Tumor Spheroid Formation Assay

Cells (1 × 10⁵ cells/well) at 10 days after retroviral transduction were seeded to Ultra-Low Attachment Surface 6 well plates (Corning Inc., Corning, NY, USA) in a serum-free DMEM medium containing 10 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan), 10 mg/mL human insulin (CSTI, Miyagi, Japan), 100 mg/mL human transferrin (Roche, Basel, Switzerland), and 100 mg/mL bovine serum albumin (BSA; Nacalai Tesque) and incubated at 37 °C in a 5% CO₂ incubator for 10 days [13 (link)]. Tumor spheroids were manually counted under an inverted phase contrast microscope (BZ-X710 Microscope and BZ-X Viewer, BZ-X Analyzer imaging system, Keyence, Osaka, Japan). All morphometric studies were performed by two examiners blinded to treatment conditions.