Abstract
Here we describe a method to study tumor angiogenesis in zebrafish (Danio rerio) based on the injection of proangiogenic mammalian tumor cells into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Within 24–48 h, proangiogenic tumor grafts induce a neovascular response originating from the developing subintestinal vessels. This can be observed at macroscopic and microscopic levels after whole-mount alkaline phosphatase staining of wild-type zebrafish embryos, or by fluorescence microscopy in transgenic VEGFR2:G-RCFP embryos in which endothelial cells express the green fluorescent protein under the control of the VEGFR2/KDR promoter. Angiogenesis inhibitors added to the injected cell suspension or to the fish water prevent tumor-induced neovascularization. The assay is rapid and inexpensive, representing a novel tool for investigating tumor angiogenesis and for antiangiogenic drug discovery. Also, gene inactivation by antisense morpholino oligonucleotides injection in zebrafish embryos may allow the identification of genes involved in tumor angiogenesis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000).
Ferrara, N. Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25, 581–611 (2004).
Presta, M. et al. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev. 16, 159–178 (2005).
Hasan, J. et al. Quantitative angiogenesis assays in vivo—a review. Angiogenesis 7, 1–16 (2004).
Lam, S.H. et al. Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression. Nat. Biotechnol. 24, 73–75 (2006).
Thisse, C. & Zon, L.I. Organogenesis—heart and blood formation from the zebrafish point of view. Science 295, 457–462 (2002).
Weinstein, B. Vascular cell biology in vivo: a new piscine paradigm? Trends Cell Biol. 12, 439–445 (2002).
Topczewska, J.M. et al. Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness. Nat. Med. 12, 925–932 (2006).
Haldi, M., Ton, C., Seng, W.L. & McGrath, P. Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis 9, 139–151 (2006).
Isogai, S., Horiguchi, M. & Weinstein, B.M. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev. Biol. 230, 278–301 (2001).
Cross, L.M., Cook, M.A., Lin, S., Chen, J.N. & Rubinstein, A.L. Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay. Arterioscler. Thromb. Vasc. Biol. 23, 911–912 (2003).
Nicoli, S., Ribatti, D., Cotelli, F. & Presta, M. Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res. 67, 2927–2931 (2007).
Pichler, F.B. et al. Chemical discovery and global gene expression analysis in zebrafish. Nat. Biotechnol. 21, 879–883 (2003).
Serbedzija, G.N., Flynn, E. & Willett, C.E. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 3, 353–359 (1999).
Nasevicius, A. & Ekker, S.C. Effective targeted gene 'knockdown' in zebrafish. Nat. Genet. 26, 216–220 (2000).
Sumanas, S. & Larson, J.D. Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics? Brief. Funct. Genomic. Proteomic. 1, 239–256 (2002).
Gilmour, D.T., Jessen, J.R. & Lin, S. in Zebrafish (eds. Nusslein-Volhard, C. & Dahm, R.) 121–143 (Oxford University Press, Oxford, 2002).
Gualandris, A. et al. Basic fibroblast growth factor overexpression in endothelial cells: an autocrine mechanism for angiogenesis and angioproliferative diseases. Cell Growth Differ. 7, 147–160 (1996).
Stoletov, K. et al. Nigh-resolution imaging of the dynamic tumor cell-vascular interface in transparent zebrafish. Proc. Natl. Acad. Sci. USA 104, 17406–17411 (2007).
Presta, M., Camozzi, M., Salvatori, G. & Rusnati, M. Role of the soluble pattern recognition receptor PTX3 in vascular biology. J. Cell. Mol. Med. 11, 723–738 (2007).
Ribatti, D. et al. Alterations of blood vessel development by endothelial cells overexpressing fibroblast growth factor-2. J. Pathol. 189, 590–599 (1999).
Giavazzi, R. et al. Distinct role of fibroblast growth factor-2 and vascular endothelial growth factor on tumor growth and angiogenesis. Am. J. Pathol. 162, 1913–1926 (2003).
Acknowledgements
This work was supported by grants from Istituto Superiore di Sanità (Oncotechnological Program), Ministero dell'Istruzione, Università e Ricerca (Centro di Eccellenza per l'Innovazione Diagnostica e Terapeutica), Associazione Italiana per la Ricerca sul Cancro, Fondazione Berlucchi and NOBEL Project Cariplo.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Nicoli, S., Presta, M. The zebrafish/tumor xenograft angiogenesis assay. Nat Protoc 2, 2918–2923 (2007). https://doi.org/10.1038/nprot.2007.412
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2007.412
This article is cited by
-
Preclinical models in head and neck squamous cell carcinoma
British Journal of Cancer (2023)
-
Using a zebrafish xenograft tumor model to compare the efficacy and safety of VEGFR-TKIs
Journal of Cancer Research and Clinical Oncology (2023)
-
Refined high-content imaging-based phenotypic drug screening in zebrafish xenografts
npj Precision Oncology (2023)
-
The inhibition of protein translation promotes tumor angiogenic switch
Molecular Biomedicine (2022)
-
Mechanosensitive turnover of phosphoribosyl pyrophosphate synthetases regulates nucleotide metabolism
Cell Death & Differentiation (2022)
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.