Fig. 3: Characterisation of monoculture and co-culture datasets using 2D features.

a Spider plot representation of the relative differences of features displayed as a percentage, acquired by annotating the same spheroids, imaged by 4 different objectives with different magnifications. Each objective was compared to the annotations that were executed on the images acquired with the 20x objective. After manual annotation, features such as Diameter, Perimeter, Area, Volume 2D, Circularity, Sphericity 2D, and Convexity were displayed (n = 50). b Beeswarm plot of the monoculture (grey) and co-culture (light blue) spheroids divided by experts (vertical distribution) and experiments (orange - Experiment 1, green - Experiment 2, purple - Experiment 3), indicating the volume and sphericity of each sample. Overall, 426 spheroids were screened. Each circle represents a spheroid on a volume axis showing the sphericity values by the size and intensity (bigger and more intensive circles represent the most spherical spheroids). The number of spheroids evaluated in each batch is displayed, with different colours representing individual experiments and black indicating the total count. c Boxplot visualisation of the monoculture (purple, n = 223) and co-culture (orange, n = 203) spheroids based on Diameter (µm), Area (µm²), and Circularity (−). Among the three measurements, statistically significant differences were observed in two cases (p < 0.001, ***), and one case showed significance with p < 0.0255 (*). d Size comparison of spheroids for each experiment (n ≥ 16, see Fig. 3b). Significant differences are not visualised. Dashed lines show the overall average of monoculture and co-culture experiments. e Shape comparison of spheroids for each experiment (n ≥ 16, see Fig. 3b). For the statistical analysis, a two-sided non-parametric Kruskal-Wallis test was conducted, followed by Dunn’s multiple comparison test. When comparing only two groups, the two-sided Kolmogorov-Smirnov test was utilised. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Box plots were visualised using Tukey’s method, with medians shown as central lines, boxes representing interquartile ranges, and whiskers extending to 1.5× interquartile range. Abbreviations: M - monoculture, C - co-culture, Exp - experiment. Source data are provided as a Source Data file.