Colorectal Tumor Sphere Formation Assay

The sphere-forming ability of colorectal tumor cells was evaluated by sphere formation assay as previously reported [22 (link)]. Briefly, one single tumor cell was sorted into one well of the Corning® Costar® ultra-low attachment 96-well plate (Sigma-Aldrich), which was filled with 200 μL of Dulbecco’s modified Eagle medium/F12 medium supplemented with 20 μg/mL fibroblast growth factor-basic, 1× B27, and 20 μg/mL epidermal growth factor (Life Technologies, Waltham, USA). Cells were cultivated in a 5% CO₂/37 °C for 7 days. The number of spheres with a diameter of >50 μm was counted and normalized to the Control group.