Figure S5 from Protumor Macrophage Polarization Mediated by BMP2/4–ACVR1 Signaling Orchestrates an Immunosuppressive Microenvironment during Tumor Initiation

Extended Data Fig.5. BMP2 and BMP4 upregulation in tumor epithelial cells while ACVR1 upregulation in macrophages, related to Figure 4. (A) A network of ligand-receptor interactions (LRIs) among different cell types after 1day of doxycycline induction, subtracting the number of interactions at week 0. Each node represents a cell type. The size of each node shows the number of LRIs occurring for each given cell type. The width of the edges represents the number of significant LRIs between the two nodes at 1day of doxycycline induction, subtracting the number of LRIs at week 0. (B) No obvious changes in the expression level of known Th2-differentiation related genes, Csf1, Il10, Il13, and Il4, in lung tissues of mouse induced by doxycycline (2mg/ml) for 0, 1, 2, and 4 weeks (measured using bulk RNA sequencing). (C) No obvious changes in the expression level of known Th2-differentiation related genes, Il10, Il13, and Il4, in lung tissues of mouse induced by doxycycline (2mg/ml) for 0, 1, 2, and 4 weeks and these genes were expressed low in the TME(measured using single cell RNA sequencing). (D) The enrichment score of the signaling pathways that are enriched with upregulated genes in human EGFR positive epithelial cells compared to EGFR negative epithelial cells (enrich 1, the left panel); The signaling pathways that are enriched with Ligand-Receptor (LR) genes with highly correlated expression levels between epithelial cells and macrophages (enrich 2, the middle panel); The enrichment score of the signaling pathways that are enriched with LR pairs between epitheliums and macrophages established after the induction of EGFR-driven LUAD (enrich 3, the right panel) The enrichment scores in the two heatmaps were standardized by taking the z-score within each row. (E) Expression levels of BMP2 and BMP4 at each lung cancer stage in t-SNE projection. (F) Quantification of ACVR1/BMP2/BMP4 protein expression by Western blot. Densitometric analysis of ACVR1/BMP2/BMP4 protein levels normalized to β-Tubulin in samples collected at 0 weeks (0W) and 4 weeks (4W). Each dot represents an individual biological replicate, and horizontal bars indicate the median value for each group.