4.14. Zebrafish Xenograft Assay

The zebrafish embryos were staged for cell xenotransplantation at 48 hpf. Embryos were de-chorionized using micro-forceps, anesthetized in a 1:10 dilution of 0.003% Tricaine-S (Western Chemicals Inc, AZ, USA) and positioned on a wet 1.0% agarose pad for cells injection. U87 cells were harvested by trypsinization and washed with PBS at room temperature. Cells were labeled with 2.5 µl/mL CellTracker™ CM-DiI Dye (Thermo Fisher Scientific, MA, USA) diluted in PBS. Cells were then incubated in the dark for 4 min at 37 °C followed by 15 min at 4 °C and washed twice with PBS. Subsequently, cells were counted, resuspended in MEM medium and approximately 125 cells per embryo were injected into the center of the yolk sac with a microinjector (FemtoJet 4i, Eppendorf, Germany) equipped with borosilicate glass capillaries (Femtotips, Eppendorf, Germany) and micromanipulator (MM-3, Narishige, Japan). Next, the injected embryos were observed under a fluorescent microscope Olympus BX51 (Olympus Corporation, Japan) and only successfully injected embryos were used further for experiments. Selected embryos were transferred to a 96-well cell culture plate (one embryo per well) containing 200 µL of embryo medium containing PTU and maintained at 32 °C

After 24h, at 1 dpi, the injected embryos were imaged with a fluorescent microscope and divided into three groups: control and groups treated with 2.5 µM Si306 or pro-Si306. Each group contained 32 embryos. After 72 h, at 4 dpi, each embryo was imaged again with a fluorescent microscope and the percentage of embryos exhibiting cancer cell dissemination from the injection site, the number of disseminated cells, and fluorescence intensity of CM-DiI-labeled xenografted cells were quantified in ImageJ software. Invasion was defined as 5 or more disseminated cells found outside of the vasculature [82,83]. The experiment was repeated three times.