Schematic of embryonic zebrafish and injections into the ducts of Cuvier. (A) Fluorescent microscopy image of a 48 h-post fertilization (hpf) Tg(fli1:GFP) zebrafish. The boxed area represents the ideal injection site. The cells will then enter into the circulatory loop, as shown in the light microscopy image. The dorsal aorta, caudal vein, and ducts of Cuvier (DoC) are emphasized with red. (B) mCherry-labelled MDA-MB-231 cells injected into the DoC immediately disseminate throughout the vasculature. Image was taken 2 h-post implantation (hpi). A magnification of the tail region, displaying the vasculature (green), and cells (red) is shown. (C) Typical images of the various stages of cell survival in the zebrafish embryo. Only cells with metastatic potential are capable of undergoing invasion and micrometastasis. MDA-MB-231 cells (red) are shown. (D) A light microscopy image of the tail fin after injection with fluorescent polystyrene beads. Beads become lodged within the end of the circulatory loop. (E) MDA-MB-231 cells (red) displayed invasion as singular cells into the collagen fibres of the tail fin (scale bar = 100 μm). M2 and M4 (shown) displayed an invasive phenotype in with cells collectively cluster together (scale bar = 50 μm). (F) Only tumourigenic cells were capable of displaying invasive properties in the fish. MDA-MB-231 cells were compared to the motile, but non-invasive 3 T3 cells, and the weakly tumorigenic 293 T cells. Scale bar = 100 μm. Data are representative of three independent experiments with at least 50 embryos per group.