Sphere-forming cells proliferated extensively and possessed resistance ability to conventional chemotherapeutics in vitro. (A) Comparison of in vitro clonogenecity of the PLC/PRF/5 parental cells and sphere-forming cells. Cells were plated at a density of 2,000 cells/well in 6-well plates and cultured for 1 week. At the end, cells were stained with crystal violet, photographed, and analyzed for their proliferation efficiency. Each experiment was performed in triplicate, and the representative examples were shown (Columns, mean; bars, SD; *, P < 0.01). (B) The PLC/PRF/5 sphere-forming cells showed a drug resistance phenotype. The parental cells and sphere-forming cells were treated with cisplatin at the beginning of plating for 12 h or 24 h and 5-Fu, gemcitabine, mitomycin and sorafenib for 36 h. Cell survival was determined by MTT assay (**, P < 0.01).