Extended Data Fig. 1: Characterization of epithelial and mesenchymal branching in PDAC organoids.
a Organoid morphologies of epithelial (upper panel mouse line ID: 9591, from 3 independent experiments-exception is the concentration 2.5 mg/mL with only 2 independent replicates included) and mesenchymal (lower panel mouse line ID 16992, from 3 independent experiments-exception is the concentration 2.5 mg/mL with only 2 independent replicates included) organoids grown in various collagen concentrations from 1.0 mg/mL up to 2.5 mg/mL, Scale bars, 500 μm. b Quantification of the organoids major axis length in the different collagen concentrations (n = 612 for the epithelial and n = 563 for the mesenchymal), graph represents mean±sem. c Organoid morphologies for epithelial (upper panel mouse line ID: 9591, 3 independent experiments) and mesenchymal (lower panel mouse line ID: 16992, 3 independent experiments) organoids grown in collagen Type I gels with the addition of the ECM proteins fibronectin (FN, concentration 40 µg/mL), laminin (LM, concentration 40 µg/mL) or the combination (FN + LM), Scale bars, 500 μm. d Quantification of the organoids major axis length in the different collagen Type I matrices with the addition of FN, LM or both (n = 378 organoids for the epithelial and n = 388 organoids for the mesenchymal), graph represents mean±sem. e-f Heatmaps of most up and down-regulated genes between epithelial and mesenchymal organoids for the “Reactome ECM degradation” and the “WP MMPs”. g Confocal IF imaging of an EMT panel, first column: E-cadherin (green) /N-cadherin (red) /DAPI (blue), second column: E-cadherin (green) /Vimentin (red) /DAPI (blue), third column: E-cadherin (green) /β-catenin (red) /DAPI (blue), fourth column: ZEB-1 (green) /ZO-1 (red) /DAPI (blue) and fifth column: YAP (green) / phalloidin (white) /DAPI (blue) for epithelial (top) and mesenchymal organoids (bottom), scale bars upper panel (10x magnification)= 200 μm, lower panel-higher magnifications (60x)= 30 μm. Images on the second and fourth row are taken on a 60x objective (higher magnification) of their respective upper panel or from the similar organoid phenotypes. h Gene set enrichment analysis (GSEA) comparing epithelial and mesenchymal 3D organoid cultures, NES= normalized enrichment scores, FDR= false discovery rate. Every bar represents individual genes for the given gene set. i Morphologies of epithelial and mesenchymal organoids in primary, secondary and tertiary structures. Scale bars primary structures= 1000 μm, secondary 1000 for the epithelial and 500 μm for the mesenchymal and tertiary= 500 μm. j Extreme limiting dilution analysis (ELDA) and log plot of nonresponding wells vs. cell dosage for the primary, secondary, and tertiary structures of epithelial and mesenchymal organoids.
