Fig. 2: Identifying the repertoire of distinct states that occur in 3D cultures. | Nature Communications

Fig. 2: Identifying the repertoire of distinct states that occur in 3D cultures.

From: Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging

Fig. 2

a Schema, analysis of heterogeneous spheroids, regardless of temporal order, enables data-driven subtype classification, visualisation of phenotypic space, and frequency relative to control. b t-SNE of states in PC3 and sublines. Plot points coloured by data-driven state classification. Black dashed lines highlight regions corresponding to data-driven states mentioned in text. Data comprised of each spheroid identified in each image of the experiment. Number of spheroids quantified in Supplementary Table 3. t-SNE analysis performed on 20,000 objects subsampled via GeoSketch, with iterations = 2000, theta = 0.5, perplexity = 50. c t-SNE of the relative distribution of spheroids for PC3 and sublines. Plot points coloured by user-defined state classifications, and data-driven state enrichment: purple-to-yellow shows per sample proportion of total objects in each data-driven state, quantified before t-SNE. Black dashed lines, highlight regions corresponding to data-driven states mentioned in text. Data comprised of each spheroid identified in each image of the experiment. Number of spheroids quantified in Supplementary Table 3. t-SNE analysis performed on 20,000 objects subsampled via GeoSketch, with iterations = 2000, theta = 0.5, perplexity = 50. d Proportion of PC3 spheroids exhibiting each data-driven state classification over time. Shaded region represents s.e.m. across experiments. n = 3 independent experiments with 3 wells/condition/experiment. Number of spheroids quantified in Supplementary Table 3. Representative outlines shown, arranged by average movement, and coloured by average size (green scale). e Quantitation of data-driven state classifications in PC3 subline pairs. Representative outlines shown, arranged and coloured (light to dark green) by average class motility and size, respectively. Heatmap shows classification of spheroids as a Log2 Fold Change from control (PC3) (blue to red). Proportion of control in each class is shown (white to black). Bubble size represents p values, Cochran-Mantel-Haenszel test (Bonferroni-adjusted), to compare each classification to control. Black dot represents p-value, Woolf test (Bonferroni-adjusted), for homogeneity of odds ratio across experiments. n = 3 independent experiments, 3 wells/condition/experiment, number of spheroids quantified in Supplementary Table 3.

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