Objectives/Hypothesis: The development of a simple, reliable, and cost-effective animal model greatly facilitates disease treatment. We aimed to establish a rapid, simple, and reproducible live zebrafish vestibular schwannoma xenograft model for antitumor drug screening. Methods: We optimized each of the following conditions for tumor cell xenografts in zebrafish larvae: larval stage, incubation temperature, and injected cell number. We used NF2-/-mouse Schwann (SC4) cells and generated mCherry fluorescent protein-expressing cells prior to injection into zebrafish larvae. SC4 cells were counted using a fluorescence microscope, suspended in 10% fetal bovine serum, and injected into the center of the yolk sac using a microinjection system. The injected embryos were transferred to E3 medium (for zebrafish embryos), and subsequent tumor formation was observed by fluorescence microscopy over a 5-day period. To validate our model, xenografted embryos were transferred into 6-well plates (5 embryos per well) and treated with everolimus, a known antitumor drug. Results: mCherry fluorescent protein-expressing SC4 cells were successfully grafted into the yolk sacs of zebrafish embryos without any immunosuppressant treatment. At 2 days postinjection, the xenografted cells had grown into tumor masses. The optimal speed of tumor formation depended on the larval stage (30 hpf), incubation temperature (31°C), and injected cell number (200 cells). In preliminary tests, everolimus treatment yielded a > 20% reduction in the number of SC4 cells in the yolk. Conclusion: Our in vivo model has the potential to greatly facilitate vestibular schwannoma treatment because of its speed, simplicity, reproducibility, and amenability to live imaging. Level of Evidence: NA Laryngoscope, 126:E409–E415, 2016.
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