Fig. 9: Single-cell analysis of co-culture spheroids.

a Cell ratio for the 114 co-culture spheroids showed 72 spheroids with MRC-5 cells in the majority (orange), 28 spheroids containing a majority of HeLa Kyoto (purple), and 14 spheroids with an equal number (grey). Ratio of classified objects with the exact number are displayed. The 114 spheroids were divided into 3 groups based on the cell type majority (i.e. HeLa Kyoto Majority, Equal, MRC-5 Majority). The size of the doughnut charts and the numbers in their centre represent the average cell numbers for each group. The average number of cells for each class is displayed with orange for HeLa Kyoto and purple for MRC-5. b Comparison of the two cell lines based on Volume 3D (µm³), the average inter-nuclear, and cell distance of the same class in µm. In this experiment, 114 co-culture spheroids were utilised, and average values were derived by aggregating the results across all constituent cells. Each group showed a statistically significant difference (p < 0.001). c Features (Solidity, Volume 3D, number of cells) collected from the spheroids with different cell ratio were displayed based on the 3 groups (HeLa Kyoto majority n = 28, Equal n = 14, MRC-5 majority n = 72). Dark grey colour represents the average value of the whole dataset (n = 114). Statistically significant differences were identified in two cases, with p-values of <0.0023 (**) and <0.001 (***), respectively. d Spearman’s correlation of the total number of cells and Volume 3D were visualised based on HeLa Kyoto majority (purple), MRC-5 majority (orange), and Equal (grey) groups separately. The variation in sample size (n) across groups was a direct outcome of the cell ratio experiment. n = 114. 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. Source data are provided as a Source Data file.