Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids

Abstract

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways1,2. Although these pathways influence intestinal stem cell niche signaling3,4,5, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system6,7 to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

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Figure 1: CRISPR-Cas9–mediated introduction of driver pathway mutations in human intestinal organoids.
Figure 2: Sequential engineering of various combinations of driver mutations.
Figure 3: Engraftment of engineered, adenoma and CRC organoids in NOG mice.
Figure 4: Generation of metastatic tumors from engineered adenoma organoids.

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Acknowledgements

This work was supported by grants from a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), by a Grant-in-Aid for Scientific Research on Innovative Areas 'Stem Cell Aging and Disease', and by Grants-in-Aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology of Japan. T.S. received a Research Grant of the Japanese Society of Gastroenterology. T.S. would like to thank H. Clevers (Hubrecht Institute) for providing discussion and comments. We also thank M. Okumura for editorial assistance, Y. Tanada for animal care and the Collaborative Research Resources, School of Medicine, Keio University for technical assistance. The R-spondin–producing cell line was a kind gift from C. Kuo (Stanford University). F. Zhang (Massachusetts Institute of Technology) kindly provided the pX330 vector.

Author information

M.M., S.D., M.S., A.T., M.F. and Y.O. performed experiments. T.W. and T.K. provided biological specimens. T.S. conceived and designed the project. T.S. wrote the manuscript.

Correspondence to Toshiro Sato.

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Competing interests

T.S. is an inventor on a patent (the Netherlands, US08643999) involving the organoid culture system. S.D. is an employee of Ohtsuka Pharmaceuticals.

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Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Tables 1, 2, and 4 (PDF 1351 kb)

Supplementary Table 3 (XLSX 20 kb)

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Matano, M., Date, S., Shimokawa, M. et al. Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids. Nat Med 21, 256–262 (2015). https://doi.org/10.1038/nm.3802

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