Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

Abstract

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein-positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/ tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis. [Cancer Res 2007;67(7):2927-31]

Catch of the day: zebrafish as a human cancer model

“…Transplantation of human cancer cells into early-stage (6-48 h post-fertilization) zebrafish embryos has also revealed important information about cancer biology as reported by several groups (Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007). The major advantage of this model is that the immune system is still immature that permits human tumor cell engraftment.…”

“…Cancer cells derived from various species (mouse and human) and tissues (adenocarcinoma, fibrosarcoma, melanoma) have been introduced into both young embryos (2-5-days old post fertilization; Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007) and juvenile 30-day-old zebrafish (Stoletov et al, 2007). Both transplantation models have their specific benefits and limitations when it comes to understanding cancer cell behavior in a living organism (Table 1).…”

“…This early study demonstrated the feasibility of using young zebrafish embryos for human cancer research and laid the foundation for future work to develop efficient throughput methods for the identification of therapeutic agents that impact human cancer. Nicoli et al (2007) showed that when injected into 2-day-old zebrafish embryos human and murine tumorigenic cell lines (mouse aortic endothelial, MDA-MB-435 human adenocarcinoma and B16 human melanoma) induced a robust angiogenic response in association with the surrounding intestinal vascular plexus. This response was significantly increased when the injected cancer cells were genetically engineered to express human FGF2 or VEGF growth factors.…”

“…Cancer progression in these animals recapitulates many aspects of human disease and opens the door for studies to identify genetic and chemical modifiers of cancer (Amatruda et al, 2002;Smolowitz et al, 2002;Stern and Zon, 2003;Berghmans et al, 2005a;Goessling et al, 2007a;Kari et al, 2007). The attention has been further fueled by the development of xenograft models that allow the propagation and visualization of human cancer cells engrafted in optically transparent zebrafish (Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007;Stoletov et al, 2007). The integration of zebrafish genetics with the large tool chest of reagents available to study human cancer cells provides a powerful new vertebrate model to visualize and dissect the mechanisms that drive cancer formation, angiogenesis and metastasis.…”

Zebrafish as a Cancer Model

“…Currently, no good in vivo model is available in which such dynamic process can be followed in real time. When transplanted into 2-day-old zebrafish embryos, human and murine tumor cell lines expressing fibroblast growth factor or vascular endothelial growth factor induced rapid neovascularization inside the tumor graft, and this could be inhibited by treatment with antiangiogenic chemicals (20). In another elegant study, researchers used i.p.…”