Fig. 3

The effect of LH on extravasation of RFP-HepG2 cells in a metastasis model in Tg(flk1:EGFP) transgenic zebrafish. (A and B) Tg(kdr1:EGFP) transgenic zebrafish at 3 days age, the arrows indicated the position where RFP-HepG2 cells were injected. A: white light; B: GFP labeled vascular system. (C) RFP -HepG2 cell entered the internode small vessels (ISV). RFP-HepG2 cells pretreated with empty liposome or LH (40 μM) for 6 hours were re-suspended in FCS-free DMEM media (2 × 10⁷/ml) and kept on ice for injection. RFP HepG2 cells were injected into the blood circulation (50–100 cells per fish) and then imaged at 0 h. Up: Image of RFP-HepG2 cells in ISV (200×). Below: Enlarge images showed RFP-HepG2 cells in ISV. (D and E) The extravasation of RFP-HepG2 cell in Tg (flk1:EGFP) zebrafish were observed and counted under a Zeiss fluorescent microscope (200×) at 4 h (D) and 24 h (E) after cell injection respectively. More than 20 fishes containing about 160 cells were analyzed for each group. D (up) showed the whole ISV image at 4 h, D (below) showed enlarged ISV image at 4 h. E (below) showed the whole ISV image at 24 h. E (UP) showed enlarged ISV image at 24 h. Apop: Apoptosis cells; EX: extravasation cells; Frag: fragmented cells. (F) The identification of extravasation, apoptosis and fragmented cells at the time point of 24 h. (G and H) Quantification of the average number of extravasation, apoptosis and fragmented cells in control and LH-pretreated cells injected zebrafish (n =20). Data represent the mean ± SE (*P < 0.05,**P < 0.01, compared with the control group).