Tumor Engraftment and Progression. (A) Zebrafish primary CNS-PNET¹⁷ tumor cells labeled with mCherry were transplanted into the fourth ventricle of 2-dpf embryos. At 3 dpf, tumor cells are seen invading into surrounding brain tissue (arrows). At 8 dpf, tumor cells are growing in the ventricle; red tumor cells are also present in the surrounding brain tissue and kidneys (asterisks). At 28 dpf, tumors cells have invaded and grow throughout the host brain. (B) Top panel. Seven 3-dpf embryos transplanted with mCherry-labeled zebrafish brain tumor cells. Tumor cells can be transplanted consistently into hundreds of embryos, with high engraftment rates (i.e., typically 85/100 embryos). Middle and bottom panels. Three representative adult fish with transplanted tumors 4-8 weeks post-fertilization (wpf). Tumors can be visualized by fluorescence microscopy.

Comparing Tumor Invasion and Drug Response. (A) CNS-PNETs¹⁷ from the optic tectum or cerebellum are labeled with mCherry or GFP, respectively, processed into a single-cell suspension, mixed together at a 1:1 ratio, and co-transplanted into 2-dpf embryos. (B) A 6-dpf embryo co-transplanted with a mixed suspension of GFP-labeled cerebellar tumor cells and mCherry-labeled tumor cells from the optic tectum. Differences in the invasive properties of each tumor in the same recipient can be observed using fluorescence microscopy. In this example, mCherry-labeled tumor cells migrate more extensively than those labeled with GFP (arrows). (C) Timeline for drug treatment on embryos transplanted with tumor cells. (D) Images of representative animals at the end of the treatment regimen (8 dpf) and at 8 weeks post-treatment (8 wpf), with either a DMSO control or a 50 µM MEK inhibitor. Tumor burden can be measured by quantifying fluorescence, as described¹⁷. In this experiment, the growth of mCherry-labeled tumor cells is reduced in MEK inhibitor-treated embryos and translates into a durable response in the adult.