Colorectal cancer metastasis requires both chromosomal instability and driver pathway mutations.
Major finding: Colorectal cancer metastasis requires both chromosomal instability and driver pathway mutations.
Approach: Human intestinal organoids were engineered to express driver gene mutations using CRISPR-Cas9.
Impact: This system enables identification of molecular lesions that contribute to colorectal carcinogenesis.
Transformation of normal colon epithelium and progression from adenoma to metastatic colorectal carcinoma has been suggested to occur via loss of the APC gene and subsequent mutations in KRAS, SMAD4, and TP53 that dysregulate driver pathways and confer a selective growth advantage to intestinal stem cells (ISC). Although these pathways are important regulators of signaling in the ISC niche, their contribution to human colorectal carcinogenesis is unknown. Matano and colleagues developed an epithelial organoid culture system in which driver gene mutations were sequentially introduced into organoids derived from normal human intestinal epithelium using CRISPR-Cas9–mediated genome editing. Isogenic epithelial organoids harboring mutations in APC, KRAS, SMAD4, TP53, and PIK3CA were selected for by modulating intestinal niche factors required for the growth of normal ISC organoids in culture, thus recapitulating the sequence of mutations in human colorectal cancer progression. Engineered organoids carrying all five mutations (referred to as AKSTP-organoids) grew independently of niche factors. Microarray analysis revealed similar gene signatures in AKSTP-organoids and organoids derived from adenomas, but not those derived from colorectal cancer, suggesting that driver pathway mutations are not sufficient for progression from adenoma to carcinoma. Introduction of driver pathway mutations in engineered AKSTP-organoids enabled tumor formation under the kidney subcapsule of immunodeficient mice and the formation of micrometastases in the spleen; however, these tumors exhibited histologic features of low-grade adenocarcinoma and impaired metastastic colonization of the liver compared with colorectal cancer organoids. Intriguingly, organoids derived from a human adenoma with a chromosomal instability phenotype that were engineered to express driver pathway mutations were capable of metastatic transformation and formed large metastatic tumors. These results suggest that driver pathway mutations contribute to niche-independent stem-cell maintenance, but that other genetic lesions, such as chromosomal instability, are required for colorectal cancer invasion and metastasis.