Figure 3.

Co-culturing increases the fraction of cancer cells with reduced proliferation and partial EMT. (A) FACS sorting of organoids and CAFs from the mono- and co-culture conditions. Organoids were selected based on their GFP expression, CAFs were selected based on their Cellbrite red expression. Organoid = P19bT, CAF = LCAF5. (B) UMAP plots of single cell RNA seq data from organoids and CAFs from the mono- and co-culture conditions. CAF co = LCAF5 originating from the co-culture droplet, CAF mono 2D = LCAF 5 grown on the plastic of the CAF mono-culture condition, CAF 3D = LCAF5 grown on the matrix droplet of the CAF mono-culture condition, Organoid co = P19bT originating from the co-culture droplet, Organoid mono = P19bT originating from the mono-culture droplet. Graph based clustering analysis defined 3 organoid clusters and 3 CAF clusters. (C) Feature plot showing EPCAM and Vimentin expression. (D) Bar charts with cell type distribution of the 3 organoid clusters across the experimental conditions. (E) Violin plots showing the expression of Hypoxia and Glycolysis hallmark gene signatures across the 3 organoid clusters. (F) Violin plots showing the expression scores of S-phase marker genes (N=43) and G2/M-phase marker genes (N=54) across the 3 organoid clusters. Cluster 2 shows the highest expression of cell cycle genes. (G) Violin plots showing the expression of epithelial and mesenchymal genes across the 3 organoid clusters. Cluster 3 shows a lower expression of epithelial genes and a higher expression of mesenchymal genes compared to cluster 1 and 2. (H) Violin plot showing the expression of the Epithelial to Mesenchymal (EMT) Hallmark gene signature across the 3 organoid clusters. Cluster 3 cancer cells show the highest EMT gene signature scores. (I) Violin plot showing the module score of the CMS4 Random Forest (CMS4 RF) classifier genes (N=143). Cluster 3 shows the highest CMS4 RF signature score. ns, non-significant, Ns = p > 0.05, ** = p < 0.01,**** = p < 0.0001.