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

Abstract

Most genetically engineered mouse models (GEMMs) 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 15 min, including preparation. In our experience, anyone with reasonable hand–eye coordination can become proficient with mouse colonoscopy and mucosal injection 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.

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Figure 1: Colonoscopy equipment.
Figure 2: Colonoscopy-guided mucosal injection technique.
Figure 3: Colorectal cancer modeling with colonoscopy-guided mucosal injection.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) (K08 CA198002 to J.R.; K99 CA187317 to T.T.; and R00 AG045144 and R01 CA211184 to Ö.H.Y.), the Department of Defense (PRCRP Career Development Award CA120198 to J.R.), the V Foundation V Scholar Award (to J.R. and Ö.H.Y.), the Sidney Kimmel Scholar Award (to Ö.H.Y.), the Pew-Stewart Trust Scholar Award (to Ö.H.Y.), the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund (to Ö.H.Y.), the American Federation of Aging Research (AFAR; to Ö.H.Y.), and the Hope Funds for Cancer Research (to T.T.), as well as by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. We thank the Swanson Biotechnology Center at the Koch Institute for technical support, specifically K. Cormier and C. Condon at the Hope Babette Tang (1983) Histology Facility; and S. Holder for histology support. The research was conducted in compliance with the Animal Welfare Act Regulations and other federal statutes relating to animals and experiments involving animals, and adheres to the principles set forth in the Guide for Care and Use of Laboratory Animals, National Research Council, 1996. We thank M. Tschurtschenthaler, R.-F. Jackstadt, J. Leach, and P. Westcott for critical review of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

J.R. designed and performed all experiments. T.T. assisted in design, construction, and production of lentiviral vectors. A.A., M.A., and S.B.S. assisted with mucosal injection experiments. T.J. participated in the interpretation of results. Ö.H.Y. supervised the experiments.

Corresponding authors

Correspondence to Jatin Roper or Ömer H Yilmaz.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Demonstration of sample preparation and transfer to injection needle.

This procedure is performed by the assistant. The sample is drawn into the syringe using the transfer needle. Then the transfer needle is removed, and the syringe is attached to the injection needle. When the operator is ready, the assistant quickly injects the sample. (MP4 23729 kb)

Demonstration of colonoscopy-guided mucosal injection.

The operator places the tip of the injection needle into the mucosa of the colon. Then, the operator asks the assistant to rapidly inject the sample. The injection forms a mucosal 'bubble' by overcoming the resistance of the tissue. Appropriate institutional regulatory board permission was obtained for these experiments. (MP4 20621 kb)

White-light colonoscopy of a colon tumor generated by somatic CRISPR–Cas9 editing of the Apc tumor suppressor gene.

Tamoxifen-treated Rosa26LSL-Cas9-eGFP/+;VillinCreER mice were injected under colonoscopy guidance with U6::sgApc-EFS::turboRFP lentivirus (titer = 10,000 TU/μl). Tumors were visualized by colonoscopy ~6 weeks after viral injection. In this video, a large tumor is seen with white-light colonoscopy ~1 year after viral injection. Appropriate institutional regulatory board permission was obtained for these experiments. (MP4 4110 kb)

GFP fluorescence colonoscopy of a colon tumor generated by somatic CRISPR–Cas9 editing of the Apc tumor suppressor gene.

Tamoxifen-treated Rosa26LSL-Cas9-eGFP/+;VillinCreER mice were injected under colonoscopy guidance with U6::sgApc-EFS::turboRFP lentivirus (titer = 10,000 TU/μl). Tumors were visualized by colonoscopy ~6 weeks after viral injection. In this video, a large tumor is seen with GFP fluorescence colonoscopy ~1 year after viral injection. Appropriate institutional regulatory board permission was obtained for these experiments. (MP4 3805 kb)

RFP fluorescence colonoscopy of a colon tumor generated by somatic CRISPR–Cas9 editing of the Apc tumor suppressor gene.

Tamoxifen-treated Rosa26LSL-Cas9-eGFP/+;VillinCreER mice were injected under colonoscopy guidance with U6::sgApc-EFS::turboRFP lentivirus (titer = 10,000 TU/μl). Tumors were visualized by colonoscopy ~6 weeks after viral injection. In this video, a large tumor is seen with RFP fluorescence colonoscopy ~1 year after viral injection. Appropriate institutional regulatory board permission was obtained for these experiments. (MP4 5375 kb)

Inflammatory polyp formation following colonoscopy-guided mucosal injection.

If mucosal injection does not result in tumorigenesis, an inflammatory polyp occasionally forms at the injection site. These polyps can be distinguished from adenomatous polyps or tumors by colonoscopy features. Inflammatory polyps are thin and small, do not grow, and exhibit a vascular pattern that is similar to that of normal mucosa. Appropriate institutional regulatory board permission was obtained for these experiments. (MP4 6014 kb)

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Roper, J., Tammela, T., Akkad, A. et al. Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation. Nat Protoc 13, 217–234 (2018). https://doi.org/10.1038/nprot.2017.136

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