Figure 1.

A, Overview of the cell‐based systems applicable to modeling of cancer driver events. Phenotypic progression for two barrier bypass‐clonal expansion (BBCE) systems (primary human p53 knock‐in [Hupki] mouse embryonic fibroblasts [MEF] and human mammary epithelial cells [HMEC]) are shown in the left and middle columns, respectively. A general schematic describing the single‐cell subcloning‐clonal expansion (scsCE) approach is depicted on the right. The immortalized cell lines used in the scsCE approach are derived from the lung (A549, BEAS‐2B), liver (HepaRG and HepG2), kidney (HK‐2) and breast (MCF10A). Other cell lines can be explored and developed for the scsCE approach. Treatment of the starting cell cultures with mutagenic carcinogens can be conducted as a one‐time, acute exposure (single red triangles, top BBCE panels), or in a chronic, repeated method (typically for 8 weeks; multiple red triangles, top scsCE panel). The resulting clonal populations (bottom panels) are subject to high‐throughput molecular and functional phenotypic analyses. AIG, anchorage‐independent growth; NGS, next‐generation sequencing. B, Cancer gene mutations in cell‐based models of carcinogen‐induced transformation. A comprehensive cancer gene list was curated from5 and8 Number shown for each exposure condition and clone indicates the number of mutations in known cancer genes. A statistically significant difference in the number of mutated cancer genes was observed between BBCE and scsCE assays (P < .0001, Mann‐Whitney U test). AA, aristolochic acid; AFB1, aflatoxin B1; AID, activation‐induced cytidine deaminase; BaP, benzo[a]pyrene; C; Cis, cisplatin; MNNG, N‐methyl‐N'‐nitro‐N‐nitrosoguanidine; Spont, spontaneous; UVC, ultraviolet light type C. *Presumed barrier bypass based on clonal outgrowth following chronic AA exposure of immortal cell lines35