Sequential cancer mutations in cultured human intestinal stem cells

Abstract

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.

Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids

“…As described, Triple SMAD4WT organoids consisted of multilayered disorganized epithelium, whereas quadruple mutant organoids mainly appeared as disorganized solid masses (SI Appendix, Fig. S2A) (7). Cell cycle analysis did not reveal major differences in proliferation rate among Triple APCWT , Triple SMAD4WT , Triple KRASWT , and quadruple mutant organoids in vitro (SI Appendix, Fig.…”

“…Importantly, the observation that cell growth depends on the deletion of P53 has consequences for the order of occurrence of mutations during the adenoma-carcinoma sequence. We and others (7,11) have demonstrated that loss of P53 function in intestinal stem cells induces chromosome instability (CIN). Because CIN can only lead to genetic alterations when cells divide (7,10), our data indicates that P53 is a gatekeeper that prevents acquisition of additional mutations.…”

“…Human intestinal stem cells can be grown "indefinitely" in vitro as 3D organoids in medium containing the stem cell niche factors Wnt, R-spondin, EGF, and Noggin while remaining genetically stable (5,6). Previously, we used CRISPR/Cas9 genome editing to engineer human colon organoids harboring mutations in four of the most frequently mutated pathways in CRC (Wnt, EGFR, P53, and TGF-β), i.e., mutations in APC [APC knock out (APC KO )], KRAS (KRAS G12D ), P53 (P53 KO ), and SMAD4 (SMAD4 KO ) (7), from here referred to as quadruple mutant organoids. Mutants were functionally selected by changing the medium composition.…”

Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models

“…Genome editing studies validating the contribution of recurrent mutations to colorectal carcinogenesis suggested that aneuploidy, as the gain of chromosomes such as 7, 13, and 20, may be associated with aggressive tumor phenotypes [97,98]. Direct confirmation of colorectal cancer exacerbation can be achieved by generating aneuploidy models by MMCT of such human chromosomes.…”

CIN and Aneuploidy: Different Concepts, Different Consequences

“…[19] Indeed, CIN has also been induced and observed in organoids, [20] but so far, side by side comparisons of CIN rates between 2D and 3D cultures in an isogenic background are still lacking.…”