Fig. 8: Patient-derived organoids develop heterogeneous phenotypes in basal branching PDO media.
a, Daily imaging of single-cell-derived organoids over the course of 13 days of development (here representing Days 3 and 8–13). The PDOs were cultured in full PDO media (top; n = 342 organoids) and in basal branching PDO media (bottom; n = 573 organoids). Scale bars, 200 µm (full PDO media); 200 µm (Days 3–11) and 500 µm (from Day 12 onwards) for the basal branching PDO media. b, Graphical summary of the organoid development in the basal branching PDO media with the media composition and the different developmental phases. c, Characteristic PDO organoid morphologies (n = 3 PDO lines) cultured in different conditions: full PDO media (top), base PDO media (middle), basal branching PDO media (bottom) and H&E staining of the corresponding primary tumours. Scale bars, 100 µm (PDO line ID B320 cultured in full PDO media and base PDO media), 200 µm (all others). d, Confocal IF imaging of phalloidin (white) and DAPI (blue). Scale bar, 200 µm, illustrating the different morphologies the PDO line ID B250 displays in basal branching PDO media. e, PCA of bulk RNA sequencing for the PDO lines (IDs: B211, B250 and B320) in full PDO media (pink colour) and basal branching PDO media (lilac colour). f, Gene set variation analysis (GSVA) of the basal/quasi-mesenchymal profile48,49,50 of PDO lines cultured in full PDO media and basal branching PDO media. Unpaired two-tailed parametric t-test with Welch’s correction, two-tailed. g, Heat map of bulk RNA sequencing for the PDO lines (IDs: B211, B250 and B320) for the hallmarks: Glycolysis, Apical Junction, Hypoxia, EMT, Oxidative phosphorylation, EMP and Cancer EMT characterizing the individual PDOs cultured in full PDO media or in basal branching PDO media. h, K-means clustering of the image-derived features of unseen data set of n = 832 organoids reveals 2 distinct morphological clusters when PDOs were cultured in full PDO media. i, K-means clustering of the image-derived features of unseen data set of n = 834 organoids reveals 2 distinct morphological clusters when PDOs were cultured in base PDO media. j, K-means clustering of the image-derived features of unseen data set of n = 928 organoids reveals 5 distinct morphological clusters when PDOs were cultured in basal branching PDO media. k, Individual PDO line (ID: B211 (left), ID: B250 (middle), ID: B320 (right)) grown in basal branching PDO media, with morphological heterogeneity as visualized by density overlays superimposed on the imaged-derived clusters. The overlays indicate which cell lines correspond to which cluster. l, Characteristic morphologies of organoids grown in basal branching PDO media as control or after pre-treatment with FFX IC50 values. Scale bars, 200 µm. m, Clustering of PDOs (superimposed on the K-means clustering from j) after pre-treatment with IC50 values of FFX and then culturing in basal branching PDO media.
