4.6. Spheroid Size and Morphology Assessment

The spheroids were generated as described above. First, 72 h after seeding, images of each spheroid were captured using a 4× objective in an OLYMPUS IX 83 inverted microscope with XC 50 camera and cellSens Dimension software. Afterwards, 100 µL of culture medium was carefully replaced with fresh medium in control spheroids, or fresh medium with 0.04, 0.05, 0.4, 0.2, 4.5, and 3.0 µM of C-2028, C-2041, C-2045, C-2053, irinotecan, and cisplatin, respectively. Images of spheroids were taken every 2–3 days up to 14 days after drug treatment, and each time the diameters of spheroids were measured using the cellSens Dimension software. Results were obtained from four independent experiments (n = 4). For each compound, at least 8 spheroids were measured during each experiment and the mean value of the spheroid growth was calculated as shown below:

where d_x is the mean diameter of at least 8 spheres at a given day of incubation and d₀ is the mean diameter of at least 8 spheres at day 0 (day of the drug treatment).