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The zebrafish/tumor xenograft angiogenesis assay

Nature Protocols volume 2, pages 2918–2923 (2007)Cite this article

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.

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Figure 1: Normal SIV development in zebrafish embryo.
Figure 2: Visualization of the site of injection of the tumor cell graft in the zebrafish embryo.
Figure 3: Angiogenic responses triggered by tumor cell grafts in the zebrafish embryo.
Figure 4: Effect of long pentraxin 3 (PTX3), an FGF2 antagonist21, on the angiogenic response induced by proangiogenic FGF2-T-MAE cells.

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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.

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  1. Department of Biomedical Sciences and Biotechnology, General Pathology and Immunology, University of Brescia Medical School, Viale Europa 11, Brescia, 25123, Italy

    Stefania Nicoli & Marco Presta

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  1. Stefania Nicoli
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  2. Marco Presta
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Correspondence to Marco Presta.

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Nicoli, S., Presta, M. The zebrafish/tumor xenograft angiogenesis assay. Nat Protoc 2, 2918–2923 (2007). https://doi.org/10.1038/nprot.2007.412

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