Tumor Sphere Formation and in vitro Limiting Dilution Assay

For tumor sphere formation assay, single-cell suspensions were seeded in 6-well ultra-low attachment plates (Corning Inc., Corning, United States) at a density of 10,000 cells per well using serum-free DMEM/F12 (Hyclone) containing 20 ng/ml of basic fibroblast growth factor (Miltenyi Biotec), 20 ng/ml of epidermal growth factor (Miltenyi Biotec, Auburn, United States), and 2 mM L-glutamine (Mediatech Inc.). After culturing for 14 days, the number of formed tumor spheres was counted and pictured using a microscope. For in vitro limiting dilution assay, dissociated single U87 and U251 cells were seeded in ultra-low attachment 96-well plates at the density of 5, 10, 20, 50, 100, or 200 cells per well. After 14 days of culture without serum, spheres formatted were counted in each well, and sphere formation frequency was calculated using online in vitro limiting dilution analysis¹.