Journal Article

Sequential cancer mutations in cultured human intestinal stem cells

van de Wetering, Marc, van Jaarsveld, Richard H., Overmeer, René M., Clevers, Hans, van Boxtel, Ruben, Kops, Geert J. P. L., Sachs, Norman, Logtenberg, Meike, Offerhaus, G. Johan, Begthel, Harry, Zimberlin, Cheryl, Drost, Jarno, Buijs, Arjan, Korving, Jeroen, Schwank, Gerald, Ponsioen, Bas, Cuppen, Edwin, Medema, Jan Paul, Snippert, Hugo J.

2015

Overview

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. Using the CRISPR/Cas9 system, up to four frequently occurring colorectal cancer mutations were introduced alone or in combination into stem cell organoids derived from human small intestinal or colon tissue, allowing an in-depth investigation of the contribution of these mutations to cancer progression. An organoid-based model of evolving tumours Using the precise genome-editing capabilities of the CRISPR/Cas9 system, Hans Clevers and colleagues have introduced four of the most common colorectal cancer mutations into human organoid cultures formed by small intestinal or colon cells. In the resulting in vitro colorectal cancer progression model, oncogenic mutation of APC , P53 , KRAS and SMAD4 removes dependency on stem-cell-niche factors and converts normal organoids into tumoroids that grow as adenocarcinomas upon xenotransplantation into mice. This system will be invaluable in the future to study the biology of human cancers and develop new therapeutic approaches.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/67/68 / Aneuploidy / Animals / Cell culture / Child / Child, Preschool / Chromosomes / Cloning / Colorectal cancer / Colorectal carcinoma / Colorectal Neoplasms - genetics / Colorectal Neoplasms - metabolism / Colorectal Neoplasms - pathology / CRISPR-Cas Systems / Female / Gene mutations / Genes, APC / Genes, p53 - genetics / Genetic aspects / Growth factors / Health aspects / Heterografts / Humanities and Social Sciences / Humans / Identification and classification / Imidazoles / Intercellular Signaling Peptides and Proteins - metabolism / Intestines - metabolism / Intestines - pathology / Mice / Middle Aged / multidisciplinary / Mutagenesis, Site-Directed / Mutation / Mutation - genetics / Neoplasm Invasiveness - genetics / Neoplasm Invasiveness - pathology / Neoplasm Transplantation / Organoids - metabolism / Organoids - pathology / Piperazines / Plasmids / Protein expression / Proteins / Proto-Oncogene Proteins p21(ras) - genetics / Proto-Oncogene Proteins p21(ras) - metabolism / Science / Smad4 Protein - deficiency / Stem Cell Niche - physiology / Stem cells / Stem Cells - metabolism / Stem Cells - pathology / Tumors