Fig. 1: PDAC subtypes give rise to morphologically distinct branching organoids.
a, Schematic representation of the preparation of PDAC organoid cultures in floating collagen gels. Image created with BioRender.com. b, Confocal IF imaging of the organoid cytoskeleton (purple) and DAPI (blue) for epithelial and mesenchymal organoids. Images are maximum projections generated with Imaris. Scale bars, 200 µm (for mesenchymal), 500 µm (for epithelial organoids). c, Daily imaging of single-cell-derived organoids from the epithelial line (ID: 9591) and the mesenchymal line (ID: 16992) over the course of 13 days of development (here displaying Days 3–13). Scale bars, mesenchymal: 200 μm (Days 3–9), 500 μm (afterwards); epithelial: 200 μm (Days 3–5), 500 μm (afterwards). d, Major axis length of the organoid development of n = 1,099 epithelial (from the mouse line ID: 9591, 3 individual experiments) and n = 904 mesenchymal (from the mouse line ID: 16992, 3 individual experiments) organoids. Plot presents mean ± s.e.m. e, Confocal IF imaging of the proliferation marker Ki67 (green) and DAPI (blue) for epithelial (from the mouse line ID: 9591, 3 individual experiments) and mesenchymal (from the mouse line ID: 16992, 3 individual experiments) organoids. Scale bars, 100 µm (Day 3) and 200 µm (Day 5 onwards) for mesenchymal panel; 50 µm (Day 3) and 200 µm (Day 5 onwards) for epithelial panel. f, Confocal IF imaging of the epithelial marker E-cadherin (green), the mesenchymal marker N-cadherin (red) and DAPI (blue) for mesenchymal (n = 3 independent mouse lines; IDs: 8028, 9091, 16992) and epithelial (n = 3 independent mouse lines; IDs: 8442, 9591, 53631) organoids. Scale bars, 200 µm. g, Hierarchical clustering of RNA sequencing data from epithelial and mesenchymal 3D organoids derived from different KrasG12D background mice. Tumour grading, Kras mRNA levels retrieved from a previous study15. Heat map of the leading-edge genes for both clusters. h, H&E staining of the primary tumours and corresponding brightfield organoid morphologies (n = 3 mouse lines for mesenchymal, n = 3 mouse lines for epithelial). Scale bars, 200 µm (H&E), 500 µm (organoids). i, ELDA of epithelial and mesenchymal organoids (left) and plot of the log fraction of non-responding wells (without organoids) versus the number of seeded cells (right). j,k, GSEA comparing 2D mesenchymal and 3D mesenchymal organoids (j) and 2D epithelial and 3D epithelial organoids (k). Every bar represents individual genes for the given gene set.
