Fig. 5

Digital Spatial Profiling revealed profound differences in compartments of the tumor microenvironment of high pSTAT3^Tyr705 cases. First row demonstrates representative images of 3 high pSTAT3 TSP^high TMA cores (pink) and 8 Low pSTAT3 TSP^high TMA cores (from patient cohort 3) (grey) stained with SYTO13 (blue), PanCK(green), aSMA(yellow) scanned on the Nanostring GeoMx® platform (A). The second row demonstrates masks used to generate PanCK + (epithelial), aSMA + (fibroblast), PanCK-SMA-(TME) segmented transcriptome compartments (A). Volcano plot demonstrating differential gene expression results comparing high pSTAT3 cores versus low pSTAT3 cores in PanCK compartment only (B). Volcano plot demonstrating differential gene expression results comparing high pSTAT3 cores versus low pSTAT3 cores in TME compartment only (C). Volcano plot demonstrating differential gene expression results comparing high pSTAT3 cores versus low pSTAT3 cores in aSMA compartment only (D). Box plots demonstrating Normalized Expression (y-axis) of common cancer signaling pathways (Nanostring WTA curated gene sets) calculated using fgsea comparing high pSTAT3 (pink) and low pSTAT3 (grey) cases in segmented compartments (x-axis) (p < 0.05*, p < 0.005**, p < 0.0005***) (E). Box plots demonstrating Normalized Expression (y-axis) of common immune signaling pathways (Nanostring WTA curated gene sets) calculated using fgsea comparing high pSTAT3 (pink) and low pSTAT3 (low) cases in segmented compartments (x-axis) (p < 0.05*, p < 0.005**, p < 0.0005***) (F). Panel 1: Heatmap demonstrating differentially expressed genes comparing high pSTAT3 and low pSTAT3 in aSMA compartment. Panel 2: Gene Set Enrichment Analysis of differentially expressed genes in the aSMA compartment comparing high pSTAT3 and low pSTAT3 in aSMA compartment generated using ClusterProfiler package to interrogate REACTOME curated pathways (G). Boxplots demonstrating results of deconvoluting transcriptomic readout of high pSTAT3 and low pSTAT3 cores into estimated immune cells counts, selected plots from TME and aSMA compartment are shown (H). Scatterplots of gene expression count of selected receptor ligand pairs (CellPhoneDB) comparing high pSTAT3 (pink) and low pSTAT3 (grey) with superimposed linear model line, well correlated gene pairs are assumed to imply a relationship between receptor and ligand in that group (I)