Abstract 1404: Bacterial infection-induced epithelial-to-mesenchymal transition (EMT) of colonic crypt cells with acquired characteristics of stem cells promotes spheroid/organoid formation in vitro and tumorigenesis in vivo

Abstract

Abstract DCLK-1 (doublecortin-like kinase-1) and LGR5 (leucine-rich-repeat-containing G-protein-coupled receptor) have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. The epithelial-to-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We aimed at delineating if bacterial infection affects colonic stemness and transdifferentiation and whether these changes facilitate mucosal priming for neoplasia. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model associated with hyperactive Wnt/β-catenin, Notch and NF-κB pathways in NIH-Swiss mice, we observed dramatic loss of DCLK-1 expression in the distal colonic crypts with significant shift towards stromal staining at peak (12-days post-infection) hyperplasia while staining for both Lgr-5 and Msi-1 increased several fold. When hyperplasia was regressing (at day 34), we observed a dramatic expansion of DCLK-1+ve cells compared to that seen in uninfected control. Colonic crypt cells when isolated from CR-infected mice at 12 days’ post-CR infection and cultured in vitro, formed monolayers, converted to mesenchymal cells, stained positive for vimentin and fibronectin but negative for E-cadherin and cytokeratin suggesting a process of EMT, and also stained positive for DCLK-1 thereby mimicking stromal staining observed in vivo. Trypsinization of these cells followed by growth in soft-agar led to formation of organoids which developed into crypt-like structures (cryptoids) that stained positive for markers of epithelial lineages. Day 34 crypt cells when deposited into the soft-agar also formed spheroids with subsequent transition into organoids/cryptoids in vitro. These changes were observed in culture conditions that did not include intestinotrophic mitogens Noggin or R-spondin. Mice exhibiting 12 or 34 days of TMCH received azoxymethane once for 8h (Gp1) or twice for two weeks (Gp2) and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as spheroids in soft-agar but failed to form tumors in athymic/nude mice. Crypt cells, isolated from Gp2 animals failed to form the monolayers but developed into huge spheroids in soft-agar and formed tumors in athymic/nude mice. These studies suggest that both hyperplasia and increased stemness promote cellular transformation in response to a second hit. Thus, the investigation of a model of bacterial infection-induced EMT and/or spheroid/organoid/cryptoid formation with acquired characteristics of stem cells represents an excellent system to study transdifferentiation, crypt regeneration or colon carcinogenesis in response to bacterial infection. 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 1404. doi:1538-7445.AM2012-1404