Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Hypoxia-induced metastasis model in embryonic zebrafish

Nature Protocols volume 5, pages 1911–1918 (2010)Cite this article

Subjects

Abstract

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average experimental time for this model is 7 d. Our protocol offers a remarkable opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Hypoxic setup for the embryonic zebrafish aquarium.
Figure 2: Critical stages for tumor cell implantation into embryonic zebrafish and for analyzing tumor cell dissemination.
Figure 3: Dissemination and quantification of disseminated mouse T241 fibrosarcoma cells.

Similar content being viewed by others

References

  1. Erler, J.T. et al. Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche. Cancer Cell 15, 35–44 (2009).

    Article  CAS  Google Scholar 

  2. Rouhi, P. et al. Pathological angiogenesis facilitates tumor cell dissemination and metastasis. Cell Cycle 9, 913–917 (2010).

    Article  CAS  Google Scholar 

  3. Cao, Y. Opinion: emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis. Nat. Rev. Cancer 5, 735–743 (2005).

    Article  CAS  Google Scholar 

  4. Lee, S.L. et al. Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model. Proc. Natl. Acad. Sci. USA 106, 19485–19490 (2009).

    Article  CAS  Google Scholar 

  5. Nissen, L.J. et al. Angiogenic factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis. J. Clin. Invest. 117, 2766–2777 (2007).

    Article  CAS  Google Scholar 

  6. Cao, R. et al. PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell 6, 333–345 (2004).

    Article  CAS  Google Scholar 

  7. Cao, Z. et al. Hypoxia-induced retinopathy model in the adult zebrafish. Nat. Protoc., 12, 1903–1910 (2010).

    Article  Google Scholar 

  8. van Rooijen, E. et al. Zebrafish mutants in the von Hippel-Lindau tumor suppressor display a hypoxic response and recapitulate key aspects of Chuvash polycythemia. Blood 113, 6449–6460 (2009).

    Article  CAS  Google Scholar 

  9. Lawson, N.D. & Weinstein, B.M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318 (2002).

    Article  CAS  Google Scholar 

  10. Jin, S.W., Beis, D., Mitchell, T., Chen, J.N. & Stainier, D.Y. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132, 5199–5209 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Y.C.'s laboratory was supported by research grants from the Swedish Research Council, the Swedish Cancer Foundation, the Karolinska Institute Foundation, the Karolinska Institute distinguished professor award, the European Union Integrated Project of Metoxia (Project no. 222741) and the European Research Council (ERC) advanced grant ANGIOFAT (Project no 250021). Z.C. is supported by the Swedish Heart and Lung Foundation.

Author information

Author notes

  1. Pegah Rouhi, Lasse D Jensen and Ziquan Cao: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

    Pegah Rouhi, Lasse D Jensen, Kayoko Hosaka & Yihai Cao

  2. Institute of Medicine and Health, Linkoping University, Linkoping, Sweden

    Ziquan Cao, Toste Länne & Eric Wahlberg

  3. Marine Biological Laboratory, Biological Institute, University of Copenhagen, Helsingor, Denmark

    John Fleng Steffensen

Authors
  1. Pegah Rouhi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lasse D Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ziquan Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kayoko Hosaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Toste Länne
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eric Wahlberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John Fleng Steffensen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yihai Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.C. designed the study. L.D.J., P.R. and Z.C. performed the experiments. L.D.J., P.R., Z.C. and Y.C. analyzed the data. K.H., T.L., J.F.S. and E.W. participated in designing and discussing this study. L.D.J., P.R., Z.C. and Y.C. wrote the paper.

Corresponding author

Correspondence to Yihai Cao.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rouhi, P., Jensen, L., Cao, Z. et al. Hypoxia-induced metastasis model in embryonic zebrafish. Nat Protoc 5, 1911–1918 (2010). https://doi.org/10.1038/nprot.2010.150

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.150

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer