Extended Data Fig. 6: Time point of CTC isolation is the main determinant of gene expression heterogeneity in CTCs.

a, Heatmap showing the Pearson’s correlation coefficient of PC1-7 eigenvectors from gene expression with technical and biological variables in BR16-CDX CTCs. P values by two-sided Pearson’s correlation test (*P < 0.01, **P < 0.001, ***P < 0.0001). b, Scatter plot showing the correlation of the fold-change between active and rest phase in single CTC (Y-axis) versus CTC clusters and CTC-WBC (X-axis), using genes with FDR ≤ 0.05 in any of the two sets (two-sided Pearson’s correlation coefficient 0.57, P value ≤ 2.22e-16). Points are colored according to the dataset where they were found with a FDR ≤ 0.05 (both, single CTC or CTC clusters and CTC-WBC clusters). The dashed red line represents the linear regression line using all the points in the plot. c, Bar plot showing the number of differentially expressed genes (absolute log₂ fold change ≥ 0.5 and FDR ≤ 0.05) using all the samples (‘All’), using clustered CTCs (CTC clusters and CTC-WBC clusters) and using single CTCs. d, Heatmap showing the pair-wise similarity matrix of enriched gene sets (gene set enrichment analysis (GSEA) adjusted P value ≤ 0.001) using differential expression between CTCs of rest and active phase from NSG-CDX-BR16 mice. Heatmap colors represent the Jaccard similarity coefficient using the set of core genes in each gene set. The heatmap on the right represents the GSEA adjusted P value. e, Plots comparing the normalized enrichment score (NES) and adjusted P value (dot size) obtained using GSEA for gene sets shown in “d”. Gene sets with an adjusted P value ≤ 0.05 in each sample set are highlighted in red. f, GSVA score for translation (yellow, n = 5) and cell division (blue, n = 17) gene sets in CTCs obtained from the NSG-LM2 time-kinetics experiment. Yellow and blue lines represent the average at each time point. Individual points represent the enrichment score for each CTC sample. The white and grey backgrounds represent environmental light (rest period) and dark conditions (active period), respectively. Adjusted F-test P values as obtained from limma are shown for each individual gene set.