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Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

Nature Medicine volume 20, pages 769–777 (2014)Cite this article

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Abstract

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (KrasG12D), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

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Figure 1: In vitro oncogenic transformation of primary pancreatic organoids and in vivo tumorigenesis.
Figure 2: In vitro oncogenic transformation of primary gastric organoids and in vivo tumorigenesis.
Figure 3: Systematic evaluation of oncogene modules of increasing complexity in primary colon organoids.
Figure 4: Serial passage and in vivo transplantation of colon four-gene AKPS organoids.
Figure 5: Comparative functional validation of miR-483 and Igf2 transforming activity in primary Apc-null colon organoid culture.

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Acknowledgements

We are grateful to members of the Kuo laboratory, M. Amieva, R. Honaker, T. Jacks and C. Chartier for helpful discussion, P. Chu and S. Michie for assistance with histology, S. Lowe (Memorial Sloan Kettering Cancer Center) for LMP-based retroviruses, B. Williams (Van Andel Research Institute) for mouse strains, R. Mulligan and J.-S. Lee (Harvard University) and J. Chen (Stanford University) for lentiviral and retroviral reagents. X.L. was supported by the Dean's Fellowship at Stanford University, A.O. was supported by a Physician-Scientist Fellowship from the California Institute for Regenerative Medicine, J.T.N. was supported by a Postdoctoral Fellowship (124574-PF-13-296-01-TBG) from the American Cancer Society, C.W.-M.C. was supported by the Croucher Foundation, and T.Y. was supported by the Royal College of Surgeons. L.N. was supported by an American Society for Clinical Oncology Young Investigator Award and US National Institutes of Health grant K08CA166512. C.-Z.C. was supported by grants from the US National Institutes of Health (1R01AI073724 and Pioneer Award DP1CA174421) and the W.M. Keck Foundation. We thank the generous support from the Innovate Foundation and the Krishnan-Shah Family Foundation to C.J.K. This work was also supported by US National Institutes of Health grants K08DK078033 and U01CA084301 to K.E.H.; US National Cancer Institute Integrative Cancer Biology Program grant U01CA151920, US National Cancer Institute Cancer Target Discovery and Development grant 1U01CA168424 and a Fidelity Foundation grant to C.J.K. and H.P.J.; and the US National Institutes of Health Transformative R01DK085720 and US National Institute of Diabetes and Digestive and Kidney Diseases Intestinal Stem Cell Consortium grant U01DK085527 to C.J.K.

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Authors and Affiliations

  1. Department of Medicine, Hematology Division, Stanford University School of Medicine, Stanford, California, USA

    Xingnan Li, Lincoln Nadauld, Akifumi Ootani, David C Corney, Michael A Cantrell, Paul G Rack, James T Neal, Carol W-M Chan, Trevor Yeung, Jenny Yuan & Calvin J Kuo

  2. Department of Internal Medicine, Saga Medical School, Saga, Japan

    Akifumi Ootani

  3. Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

    Reetesh K Pai

  4. Department of Radiology, Stanford University School of Medicine, Stanford, California, USA

    Olivier Gevaert & Sylvia K Plevritis

  5. Baxter Laboratories, Stanford University School of Medicine, Stanford, California, USA

    Xue Gong & Chang-Zheng Chen

  6. Division of Oncology, Stanford University School of Medicine, Stanford, California, USA

    Julie Wilhelmy & Hanlee P Ji

  7. Equipe de Morphogenèse et Signalisation Cellulaires, UMR 144 CNRS/Institut Curie, Paris, France

    Sylvie Robine

  8. Division of Radiation Biology, Stanford University School of Medicine, Stanford, California, USA

    Laura D Attardi

  9. Department of Medicine, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, USA

    Kenneth E Hung

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  1. Xingnan Li
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Contributions

X.L., L.N., A.O., D.C.C., M.A.C., P.G.R. and J.T.N. designed and executed organoid experiments. R.K.P. performed blinded histologic interpretation. O.G. and X.G. performed bioinformatic analyses. C.W.-M.C., T.Y., J.Y. and J.W. performed organoid experiments and produced viruses. S.R., L.D.A. and K.E.H. provided reagents. K.E.H., C.-Z.C., S.K.P., H.P.J. and C.J.K. designed experiments. X.L., L.N., D.C.C. and C.J.K. wrote the manuscript.

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Correspondence to Calvin J Kuo.

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Competing interests

C.J.K., L.N. and A.O. have filed patents with the United States Patent and Trademark Office to cover the use of this organoid technique for cancer modeling.

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Li, X., Nadauld, L., Ootani, A. et al. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nat Med 20, 769–777 (2014). https://doi.org/10.1038/nm.3585

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