Morphological analysis of spheroids

Morphological features of treated spheroids were analyzed based on phase-contrast images of spheroids captured using Olympus BX-51 light microscope with a × 10 magnification objective, equipped with an Olympus E-410 digital camera (Olympus® Corporation, Tokyo, Japan). ImageJ software was used for determination of changes in morphology and area of spheroids after 24 h and 48 h of treatment. Images were converted to 8-bit grayscale mode, and modification of contrast and brightness was done. The area (A) of pseudo-circular spheroids was obtained by manualy drawing a border around spheroids (two spheroids for each treatment and control) three times, and determined as an average value of three measurements of duplicates. Diameter and volume of spheroids were then calculated using the formula for area of the 2D projection (determined by ImageJ) of the spheroids (A=r2π, r=A/π, V=4/3πr3). Effects of the extracts were compared to the control sample and the effect of cisplatin. The statistical significance of differences between the control spheroid volume change and volume change of spheroids treated with extracts over time was evaluated using two-way ANOVA with Dunnett’s post test (GraphPad Prism version 9.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com). p values below 0.05 were considered statistically significant.