Spheroids Assay for Synergistic Drug Effects

HCT116 cells were resuspended in serum-free stem cell culture media consisting of DMEM/F12 supplemented with 10 ng·mL⁻¹ of bFGF, 20 ng·mL⁻¹ of EGF from Biotechne (Citomed Lda, Lisboa, Portugal), 1 × B27 from Life Technologies (Porto, Portugal), and 5 μg·mL⁻¹ of insulin (Sigma-Aldrich). HCT116 cells were plated in 24-well ultra-low attachment plates (one spheroid per well; Corning Inc., Sigma-Aldrich), at a density of 1 × 10³ cells/well [48 (link)]. To assess the synergistic effect of SLMP53-1 with DCA in spheroids development, colonospheres were allowed to form for 3 days, followed by treatment with 20 μM SLMP53-1 and/or 12 mM DCA, for an additional 9 days. During this period of time, new medium with the drugs (or DMSO only) was added to the wells each three days. Spheroids were photographed by using an inverted Nikon TE 2000-U microscope at ×100 magnification, with a DXM1200F digital camera and NIS-Elements microscope imaging software (VWR). Determination of spheroids diameter was performed by using Image J software.