Extended Data Fig. 10: Endothelial and epithelial cell identification by scRNA-seq from co-cultures of CRCOs with R-VECs.

a, Schematic of 10X Chromium scRNA-seq experiments of R-VECs alone, R-VECs co-cultured with human CRCOs or CRCOs alone. Samples were analysed 7 days after co-culture. The same compatible medium was used across all three conditions. b, UMAP of cells from each condition alone and the three conditions merged. c, Endothelial cells were identified as cells expressing either VEcad, CD31 or VEGFR2 and negative for the epithelial marker EPCAM. Epithelial cells were defined as positive for EPCAM and negative for any EC markers VEcad, CD31 or VEGFR2. d, UMAP of the 9 unique clusters identified in the merged samples. e, Endothelial and epithelial cell-specific markers were used to confirm the endothelial cell clusters (clusters 6, 7, 8) vs. epithelial cell clusters (clusters 1, 2, 3, 4, 5, 9). f, UMAP of merged epithelial cell fractions from hCRCO cultured alone or co-cultured with R-VECs. Six unique clusters were identified. g, h, Heat map (g) and dot plot (h) of differentially expressed genes in tumour epithelial cells in cluster 2 and cluster 5 that are enriched in co-culture with R-VECs. Differential expression was performed using the Wilcoxon rank-sum test; FDR-adjusted P < 0.05. For statistics, see Supplementary Data 1. For medium formulations, see Supplementary Data 2.