Abstract 3301: Multiple cytokeratin-19 positive stem cells contribute to the clonal origin of colonic tumors in mice

Abstract

Abstract Introduction: The clonal origin of tumors is not well understood. Previous studies using chimeric mice have suggested that the origin of tumors in the mammalian small intestine may be polyclonal, with 2 or more progenitor cells contributing to intestinal adenomas. In the intestine, tissue stem cells are believed to be present in small numbers, and either quiescent or actively dividing, as previously shown with Lgr-5+ cells. Cytokeratin 19 (K19) is a potential progenitor marker expressed in the intestinal isthmus. Using the K19 gene promoter to drive Cre recombinase expression, we tracked the lineage of K19+ cells in an inducible fashion. Here, we used K19-CreERT2;ROSA26rLacZ mice to examine whether K19+ cells contribute to colonic tumor formation. Methods: In order to recapitulate the endogenous expression pattern of K19, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the K19 promoter (K19-BAC-Cre-ERT2). K19CreERT2 mice were crossed to ROSA26rLacZ (or GFP) reporter mice and treated with tamoxifen (6 mg/ day p.o. for 3 days) to assess K19+ lineage tracing by X-gal or GFP staining. To examine the contribution of the K19+ cells to colonic tumorigenesis, we used the AOM/DSS inflammation-associated model of colonic carcinogenesis. Mice were sacrificed 20 weeks following AOM induction and X-gal staining performed to stain for the K19+ cell lineage. Results: Treatment of control K19CreERT2/Rosa26r mice with tamoxifen resulted in the stochastic labelling of about 20-50% of colonic and intestinal glands, respectively. Consistent with the labeling of stem cells, marked glands stained positive beyond 52 weeks following tamoxifen induction. K19CreERT2/Rosa26r mice treated with AOM or DSS alone following tamoxifen labelling of K19+ glands displayed evidence of K19+ cell expansion and crypt fission, as determined by increased labelling of contiguous X-gal positive glands. K19CreERT2/Rosa26r mice were then treated with a single dose of AOM followed by DSS. When K19+ cells were labelled by tamoxifen administration prior to AOM, we observed that ∼10% tumors were entirely X-gal positive, suggesting derivation from a single K19+ cell. Interestingly, when K19+ cells were labelled by tamoxifen shortly after tumor initiation with AOM, some tumors were derived from both K19 recombined and non-recombined cells in a clonal fashion, suggesting a model whereby a K19+ cell gives rise to multiple K19+ daughter cells that each contribute to the tumor. Conclusions. Using K19CreERT2/Rosa26r mice, we show that K19 marks stem cells within the intestinal isthmus of the small intestine and colon. Using a carcinogen induced model of inflammation-associated carcinogenesis, we demonstrate that K19 expressing cells contribute to tumor formation in a manner consistent with tumor initiation following division of K19+ cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3301. doi:1538-7445.AM2012-3301