Initiate Tumors with Single Cell Spheres Formed in Serum-Containing Medium

Background: It is difficult to prospectively identify and maintain putative tumor-initiating cells (TICs). Spheres that formed in serum-free media contained more TICs while spheres formed in serum-containing media were not used in tumor-initiating. Methods: Soft-agar was used to isolate colonies. A continuous, static suspension culture using serum-containing media was modified from liquid overlay technique and tumor cell spheres could be maintained by this method for >90 days. Tumor-initiating capacity of these spheres was tested in nude mice. The nuclear staining of OCT4 in sphere cells and the expression profile of hepatic cell lineage related genes were examined. Results: Soft-agar derived HepG2 colonies indicated different properties from their parental cells. The suspension cells of A549 and MCF7 could initiate tumors at 10(4) cells level. The growth proportions of individual A549, MCF7 and HepG2 spheres with diameter of 101-150 µm were significantly higher than smaller spheres. After suspension culture for 15-27 days, HepG2 and MCF7 spheres could initiate tumors with diameter up to 200 µm; the estimated TIC frequency was 1/1500-1/400. The HepG2 and MCF7 spheres retain tumor-initiating potential for at least 52 days. Conclusion: After 15 days' serum-containing suspension culture small HepG2 and MCF7 cell spheres (diameter ~200 µm) could initiate tumors in nude mice.