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A high-throughput in vivo screening method in the mouse for identifying regulators of metastatic colonization

Nature Protocols volume 12, pages 2465–2477 (2017)Cite this article

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Abstract

We describe a sensitive, robust, high-throughput method for quantifying the ability of metastatic tumor cells to colonize a secondary organ. Metastasis is the leading cause of death in cancer patients, and successful colonization of the secondary organ is the rate-limiting step in the metastatic process; thus, experimental methods that can be used to interrogate the key factors required for this critical step are of great importance. The experimental metastasis assay we detail here includes tail-vein injection of cancer cells into the mouse and determination of the resulting secondary organ colonization, primarily in the lung, 10 d post dosing. This assay can be used to investigate factors that regulate metastatic colonization both at the tumor-cell-intrinsic level (via manipulation of the tumor cells before injection) and at the tumor-cell-extrinsic level (such as the tissue microenvironment, via the use of genetically modified (GM) mice or agents such as antibodies or drugs). Using this method, we have robustly screened more than 950 GM mouse lines to identify novel microenvironmental regulators of metastatic colonization. The experimental details discussed here include choosing of appropriate cell numbers, handling of the cells, selection of recipient animals and injection techniques. Furthermore, we discuss key experimental design considerations, including the choice of the method used to determine metastatic burden and statistical analysis of the results, as well as provide troubleshooting tips and identification of factors that contribute to experimental variability.

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Figure 1: Schematic of the metastatic process and experimental procedures to model metastasis in the mouse.
Figure 2: Schematic of the experimental metastasis assay using tail-vein injection.
Figure 3: Applications for the experimental metastasis assay.
Figure 4: Effect of sex, genetic background and age on the experimental metastasis assay, metastatic colonization and variation of the results of the assay over time.
Figure 5: Methods for determining metastatic burden of a tissue.

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Acknowledgements

This work was supported by grants from Cancer Research UK (C20510/A13031 to D.J.A.), the Wellcome Trust (WT098051 to D.J.A.), ERC Combat Cancer (319661 to D.J.A.) and the National Institutes of Health (U54HG004028 to N.A.K.). The authors thank the members of the Research Support Facility (Wellcome Trust Genome Campus) for their care of the mice, and M. Nohadani for histological processing of the lung samples.

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Authors and Affiliations

  1. Experimental Cancer Genetics Group, Wellcome Trust Sanger Institute,, Hinxton, UK

    Anneliese O Speak, Agnieszka Swiatkowska, Natasha A Karp, David J Adams & Louise van der Weyden

  2. Quantitative Biology, Innovative Medicines and Early Development (IMED), AstraZeneca,, Cambridge, UK

    Natasha A Karp

  3. Cancer Research UK Edinburgh Centre, Institute of Genetics & Molecular Medicine (IGMM),

    Mark J Arends

  4. University of Edinburgh, Edinburgh, UK

    Mark J Arends

Authors
  1. Anneliese O Speak
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  2. Agnieszka Swiatkowska
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  4. Mark J Arends
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  5. David J Adams
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  6. Louise van der Weyden
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Contributions

L.v.d.W. and D.J.A. conceived the idea of screening mice from large-scale mouse phenotyping pipelines for metastatic colonization. L.v.d.W., A.S. and A.O.S. performed the experiments. N.A.K. performed statistical analysis. M.J.A. performed histopathology analysis. L.v.d.W. and A.O.S. wrote the manuscript with contributions from all authors.

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Correspondence to Louise van der Weyden.

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The authors declare no competing financial interests.

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Speak, A., Swiatkowska, A., Karp, N. et al. A high-throughput in vivo screening method in the mouse for identifying regulators of metastatic colonization. Nat Protoc 12, 2465–2477 (2017). https://doi.org/10.1038/nprot.2017.118

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