Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation

Abstract

Most genetically engineered mouse models of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations, such as the subcutaneous space, kidney capsule, or cecal wall, that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection. These techniques can be adapted to deliver viral vectors carrying Cre recombinase, CRISPR-Cas9 components, CRISPR-engineered mouse tumor organoids, or human cancer organoids to mice to model the adenoma-carcinoma-metastasis sequence of tumor progression. The colonoscopy injection procedure takes approximately 15 minutes, including preparation. In our experience, anyone with reasonable hand-eye coordination can become proficient with basic mouse colonoscopy with a few hours of practice. These approaches are ideal for a wide range of applications, including assessment of gene function in tumorigenesis, examination of tumor-stroma interactions, studies of cancer metastasis, and translational research with patient-derived cancers.

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

“…and gene modification of oncogenes (KRAS, PI3K, etc.). 83 Moreover, guided by colonoscopy, through mucosal injection, Roper et al 84 established CRISPR engineered mouse tumor organoids by delivering viral vectors carrying CRISPR/Cas9 components to the distal colon of mice. Such an approach has already been applied in a study modeling tumor progression with an adenoma-carcinoma-metastasis sequence.…”

Colorectal Cancer: Genetic Abnormalities, Tumor Progression, Tumor Heterogeneity, Clonal Evolution and Tumor-Initiating Cells

“…These findings suggest that the metastatic process is a direct consequence of the loss of dependency of specific niche signals [ 295 ]. Roper and coworkers reported a CRIPR-Cas9-based gene editing and organoid transplantation approach that employs colonoscopy-guided mucosal injection for primary and metastatic tumor induction in mice [ 296 , 297 ]. APC -deleted and TP53 -deleted and KRAS G12D/+ mouse colon organoids and human colorectal cancer organoids engraft in the distal colon and metastasize to the liver [ 297 ].…”

Antitumor immunity in dMMR colorectal cancers requires interferon-induced CCL5 and CXCL10

“…Orthotopic CRC experiments were performed by injecting 1×10 5 MC38 CRC cells in 50ul PBS into the wall of the descending colon using a flexible needle (Hamilton) inserted through the working channel of a Wolfe endoscope and visualized via the ColoView imaging system (Storz). 25 Orthotopic tumour growth was monitored by endoscopy and tumours were harvested after 2-3 weeks. Resected tumours were minced and digested in enzyme cocktail (RPMI containing 0.5mg/ml collagenase IV, 10μg/ml DNaseI, 10% FBS, 1% penicillin-streptomycin and 1% HEPES buffer) for 30 minutes at 37°C in a shaking incubator.…”

“…Primary dMMR mouse or human organoids were generated using lentiviral transduction as described previously. 25,30 Lentivirus was prepared as previously described using the pLKO.1 system (Addgene #10878) and containing the shRNA sequences in Supplementary Table 1. 24 Lentivirus was concentrated 100X by ultracentrifugation.…”