Fig. 7: Targeted therapy reduces phenotypic heterogeneity via phenotypic reprogramming.
Targeted therapy treatment and transcriptomic analysis of individual organoid phenotypes isolated from the E-mouse line ID: 9591 and the M-mouse line ID: 16992. a, Pie chart of the library design (n = 102 drugs) with the drug approval status: preclinical, phase 1, 2, 3, 4 and FDA approved. b, Pie chart of the specific targeted pathways by the 102 drugs. c, Schematic summary of the drug-treatment workflow; image created with BioRender.com. d, Heat maps of the z-score for specific drugs from the 102-drug screening of the 2D epithelial (4 phenotype clones and the bulk population from the mouse line ID: 9591, n = 2 individual experiments) and mesenchymal (3 phenotype clones and the bulk population from the mouse line ID: 16992, n = 2 individual experiments) cells. e, Brightfield imaging of bulk organoid morphologies post treatment with selective drugs using the IC50 values of the most sensitive 2D clones in 3D. Scale bars, 200 µm (mesenchymal treated with JIB-04 ), 500 µm (all others). f, Manual phenotypic analysis of epithelial bulk (n = 397 organoids from 3 individual experiments) and mesenchymal bulk (n = 246 organoids from 3 individual experiments) organoid populations after the selective treatment. Bar plot represents the average number of organoid phenotypes (%). g, Brightfield images of epithelial control (n = 334 organoids, 3 individual experiments) and combinatory treatment with AZD5153+Poziotinib (n = 321 organoids, 3 individual experiments) organoids from the bulk population, TEBBO, cystic branched, thick branched and tree-like phenotypes. Scale bars, 500 µm. h, Major axis length (µm) of bulk epithelial organoids as control (n = 51 organoids from 3 individual experiments) and organoids treated with AZD5153+Poziotinib (n = 49 organoids from 3 individual experiments). Graph represents mean ± s.e.m., unpaired two-tailed parametric t-test with Welch’s correction, two-tailed. i, O-SFUs per gel of control and AZD5153+Poziotinib-treated (in pink) epithelial organoid phenotypes; graph represents mean ± s.e.m. of 3 individual experiments. j, Manual phenotypic analysis of control and AZD5153+Poziotinib-treated bulk organoid populations of bulk epithelial organoids (n = 107 organoids). Pie chart represents the average number of organoid phenotypes (%). k, PCA analysis of the bulk RNA sequencing from control and AZD5153+Pozitionib-treated bulk, TEBBO, cystic branched, thick branched and tree-like organoids. Each dot represents the mean of 3 individual experiments. Dashed circles highlight the organoid phenotypes under the combinatory treatments. l, Dot plot of the GSEA comparing the epithelial control (from all phenotypes) and AZD5153+Poziotinib-treated (from all phenotypes) epithelial organoids. Padj, Benjamini–Hochberg adjusted P values. m, Mesenchymal control and RO5126766-treated organoid morphologies for the bulk population, branched mesenchymal, firework and star-like phenotypes. Scale bars, 500 µm. n, Major axis length (µm) of bulk mesenchymal organoids as control (n = 93 organoids from 3 individual experiments) and after treatment with RO5126766 (n = 67 organoids from 3 individual experiments). Graph represents mean ± s.e.m., unpaired two-tailed parametric t-test with Welch’s correction, two-tailed. o, PCA analysis of the bulk RNA sequencing from control and bulk, branched mesenchymal, firework and star-like organoids treated with Birinapant, Poziotinib, Saracatinib or RO5126766. In addition, we included an earlier time point for the star-like organoids (Day 10). Dashed circles highlight the organoid phenotype transitions under specific treatments. Each dot represents the mean of 3 individual experiments (except for the branched mesenchymal+RO5126766 with 1 replicate). p, Dot plot of GSEA comparing the mesenchymal control (from all phenotypes) with the RO5126766-treated (from all phenotypes) mesenchymal organoids. q, Graphical summary of the distinct epithelial and mesenchymal organoid phenotypes and how mono or combinational treatments can either plastically switch morphologies or reveal persister phenotypes. Br. mesenchymal, branched mesenchymal.
