Extended Data Fig. 2: Lung TOs preserve the histological, proteome, and phenotypic landscape of their parental tumours and enable individualized proteome profiling.
From: Lung tumouroids as a testing platform for precision CAR T cell therapy
a, Immunohistochemical (IHC) staining of lung cancer-associated markers in squamous cell carcinoma TT and matched TOs from patient 12, patient 17 and patient 18. Antibodies include TTF1, p40, p63, CK5/6, and CK7. Positive staining is shown in brown, with hematoxylin counterstaining in blue. IHC staining was performed on n = 2 independent TO passages and n = 1 TT tissue section. Scale bars, 100 μm. b, Flow cytometry analysis. Left: Gating strategy for TT, HT and matched TO and HO to identify epithelial cells (CD45−, EPCAM+), leukocytes (CD45+, EPCAM−), and stromal cells (CD45−EPCAM−). Right: Bar plot showing mean expression + SD of lineage markers for epithelial cells, leukocytes, and stromal cells; points indicate individual samples. c, Bar plot displaying combinations of cancer stem cell markers in TO as percentages of EPCAM+ cells. d, Venn diagrams showing shared and unique protein identifications in TO (top) and HO (bottom). e, Dot plots of the top 20 significantly enriched Gene Ontology (GO) pathways based on distinct protein subsets. Statistical analysis: GO enrichment performed using clusterProfiler with hypergeometric test (enrichGO function), adjusted for multiple comparisons using Benjamini-Hochberg method. Significance threshold: adjusted P value (q-value) < 0.05 f, UpSet plot illustrating protein distribution after exclusion of TT-, HT-, and TT/HT-unique proteins. g, Single-sample gene set enrichment analysis (ssGSEA) of ranked protein data in HO and HT, represented as normalized enrichment scores (NES); HO indicated by purple dots, HT by green bars. h, Volcano plots showing differential protein expression between TT vs. HT (top) and TO vs. HO (bottom) using the NSCLC Protein Atlas subset. Statistical analysis: limma moderated t-test with empirical Bayes variance estimation, two-sided, adjusted for multiple comparisons using Benjamini-Hochberg method. Significance threshold: adjusted P value < 0.005 and absolute fold change > 0.5 log2. i, Heatmap of enriched pathways from ssGSEA using the NSCLC Protein Atlas subset for TT vs. HT and TO vs. HO comparisons (log2 fold change > 0.5), referenced against the Molecular Signatures Database. j, Pathway enrichment analysis comparing TT vs. HT (blue) and TO vs. HO (green), integrated with the top 20 mutations identified by targeted DNA sequencing, with variant allele frequencies (VAFs) color-coded and enrichment shown as mean absolute ssGSEA scores. k, Heatmaps of log2-transformed protein intensities for the NSCLC Protein Atlas (top) and Cancer Hallmark (bottom) gene subsets, clustered by Euclidean distance and Ward’s D2 linkage.
