The zebrafish/tumor xenograft angiogenesis assay

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.

[1]  Z. Nagy,et al.  of Identification of novel vascular markers through gene expression profiling of tumor-derived endothelium , 2008 .

[2]  G. Jayson,et al.  Quantitative Angiogenesis Assays in vivo – A Review , 2004, Angiogenesis.

[3]  D. Ribatti,et al.  Antiangiogenic and Vascular-Targeting Activity of the Microtubule-Destabilizing trans-Resveratrol Derivative 3,5,4′-Trimethoxystilbene , 2005, Molecular Pharmacology.

[4]  J. Spitsbergen.  Imaging neoplasia in zebrafish , 2007, Nature Methods.

[5]  Rajan P. Kulkarni,et al.  Quantum dots are powerful multipurpose vital labeling agents in zebrafish embryos , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[6]  M. Hendrix,et al.  Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness , 2006, Nature Medicine.

[7]  B. Weinstein.  Vascular cell biology in vivo: a new piscine paradigm? , 2002, Trends in cell biology.

[8]  Jon D Larson,et al.  Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics? , 2002, Briefings in functional genomics & proteomics.

[9]  M. Presta,et al.  Calcitonin receptor-like receptor guides arterial differentiation in zebrafish. , 2008, Blood.

[10]  Michael Brand,et al.  Micro fluid segment technique for screening and development studies on Danio rerio embryos. , 2007, Lab on a chip.

[11]  Napoleone Ferrara,et al.  Vascular endothelial growth factor: basic science and clinical progress. , 2004, Endocrine reviews.

[12]  R. Klemke,et al.  Catch of the day: zebrafish as a human cancer model , 2008, Oncogene.

[13]  P. Dell’Era,et al.  Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. , 2005, Cytokine & growth factor reviews.

[14]  Wolfram Goessling,et al.  Ultrasound biomicroscopy permits in vivo characterization of zebrafish liver tumors , 2007, Nature Methods.

[15]  Shuo Lin,et al.  Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay. , 2003, Arteriosclerosis, thrombosis, and vascular biology.

[16]  S. Ekker,et al.  Effective targeted gene ‘knockdown’ in zebrafish , 2000, Nature Genetics.

[17]  R. Giavazzi,et al.  Copyright © American Society for Investigative Pathology Distinct Role of Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor on Tumor Growth and Angiogenesis , 2022 .

[18]  L. Zon,et al.  Transparent adult zebrafish as a tool for in vivo transplantation analysis. , 2008, Cell stem cell.

[19]  S. L. Gonias,et al.  High-resolution imaging of the dynamic tumor cell–vascular interface in transparent zebrafish , 2007, Proceedings of the National Academy of Sciences.

[20]  B. Weinstein,et al.  The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. , 2001, Developmental biology.

[21]  S. Revskoy,et al.  Transplantable tumor lines generated in clonal zebrafish. , 2006, Cancer research.

[22]  P. Ratcliffe,et al.  Regulation of angiogenesis by hypoxia: role of the HIF system , 2003, Nature Medicine.

[23]  Marco Presta,et al.  Role of the soluble pattern recognition receptor PTX3 in vascular biology , 2007, Journal of cellular and molecular medicine.

[24]  Huiqing Zhan,et al.  Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression , 2006, Nature Biotechnology.

[25]  P. Carmeliet,et al.  Angiogenesis in cancer and other diseases , 2000, Nature.

[26]  Marina Mione,et al.  How to create the vascular tree? (Latest) help from the zebrafish. , 2008, Pharmacology & therapeutics.

[27]  A Vacca,et al.  Basic fibroblast growth factor overexpression in endothelial cells: an autocrine mechanism for angiogenesis and angioproliferative diseases. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[28]  S. Laurenson,et al.  Chemical discovery and global gene expression analysis in zebrafish , 2003, Nature Biotechnology.

[29]  G. Serbedzija,et al.  Zebrafish angiogenesis: A new model for drug screening , 2004, Angiogenesis.

[30]  M. Noyes,et al.  Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases , 2008, Nature Biotechnology.

[31]  D. Ribatti,et al.  Mammalian tumor xenografts induce neovascularization in zebrafish embryos. , 2007, Cancer research.

[32]  M. Presta,et al.  Fibroblast growth factor 2‐induced angiogenesis in zebrafish: the zebrafish yolk membrane (ZFYM) angiogenesis assay , 2009, Journal of cellular and molecular medicine.

[33]  K. Kinzler,et al.  Genes expressed in human tumor endothelium. , 2000, Science.

[34]  M. Haldi,et al.  Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish , 2006, Angiogenesis.

[35]  Edwin Cuppen,et al.  Zebrafish as a Cancer Model , 2008, Molecular Cancer Research.

[36]  D. Ribatti,et al.  Alterations of blood vessel development by endothelial cells overexpressing fibroblast growth factor‐2 , 1999, The Journal of pathology.

[37]  Leonard I. Zon,et al.  Organogenesis--Heart and Blood Formation from the Zebrafish Point of View , 2002, Science.