Fig. 1: Alternate phenotypes occur in parallel within 3D cultures.

a Schema, heterogeneous spheroids imaged in high-throughput over time. Size, shape and movement characteristics extracted for thousands of spheroids. Machine learning used to classify user-defined phenotypic states, frequency of which was quantified over time. b Representative phase images of spheroids exhibiting variable morphology over time. n = 3 independent experiments, 3 wells/condition/experiment. Scale bars, 50μm. c Proportion of PC3 spheroids exhibiting user-defined classification states. Shaded region, s.e.m. across experiments. n = 3 independent experiments, 3 replicates/condition/experiment. Total number of spheroids quantified in Supplementary Table 3. d Representative phase images of spheroids. Outlines, user-defined state classification. Scale bar, 100μm. Time-lapse of boxed regions shown. Arrowheads and schema indicate changes in classification over time. Scale bars, 50 μm. n = 3 independent experiments, 3 wells/condition/experiment. e Schema of PC3 subline derivation. PC3 were selected in vitro for epithelial shape (PC3-Epi), high surface E-Cadherin (E-cad+) or mesenchymal characteristics after macrophage co-culture (PC3-EMT). PC3 were injected into murine tail veins and harvested from alternate metastatic sites; GS689.Li (liver), GS672.Ug (urogenital tract) and GS694.Lad (adrenal gland, after in vivo injection of PC3 JD549.Ki). Sublines were isolated after serial passage across endothelial barriers (TEM2-5 vs TEM4-18). TEM4-18 were injected into tail veins and cells harvested from lymph node (GS683.LALN) and lung mets (JD1203.Lu). f Representative phase images of PC3 subline spheroids, 72 h. Outlines, user-defined state classification. n = 3 independent experiments, 3 wells/condition/experiment. Scale bar, 100μm. g Representative outlines of phenotypes formed by PC3 sublines. h Quantitation of PC3 and sublines. Heatmap shows Area as mean of Z-score normalised values (purple to yellow), and classification into Round, Spread or Spindle as a Log2 Fold Change from control (PC3) (blue to red). Proportion of control at each timepoint is also Z-score normalised (white to black). Bubble size represents p-values, Student’s t-test (two-sided) and Cochran-Mantel-Haenszel test, Bonferroni adjusted, to compare Area and proportion of each classification to control respectively. Dot represents p-value, Breslow-Day test, Bonferroni-adjusted for homogeneity of odds ratio across experimental replicates. n = 3 independent experiments, 3 wells/condition/experiment. Number of spheroids quantified in Supplementary Table 3.