Fig. 2: Canonical TGFβ signalling is essential for the formation of branching PDAC organoids for both epithelial and mesenchymal subtypes.

a, Morphological effect of major developmental pathways: EGFR by AG1478 (EGFR inhibitor) and EGF, Wnt/β-catenin by Wnt3a, XAV393 (Tankyrase inhibitor), iCRT14 (β-catenin-Tcf inhibitor), Notch (γ-secretase inhibitor), HGF, and Hedgehog by Ihh, Shh, GANT61 (GLI antagonist) and SANT-1 (Smo antagonist) on epithelial (n = 1,045 organoids, line ID: 9591, 3 individual experiments) and mesenchymal organoids (n = 873 organoids, line ID: 16992, 3 individual experiments). Treatments were performed with 10 µM AG1478, 5 ng EGF, 100 ng Wnt3a, 5 µM XAV939, 5 µM iCRT14, 5 ng HGF, 10 µM DAPT, 10 µM GANT61, 100 ng Ihh, 100 ng Shh, 2 µM Sant-1. Scale bars, 500 µm. b, GSEA of epithelial (n = 3 mouse lines, from 3 individual experiments) and mesenchymal (n = 3 mouse lines, from 3 individual experiments) 2D monolayers and 3D organoids for the TGFβ pathway. c, Heat maps of most up and downregulated genes between 2D monolayers and 3D cells for epithelial and mesenchymal organoids (from left to right). d, Monotreatments of Kras^G12D epithelial (n = 468 organoids, line ID: 9591, 3 individual experiments) or mesenchymal (n = 618 organoids, line ID:16992, 3 individual experiments) organoids with 5 ng of TGFβ-1; treatment administration from Day 0, Day 7 or Day 11 after seeding, 1× StemXVivo EMT-inducing media supplement (termed STEMX) treatment from Day 0, or 5 µM TGFβ-RI inhibitor A83-01 treatment from Day 0. All organoids were imaged on Day 13. Scale bars, epithelial organoids: 200 µm (all A83-01), 500 µm (all others); mesenchymal organoids: 200 µm (all STEMX and the bottom A83-01), 500 µm (all others).