Utilization of murine colonoscopy for orthotopic implantation of colorectal cancer

Abstract

Background: Colorectal-cancer (CRC) research has greatly benefited from the availability of small animal tumor models. Spontaneous and chemically-induced CRC models are widely used yet limited in their resemblance to human disease and are often prolonged, not accurately repetitive, and associated with inflammatory side effects. In-situ murine or human tumor implantation in the gastrointestinal tract of mice is extremely challenging, and limited by inter-animal variability and procedure-related complications and mortality. As a result, in frequent studies CRC is implanted in distal sites, most commonly the subcutaneous region, an approach that is greatly limited by the absence of normal gastrointestinal tumor milieu and has substantial effects on tumor development.

Aims: In this study we aimed to develop a well-tolerated repetitive tool to study CRC in small animals by adapting the murine colonoscopy system to serve as a platform for colonic sub-mucosal orthotopic implantation of human and murine CRC tumor cells.

Results: We report the establishment of a novel small-animal CRC model that is minimally invasive, rapid, well-tolerated, highly reproducible, and confers precise control of tumor number, location and growth rate. Moreover, we show that this model uniquely allows the side-by-side induction of distinct genetically manipulated tumors, enabling the mechanistic study of tumor interaction and cross-talk within the native intestinal microenvironment.

Conclusions: Employment of this new approach may represent a major technical advance for the in-vivo study of CRC.

Figure 1. Orthotopic induction of CRC using the mouse colonoscopy system.

(A) Colonoscopy image presenting colonic sub-mucosal injection of India-ink dye in C57BL/6 mouse for the assessment of the appropriate injection volume and its distribution at the colonic lamina propria. (B) Representative colonoscopy images indicating the progressive development of CRC tumor within the same C57BL/6 mouse following orthotopic implantation of 1×10⁵ MC38 CRC cells from day 5, through day 12 to day 19. (C–D) Representative image of histology specimen stained with hematoxyline and eosin (H&E) and isolated from colonic injection site at day 14 following injection of 1×10⁵ MC38 CRC cells. Magnifications: C- [x40], D – [x100]. Note the boundary area between the tumor and the adjacent normal mucosa and the projection of the tumor through the epithelial layer into the lumen. (E) Representative image showing H&E staining of histology specimen of murine-CRC tumor generated 5 days following orthotopic implantation of MC38 CRC tumor cells into C57BL/6 mice (Magnification x40). (F) Fluorescent microscopic image showing GFP labeled tumor cells and their dissemination through the epithelial layer toward the lumen (Magnification x100). Data are representative of three independent experiments.

Figure 2. Absolute control on growth kinetics of orthotopically induced tumors.

(A) Representative colonoscopy images demonstrating the correlation between amount of injected MC38 tumor cells and the size of the established CRC tumor 3 weeks following orthotopic tumor cell implantation. (B) Graphs summarizing the growth kinetics, circumference percentage and developmental grade of colonic tumor in correlation to the amount of injected MC38 CRC cells. Note the rapid establishment of grade 5 tumors already 2 weeks following implantation of only 1×10⁵ MC38-CRC cells. Each group consisted of 5 mice.

Figure 3. Orthotopic induction of distinct genetically manipulated CRC tumors.

(A) Color-coded representative image of whole body bioluminescence optical imaging of BALB/c mice 2 weeks following sub-mucosal injection of PBS^-/- (left mouse) or 1×10⁵ CT26 CRC tumor cells expressing luciferase and 10 minutes following i.p injection of D-Luciferin. Note the luminescence emission specifically from the area of injected CRC tumor cells (right mouse). (B) Upper left panel – fluorescent stereo-microscopic image overlaid on a photo graphic image of recipient C57BL/6 mouse colon 3 weeks following its orthotopic injection with MC38 CRC cells that were genetically manipulated by lentiviral reporter system to express RFP, magnification x10. Down left panel – colonoscopy image of C57BL/6 mouse colon following orthotopic implantation of 2 adjacent MC38 CRC tumor cells; MC38-RFP (red arrow) and MC38-GFP (green arrow). Right panel - fluorescent stereo-microscopic image overlaid on a photo graphic image of recipient C57BL/6 mouse opened colon 2 weeks following side-by-side orthotopic injection of MC38-RFP (distal) and MC38-GFP (proximal) CRC tumor cells, magnification x10. Note the induction of 2 adjacent CRC tumors that differ only by the expression of the transduced reporter gene. Data are representative of three independent experiments.