Supplementary Material for: Isolation and Characterization of Spheroid Cells from Human Malignant Melanoma Cell Line WM-266-4

Background/Aims: Spheroid cells which can grow as nonattached spheroids in vitro culture condition are considered as tumor-initiating cells that have properties similar to those of stem cells. However, the existence of spheroid cells in WM-266-4, a human malignant metastatic melanoma cell line, has not yet been reported. Methods: Accordingly, we investigated whether WM-266-4 can form spheroids, and characterized these spheroids using qRT-PCR, histology, immunohistochemistry and xenograft. Results: WM-266-4 contains a small subpopulation of cells that grow as spheroids and express genes strongly related to tumor malignancy and stem-like factors. Second, histological analysis of the spheres revealed that they consist of 300–400 round cells per sphere with a high karyoplasmic ratio. They have a basophilic cytoplasm and are highly pleomorphic in size, and sometimes multinucleated and giant. Third, although there were differences between the spheroid and bulk cells, they both have high tumorigenic potential, as both cell types formed a tumor mass upon injection of only 100 cells in nude mice. Conclusion: We characterized the spheroid cells in an established melanoma cell line. We suggest that enriched spheroid cells might contain more dedifferentiated progenitor cells, but we could not conclude spheroid cells are cancer stem cells.