Generating Metastatic Tumor Cells from B16 Melanoma

Mouse B16-F10 cells were purchased from Wuhan Boster Biology Technology, Ltd. (Wuhan, China); mouse B16-F1 cells and mouse 3T3 cells were from China Center for Type Culture Collection (Wuhan, China). tfRFP-expressing B16-F10 cells were obtained by stably transfecting B16-F10 cells with a plasmid containing the KatushkaS158A gene17 (link). B16-F1, tfRFP B16-F10, and 3T3 cells were cultured on rigid dishes with MEM, 1640 and DMEM cell culture medium (HyClone) respectively supplemented with 10% fetal bovine serum (Invitrogen), and 1% penicillin/streptomycin at 37 °C with 5% CO₂. Cells were passaged every 2–3 days using TrypLE (Invitrogen). TRCs were mechanically selected by being cultured in the 3D soft fibrin gels (90-Pa) for 5 days12 (link) from B16 cell lines and both were more metastatic than their respective counterpart control cells. Dexamethasone (Dex), Latrunculin A (Lat A), retinoic acid (RA), dimethyl sulfoxide (DMSO), and ethanol were from Sigma. Angiogenic and vasculogenic blocker SU5416 was purchased from Selleck. Its stock solution was 10 mM in DMSO. The drug was diluted 5000 times such that its final working concentration was 2 μM with 0.02% DMSO in fish medium. After 1 hr incubation with the drug, fresh medium was added to wash out the drug. The drug-treated embryos appeared slightly yellowish, suggesting good absorption.