2-O-Bn-InsP₅ inhibits MDA-MB-231 cells dissemination in zebrafish xenotransplant.

(A) Representative tracks showing the swimming behaviour of 6dpf embryos, untreated or treated with 2-O-Bn-InsP₅ (100 µM) and corresponding quantification (B) of the locomotor activity measured. Graph shows the average of the total distance ± SEM covered by each embryos (n = 15 untreated; n = 16 treated). (C) Representative image of 48 h post fertilisation Tg(kdrl:HsHRAS-mCherry)^s896 zebrafish embryos injected with MDA-MB-231 cells stably expressing GFP. Embryos express Cherry fluorescent protein specifically in endothelial cells. Arrows indicate the injected cancer cells into the cardiac chamber. Arrowheads indicate the heart. (D) Zebrafish embryos injected with MDA-MB-231 and treated with or without 2-O-Bn-InsP₅ (100 µM) immunostained using anti-Cherry and anti-GFP antibodies and imaged by high resolution confocal microscopy. GFP-derived fluorescence was quantified in maximal z-stack projections of untreated and treated embryos (untreated n = 7; treated n = 7). (E) Image shows maximal z-stack projections and 3D-rendered surface of the vasculature (red) of zebrafish embryos and metastasis (green) in control and treated embryos. Implantation of MDA-MB-231 cells in zebrafish embryos resulted in widespread tumour cell dissemination and metastasis at 5dpf (white arrows). (F,G) The volume of metastasis and number of disseminated tumour cells (DTC) were evaluated using Imaris. Graphs indicate means ± SEM from n = 6 control and n = 10 treated embryos. Asterisks indicate **P value ≤ 0.01. (H) MDA-MB-231 cells stably expressing GFP were injected into the perivitelline cavity of 48 h zebrafish embryos. 2-O-Bn-InsP₅ (100 µM) was added 3 h after injection into the marine salt water. Graph shows the number of DTC counted at 5dpf and indicates means±SEM from n = 10 control and n = 7 treated embryos.