title: Genetic reconstitution of tumorigenesis in primary intestinal cells.
abstract: Animal models for human colorectal cancer recapitulate multistep carcinogenesis that is typically initiated by activation of the Wnt pathway. Although potential roles of both genetic and environmental modifiers have been extensively investigated in vivo, it remains elusive whether epithelial cells definitely require interaction with stromal cells or microflora for tumor development. Here we show that tumor development could be simply induced independently of intestinal microenvironment, even with WT murine primary intestinal cells alone. We developed an efficient method for lentiviral transduction of intestinal organoids in 3D culture. Despite seemingly antiproliferative effects by knockdown of adenomatous polyposis coli (APC), we managed to reproducibly induce APC-inactivated intestinal organoids. As predicted, these organoids were constitutively active in the Wnt signaling pathway and proved tumorigenic when injected into nude mice, yielding highly proliferative tubular epithelial glands accompanied by prominent stromal tissue. Consistent with cellular transformation, tumor-derived epithelial cells acquired sphere formation potential, gave rise to secondary tumors on retransplantation, and highly expressed cancer stem cell markers. Inactivation of p53 or phosphatase and tensin homolog deleted from chromosome 10, or activation of Kras, promoted tumor development only in the context of APC suppression, consistent with earlier genetic studies. These findings clearly indicated that genetic cooperation for intestinal tumorigenesis could be essentially recapitulated in intestinal organoids without generating gene-modified mice. Taken together, this in vitro model for colon cancer described herein could potentially provide unique opportunities for carcinogenesis studies by serving as a substitute or complement to the currently standard approaches.
authors.affiliation: Division of Cancer Development System, National Cancer Center Research Institute, Tokyo 104-0045, Japan.
authors.forename: Kunishige
authors.initials: K
authors.lastname: Onuma
authors.forename: Masako
authors.initials: M
authors.lastname: Ochiai
authors.forename: Kaoru
authors.initials: K
authors.lastname: Orihashi
authors.forename: Mami
authors.initials: M
authors.lastname: Takahashi
authors.forename: Toshio
authors.initials: T
authors.lastname: Imai
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakagama
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hippo
mesh_terms: D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000473:pathology*; D015179:Colorectal Neoplasms / Q000209:etiology* / Q000235:genetics* / Q000473:pathology; D055785:Gene Knockdown Techniques; D017491:Genes, APC; D016158:Genes, p53; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D034741:RNA, Small Interfering / Q000235:genetics; D014161:Transduction, Genetic; D060449:Wnt Signaling Pathway
chemical_list: D034741:RNA, Small Interfering
keywords: Matrigel; colon carcinogenesis; primary culture; shRNA; validation