Zebrafish embryo extracts promote sphere-forming abilities of human melanoma cell line

Sphere-forming abilities in culture condition are considered a hallmark of cancer stem-like cells, which represents tumor cell invasiveness and stem-like characteristics. We aimed to show that the sphere-forming subpopulation of human malignant melanoma cell line WM-266-4 acts differently to zebrafish embryo extracts compared with their bulk counterpart. Spheres were maintained in neural stem cell culture conditions. The embryos of zebrafish at specific developmental stages were collected and the extracts were purified under 100 kDa. Spheres were treated with embyo extracts and proliferation assay and immunocytochemistry were conducted. Spheroid cells expressed nestin and epidermal growth factor receptor (EGFR) but not melanoma antigen recognized by T-cells (MART)1, indicating their stem-like character. Zebrafish embryo extracts at 50% epiboly stage inhibited melanoma bulk cell proliferation in a dose-dependent manner. However, sphere-forming abilities were significantly enhanced under 1 µg/mL concentration of 50% epiboly stage embryo extract treatment. Our findings implicate that we should consider cell subsets of a different character from the tumor origin that can respond differently to exogenous substances or tumor microenvironments. We suggest that cancer research should consider both minor stem-like subpopulations and the other major bulk tumor cells. (Cancer Sci 2009)