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.colon carcinogenesis | shRNA | primary culture | Matrigel | validation

Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer

“…Consistent with previous work (Haigis et al, 2008; Hung et al, 2010; Janssen et al, 2006; Onuma et al, 2013), mutation of Kras (G12D) strongly synergized with Apc suppression to promote rapid and widespread tumor growth in both the small and large intestine of shApc/Kras G12D /Lgr5 mice (Figure 6B, not shown), reducing median survival to 63 days (n=15) (Figure 6D). Tumors were highly proliferative and showed no evidence of differentiation or cell death (Figure 6C).…”

miR-137 Regulates the Tumorigenicity of Colon Cancer Stem Cells through the Inhibition of DCLK1

“…In addition, intestinal tumor organoids also recapitulate intestinal tumorigenesis (30). We applied this system to evaluate the function of miR-137 in a human cancer cell line, hPCCs and the murine small intestine.…”

Organoid Models of Human Gastrointestinal Development and Disease

“…In epithelial organoids generated from intestinal adenomas of APC-deficient mice, R-spondin-1 was not required for constitutive Wnt signaling 60 . Lentiviral knockdown of APC in mouse small intestine epithelial organoids confirmed Wnt activation and perturbed differentiation; these organoids generated subcutaneous tumors when injected into immunocompromised mice 149 . Activation of Kras in these organoids further increased the tumor burden 149 .…”

“…Lentiviral knockdown of APC in mouse small intestine epithelial organoids confirmed Wnt activation and perturbed differentiation; these organoids generated subcutaneous tumors when injected into immunocompromised mice 149 . Activation of Kras in these organoids further increased the tumor burden 149 .…”