LLC cell dissemination both in mouse and in Tg(fli1:EGFP) zebrafish tumour models.

(a,b) Tumour cell dissemination and metastases detected after 4 weeks of mice treatments. Representative image of lung and liver metastases are shown in (panel A,B and C,D), respectively. Arrows indicate metastases in lung. (b) Quantification of number of LLC metastases in lung and liver (15–18 mice/group). The data shown are means ± SEM of 5 independent experiments (n = 5). (*p ≤ 0.05, **p ≤ 0.01). (c) Panel A, LLC cells were implanted into 48 h post-fertilization zebrafish embryos. Tumour cell dissemination and metastases were detected at day 4 after injection. Arrows indicate primary tumours, white arrowheads indicate disseminated tumour foci (bar = 500 µm). BF: bright field; GF: green fluorescence; RF: red fluorescence. B, representative 3-D micrographs of confocal images of tumours (red) and vasculature (green). Yellow signal show the vasculature overlapping with tumour cells (Scale bar, 100 µm). (d) Quantification of tumour volume (n = 20/group). (e) Quantification of number of disseminated tumour foci (n = 20/group). (f) Average of maximal distances of metastatic foci (n = 20/group). Data shown are means ± SEM of 3 independent experiments (n = 3). (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001).

DHS inhibits metastasis of LLC cells in a dose-dependent manner.

(a) Fluorescent micrographs of DiI-labeled LLC cells (red) and host endothelial cells (green) in 5 days old fli1:EGFP transgenic zebrafish embryos, 3 days following tumour cell implantation and treatment with vehicle, DHS at 0.01 µM, DHS at 0.1 µM or DHS at 1 µM. The tumour and main metastatic site in the caudal venous plexus are indicated by white boxes and shown in higher magnification below. White arrows indicate metastasized tumour cells. (b) Quantification of the number of metastatic foci in the caudal venous plexus, 3 days after LLC implantation. *p ≤ 0.05, **p ≤ 0.01. n = 20, 15, 22 and 15 for vehicle-treated, DHS 1 µM, DHS 0.1 µM and DHS 0.01 µM groups respectively.