Intestinal carcinogenicity screening of environmental pollutants using organoid-based cell transformation assay

Abstract

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.

Keywords: Carcinogenicity testing; Cell transformation assay; Colorectal cancer; Environmental pollutants; Intestinal organoids.