Sequential cancer mutations in cultured human intestinal stem cells
Menée à l'aide d'une technologie de modification ciblée des gènes (CRISPR/Cas9) sur des cellules souches intestinales humaines, puis à l'aide de xénogreffes sur des modèles murins, cette étude met en évidence le rôle joué par quatre gènes mutés (APC, P53, KRAS et SMAD4) dans le développement d'un carcinome colorectal invasif
Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.