Fig. 2

A TEAZ platform to screen for metastasis modulators in vivo. A, Generation of a baseline BRAFV600E tp53^−/−pten^−/− metastatic melanoma model using TEAZ. MAZERATI 2X (MAS2X) sgpten plasmid was coelectroporated with a mitfa:GFP plasmid to generate loss of pten in a melanocyte-specific manner. mets, metastases. B, Representative fluorescent image of an adult zebrafish with a BRAFV600E tp53^−/−pten^−/− tumor 16 weeks after TEAZ. C, Quantification of tumor incidence in animals expressing a nontargeting (NT) sgRNA or pten sgRNAs represented as percent of fish with tumors. Fisher two-sided t test was performed on data from n = 3 independent experiments. ***, P = 0.003. D, Sagittal sectioning of animals with melanomas for IHC. E, Histology of BRAFV600E tp53^−/−pten^−/− melanomas demonstrating tumor cell expression of GFP, BRAFV600E, and activated Akt signaling. Scale bar, 60 μm. H&E, hematoxylin and eosin. F, IHC staining for GFP in muscle (left images) and kidney marrow (right images) of animals bearing BRAFV600E tp53^−/−pten^−/− showing the presence of invading tumor cells (scale bar, 100 μm) and distal metastasis (scale bar, 60 μm), respectively. Arrows indicate metastatic tumor cells. G, Representative images of animals with NT or alb KO melanomas at 6 weeks after TEAZ (top, fluorescent GFP imaging at 25×) and 12 weeks after TEAZ (bottom, brightfield image at 3.5×). Tumor is indicated by a black arrow in each group. Scale bar on the graph. BF, Brightfield. H, Quantification of the presence of melanin in animals expressing a NT sgRNA or alb sgRNAs represented as percent of fish with melanin. Fisher two-sided t test was performed on data from n = 3 independent experiments. *, P < 0.05.