• A Corrigendum to this article was published on 08 December 2017

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Abstract

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.

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Change history

  • 06 July 2017

    In the version of this article initially published, the initial “J” was omitted from an author's name, which should appear as Francisco J Sánchez-Rivera. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by the Howard Hughes Medical Institute (T.J., R.O.H.), NIH (K08 CA198002, J.R.; K99 CA187317, T.T.; Ö.H.Y.; R01CA211184, Ö.H.Y.; U54-CA163109, R.O.H.), the Sigrid Juselius Foundation (Ö.H.Y), the Maud Kuistila Foundation and the Hope Funds for Cancer Research (Ö.H.Y.), Department of Defense (PRCRP Career Development Award CA120198; J.R.), and the V Foundation V Scholar Award (J.R. and Ö.H.Y.), the Sidney Kimmel Scholar Award (Ö.H.Y.), the Pew-Stewart Trust Scholar Award (Ö.H.Y.), the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund (Ö.H.Y.), American Federation of Aging Research (AFAR, Ö.H.Y.), the Hope Funds for Cancer Research (T.T.), the Metastasis/Cancer Research Postdoc fellowship from the MIT Ludwig Center for Molecular Oncology Research (S.R.), the Bloodwise (UK) Visiting Fellowship Grant 14043 (A.R.), and by the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. We thank T. Papagiannakopoulos for helpful discussions; Y. Soto-Feliciano for help and expertise with massively parallel sequencing; K. Bedrossian for assistance with human colorectal cancer sample collection at Tufts Medical Center; 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; S. Holder for histology support; and Y.D. Soo and the Peterson (1957) Nanotechnology Materials Core Facility for assistance with electron microscopy. L-WRN cells were a kind gift from T. Stappenbeck, Washington University.

Author information

Author notes

    • Jatin Roper
    •  & Tuomas Tammela

    These authors contributed equally to this work.

Affiliations

  1. The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts, USA.

    • Jatin Roper
    • , Tuomas Tammela
    • , Naniye Malli Cetinbas
    • , Adam Akkad
    • , Ali Roghanian
    • , Steffen Rickelt
    • , Mohammad Almeqdadi
    • , Katherine Wu
    • , Matthias A Oberli
    • , Francisco J Sánchez-Rivera
    • , Yoona K Park
    • , Xu Liang
    • , George Eng
    • , Roxana Azimi
    • , Dmitriy Kedrin
    • , Rachit Neupane
    • , Semir Beyaz
    • , Jacqueline Lees
    • , Robert Langer
    • , Richard O Hynes
    • , Jianzhu Chen
    • , Arjun Bhutkar
    • , Tyler Jacks
    •  & Ömer H Yilmaz
  2. Division of Gastroenterology, Tufts Medical Center, Boston, Massachusetts, USA.

    • Jatin Roper
  3. Molecular Oncology Research Institute, Tufts Medical Center, Boston, Massachusetts, USA.

    • Jatin Roper
    • , James Yoo
    •  & Philip N Tsichlis
  4. Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, United Kingdom.

    • Ali Roghanian
  5. Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • George Eng
    • , Martin S Taylor
    • , Lawrence Zukerberg
    • , Vikram Deshpande
    •  & Ömer H Yilmaz
  6. Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, USA.

    • Ewa T Sicinska
    •  & Adam J Bass
  7. Department of Pathology, Tufts Medical Center, Boston, Massachusetts, USA.

    • Yvelisse Suarez
  8. Department of Surgery, Tufts Medical Center, Boston, Massachusetts, USA.

    • James Yoo
    •  & Lillian Chen
  9. Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

    • Pekka Katajisto
  10. Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

    • Pekka Katajisto
  11. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Richard O Hynes
    •  & Tyler Jacks

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Contributions

J.R. and T.T. performed all experiments and participated in their design and interpretation with T.J. and Ö.H.Y. J.R. and Ö.H.Y. developed and optimized the colonoscopy mucosal injection technique, with assistance from D.K. and P.K. J.R. wrote the paper with support from T.T. and Ö.H.Y. N.M.C. contributed to study design, plasmid design, lentivirus production, and mucosal injections. F.J.S.-R. contributed to plasmid and study design, and performed massively parallel sequencing. M.A., Y.K.P., R.N., R.A., X.L., D.K., K.W., S.R., and A.A. assisted with mucosal injections, mouse and human organoid derivation, molecular biology, and immunohistochemistry. A.R. assisted with humanized mouse experiments. M.A.O. designed and synthesized lipid nanoparticles for mRNA encapsulation. G.E., E.T.S., M.S.T., A.J.B., Y.S., J.Y., L.C., V.D., and L.Z. assisted with human CRC specimen collection. S.B. performed organoid qRT-PCR. A.B. performed bioinformatics analysis. R.L., J.L., J.C., P.N.T., R.O.H., and T.J. participated in interpretation of results. Ö.H.Y. supervised all aspects of the study.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ömer H Yilmaz.

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  1. 1.

    Colonoscopy-guided mucosal injection.

    During optical colonoscopy, a 33-gauge needle with 45-degree bevel is inserted into the working channel of the endoscopy and directed to the colonic mucosa without passing through the muscularis propria or serosa. 50-100 μl of liquid (containing virus or organoids, for example) is then rapidly injected to produce a mucosal bubble.

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https://doi.org/10.1038/nbt.3836

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