Tumorigenic Spheroid Formation Assay

For tumorigenic spheroid formation, approximately 100 cells per well were seeded in an ultra-low attachment 96-well plate (Corning Inc., Lowell, MA, USA). The tumorigenic spheroids were photographed with the HoloMonitoring and confocal microscopy as live images. Cells were suspended in serum-free DMEM/F12 (1:1) culture medium supplemented with B27. Approximately 100–150 cells per well were seeded in an ultra-low attachment 96-well plate (Corning Inc., Lowell, MA, USA). The effect of carcinogenic regimen of 17β-estradiol (E2) was evaluated by E2 treatment (100 pg/mL) on the day of seeding cells. Spheroids were grown for 27 days in liquid culture. A total of 15 spheroids with a minimum diameter of 50 mm were counted in each experimental group. Data were analyzed by ANOVA; Tukey’s HSD test was used for multiple comparisons. Cells obtained from spheroids were analyzed by immunofluorescence, FACS, or immunoblotting, as described previously [7 ,8 (link)].