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Inducible mouse models of colon cancer for the analysis of sporadic and inflammation-driven tumor progression and lymph node metastasis

Nature Protocols volume 16, pages 61–85 (2021)Cite this article

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Abstract

Despite advances in the detection and therapy of colorectal cancer (CRC) in recent years, CRC has remained a major challenge in clinical practice. Although alternative methods for modeling CRC have been developed, animal models of CRC remain helpful when analyzing molecular aspects of pathogenesis and are often used to perform preclinical in vivo studies of potential therapeutics. This protocol updates our protocol published in 2007, which provided an azoxymethane (AOM)-based setup for investigations into sporadic (Step 5A) and, when combined with dextran sodium sulfate (Step 5B), inflammation-associated tumor growth. This update also extends the applications beyond those of the original protocol by including an option in which AOM is serially applied to mice with p53 deficiency in the intestinal epithelium (Step 5C). In this model, the combination of p53 deficiency and AOM promotes tumor development, including growth of invasive cancers and lymph node metastasis. It also provides details on analysis of colorectal tumor growth and metastasis, including analysis of partial epithelial-to-mesenchymal transition, cell isolation and co-culture studies, high-resolution mini-endoscopy, light-sheet fluorescence microscopy and micro-CT imaging in mice. The target audience for our protocol is researchers who plan in vivo studies to address mechanisms influencing sporadic or inflammation-driven tumor development, including the analysis of local invasiveness and lymph node metastasis. It is suitable for preclinical in vivo testing of novel drugs and other interventional strategies for clinical translation, plus the evaluation of emerging imaging devices/modalities. It can be completed within 24 weeks (using Step 5A/C) or 10 weeks (using Step 5B).

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Fig. 1: Schemes of AOM-based colon tumor induction in mice.
Fig. 2: Step-by-step preparation of the colon for tumor analysis.
Fig. 3: Development of colon tumors in mice and in vivo monitoring by mini-endoscopy.
Fig. 4: Advanced in vivo and ex vivo imaging of colon tumors by micro-CT and LSFM.
Fig. 5: Ex vivo evaluation of colon tumor development in the AOM–DSS model.
Fig. 6: Ex vivo evaluation of tumor invasiveness in the Tp53ΔIEC–AOM model.
Fig. 7: Partial EMT of invasive tumor cells.
Fig. 8: Analysis and in vitro studies of the tumor stroma.
Fig. 9: Studies of the CAF-associated crosstalk with epithelial cells.

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Data availability

All data generated during this study are included in this published article (Fig. 2, Fig. 4a, Fig. 7, Fig. 9b, images in Box 2). Some experiments were performed during previous studies, and similar data were published earlier in different formats (Fig. 3 in ref. 24, Fig. 4b,c and Fig. 5 in ref. 23, Fig. 6 in ref. 20, Fig. 8 and Fig. 9a in ref. 23). Additional information can be provided by the authors upon request.

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Acknowledgements

We thank K. Enderle, K. Hofmann, and I. Zoeller-Utz for excellent technical assistance. This work was funded by the DFG (FOR2438/project 9 to C.N. and M.F.N.; NE1927/2-2, SFB1181-C02 and TRR241-A08 to C.N.) and by the Bartling Stiftung (to C.N.). T.B. was supported by grants from German Research Foundation (FOR 2438/project 4; BR 1399/9-1; 1399/10-1; 1399/13-1).

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Author notes

  1. These authors contributed equally: Clemens Neufert, Christina Heichler.

Authors and Affiliations

  1. First Department of Medicine, Friedrich-Alexander-Universität, Erlangen-Nürnberg, Universitätsklinikum Erlangen, Erlangen, Germany

    Clemens Neufert, Christina Heichler, Kristina Scheibe & Markus F. Neurath

  2. Deutsches Zentrum Immuntherapie (DZI), Erlangen, Germany

    Clemens Neufert & Markus F. Neurath

  3. Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-Universität, Erlangen-Nürnberg, Universitätsklinikum Erlangen, Erlangen, Germany

    Thomas Brabletz & Florian R. Greten

  4. Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany

    Verawan Boonsanay

  5. Frankfurt Cancer Institute, Goethe University Frankfurt, Frankfurt am Main, Germany

    Florian R. Greten

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Contributions

C.N., C.H., T.B., K.S. and V.B. designed and performed the experiments. C.N., C.H., T.B., V.B., F.R.G. and M.F.N. analyzed and discussed the data. C.N., C.H. and T.B. wrote the manuscript.

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Correspondence to Clemens Neufert.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Jarom Heijmans and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Neufert, C. et al. J. Clin. Invest. 123, 1428–1443 (2013): https://doi.org/10.1172/JCI63748

Schwitalla, S. et al. Cancer Cell 23, 93–103 (2013): https://doi.org/10.1016/j.ccr.2012.11.014

Heichler, C. et al. Gut 69, 1269–1282 (2020): https://doi.org/10.1136/gutjnl-2019-319200

This protocol is an update to: Nat. Protoc. 2, 1998–2004 (2007): https://doi.org/10.1038/nprot.2007.279

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Neufert, C., Heichler, C., Brabletz, T. et al. Inducible mouse models of colon cancer for the analysis of sporadic and inflammation-driven tumor progression and lymph node metastasis. Nat Protoc 16, 61–85 (2021). https://doi.org/10.1038/s41596-020-00412-1

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