Fig. 6

TRCs extravasate more effectively than control melanoma cells in vivo.(a) Schematic of injecting tumor cells into the pericardium of the fish. (b) A low resolution confocal image showing injected tumor cells (white arrow) being moved to the tail (yellow dashed-line box) 1 hr after injection. Scale bar, 1 mm. (c) TRCs and Cont were injected into the pericardium of 48 hpf embryos respectively. Images in each panel (246 µm × 246 µm) corresponding to the yellow dashed-line box area in (b) were acquired with high resolution confocal microscopy (63 × objective), showing vessel penetration of Cont (left panels) or TRCs (right panels) at 1 (yet to penetrate), 12 and 24 hpi respectively and summarized in (d). Acquired images were stacked in z-axis and vessel boundaries were outlined to quantify the cell areas that were out of the vessels as extravasated areas and that were inside the vessels. Dashed white lines mark the tumor extravasated areas (i.e., various sizes of micrometastases) from vessels to surrounding tissues. Insets were 3D-images reconstructed using Imaris software that correspond to respective blue dashed boxes, showing tumor cells stay inside the vessels at 1 hpi and extravasated gradually at 12 and 24 hpi. Scale bars, 100 µm; Insets scale bars, 20 µm. It is of interest to note that the extravasated cells are in general cell aggregates. (d) Quantification of extravasated tumor area relative to the total tumor area at different time points: 1, 12, and 24 hpi. Color code: Zebrafish blood vessels are green, and mouse tumor cells are red. TRCs exhibit higher penetration rates than Cont. (n >6 fish per group; e3 independent experiments). Mean + s.e.m.; *p < 0.05.