Abstract. Zebrafish (Danio rerio) is becoming an increasingly popular vertebrate cancer model. In this study, we established a xenotransplanted zebrafish embryo glioma model to further investigate the molecular mechanisms of tumor angiogenesis. We find that the glioma cell line U87 can survive, proliferate and induce additional SIV branches in zebrafish embryos. In addition, by the means of in situ hybridization and quantitive RT-PCR analyses we find that the transplanted U87 cells can induce the ectopic zebrafish vascular endothelial growth factor A (VEGF A) and its receptor VEGFR2/KDR mRNA expression and increase their expression levels, resulting in additional SIV branches. IntroductionGlioma is the most common primary brain tumor in adults and the median survival for patients without therapy is up to 3 months (1). Currently the traditional treatment such as resection, chemotherapy and radiotherapy has not greatly improved the median survival for patients. Malignant glioma is notorious for its behavior of rapid proliferation, great invasion and metastasis and it is also among the best vascularized tumors in humans. It is well known that angiogenesis plays a critical role in tumor progression. Therefore new anti-angiogenic treatment strategies are required (2-6). Revealing of various signaling pathways that lead to activation of angiogenesis will provide molecular insight into developing therapeutic agents to treat glioma. In the past decades, researchers have discovered many molecular mechanisms related to tumor angiogenesis (7-9).Traditional rodent cancer models have disadvantages, e.g., the high cost, long developmental phase, and limited availability for high-throughput assays (10), which limited effective ways to find the new molecular mechanisms and monitor the tumor in vivo in real time.The teleost zebrafish (Danio rerio), a promising alternative vertebrate cancer model has attracted considerable attention in recent years because of compelling advantages of transparency of the embryo, rapid development, fecundity, tractable genetics, great screening efficiency, high levels of physiologic and genetic homology with higher vertebrates (11-13). Moreover, the zebrafish embryo within 1 week is immune-free, and showed no immunosuppression to transplanted human glioma cells (14). The transgenic zebrafish strains, e.g., casper transparent mutant line (15), and VEGFR2:G-RCFP line with green fluorescence specifically in blood vessels (16), offered new tools for cancer research.In this study, we microinjected human U87 glioma cells into zebrafish embryos and developed a xenograft zebrafish glioma model. The U87 cells are first labeled with a red fluorescence protein and then microinjected into perivitelline space of VEGFR2:G-RCFP transgenic zebrafish embryos at 48 h post-fertilization. Then in order to investigate the angiogenesis mechanisms in details, we make use of the staining of endogenous alkaline phosphatase, fluorescent microscopy monitoring, in situ hybridization and quantitive RT-PCR to investigate the ch...

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