2.5. Colony-Forming and Tumor Sphere-Forming Assays

The colony-forming assay was carried out in the following conditions. Briefly, colon cancer cell lines, HCT116 and DLD-1 cells were seeded in six-well plates with (500 cells, 2.8 μM ovatodiolide, equivalent of IC10 values) and without ovatodiolide. The plates were then stained using 0.005% crystal violet, and the colonies were counted. The cells were allowed to grow for another week. The cells were then harvested, fixed, and counted. The migratory ability of the cells was examined using the Transwell migration assay (ThermoFisher, Taipei, Taiwan). To evaluate the self-renewal ability of cancer cells, we used a sphere-forming assay. Colon cancer cells were cultured under serum-deprived conditions and using Ultra-Low Attachment Plates (Corning Inc., Taipei, Taiwan). The culture conditions were modified slightly from Lo et al. [18]. Colon cancer cells (density: 10⁴ cells/mL) were cultured in a medium composed of 20 ng/mL epidermal growth factor (EGF), 10 ng/mL basic fibroblast growth factor (BFGF), 5 μg/mL insulin, and 0.4% Bovine Serum Albumin (BSA). After approximately 5–7 days of culture (depending on the cell type), tumor spheres were formed, and the numbers were counted under a phase-contrast microscope (40× magnification). The self-renewal ability was represented by the average number of spheres generated. The average sphere number formed was obtained from three different views.