意的是，14 种化学物质的细胞转化结果与流行病学发现 (8/10) 和小鼠体内研究 (12/14) 高度一致。此外，我们发现锚定非依赖性生长的增加与测试化学品的致瘤性呈正相关。通过基准剂量（BMD）模型分析锚定非依赖性生长的剂量反应关系，鉴定出强效肠道致癌物，其致癌效力从高到低依次为AOM、Cd、MEHP、Cr-VI、iAs-III和DMH。重要的是，化学转化的 mASC-IO 的活性与皮下肿瘤的细胞分化程度、致癌基因转录的改变以及与 CRC 发展相关的激活途径相关，包括 Apc、Trp53、Kras、Pik3ca、Smad4 基因，如以及 WNT 和 BMP 信号通路。总而言之，我们成功开发了基于 mASC-IO 的 CTA，它可能作为化学品肠道致癌性筛选的潜在替代方案。