title: Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.
abstract: Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.
authors.affiliation: Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Matano
authors.affiliation: 1] Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan. [2] Fujii Memorial Research Institute, Otsuka Pharmaceutical Co., Ltd., Shiga, Japan.
authors.forename: Shoichi
authors.initials: S
authors.lastname: Date
authors.affiliation: Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Shimokawa
authors.affiliation: Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Takano
authors.affiliation: 1] Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan. [2] Department of Surgical Oncology, The University of Tokyo, Tokyo, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Fujii
authors.affiliation: Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ohta
authors.affiliation: Department of Surgical Oncology, The University of Tokyo, Tokyo, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Kanai
authors.affiliation: Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Sato
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D000236:Adenoma / Q000235:genetics*; D000818:Animals; D058534:Class I Phosphatidylinositol 3-Kinases; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D015179:Colorectal Neoplasms / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D017491:Genes, APC; D016158:Genes, p53; D011905:Genes, ras; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa; D051379:Mice; D009154:Mutation; D009940:Organoids; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D051901:Smad4 Protein / Q000235:genetics
chemical_list: C497796:SMAD4 protein, human; D051901:Smad4 Protein; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human