Figure S6 from Protumor Macrophage Polarization Mediated by BMP2/4–ACVR1 Signaling Orchestrates an Immunosuppressive Microenvironment during Tumor Initiation

Extended Data Fig.6. BMP2 and BMP4 upregulation correlates with poor prognosis, related to Figure 4. (A) Immunofluorescence staining for CD68 (yellow) along with DNA visualized by DAPI staining. The upper four represent adjacent non-cancerous tissues, while the lower four are the corresponding cancerous tissues. Scale bar :50um. (B) Quantification of CD68⁺ cells (macrophages) in equal-area regions of matched tumor and adjacent non-tumor tissues using immunofluorescence imaging. Each pair of data points represents tumor and adjacent tissue from the same individual, and different pairs represent different individuals. The number of macrophages was significantly higher in tumor regions compared to adjacent tissues (P < 0.01, paired t test). (C, D) Box plot quantifying the proportions of CD8+ T cells in nonEGFR and EGFR-mutant patients from scRNA-seq dataset (C), and TCGA database (D), highlighting an increase in the proportion of CD8+ T cells in EGFR-mutant NSCLC patients. (E) Kaplan–Meier plots for overall survival (OS), with log-rank tests between cases with high and low ACVR1 gene expression in the LUAD TCGA database. The Kaplan–Meier curves were used to visualize and compare the OS of the low-risk (blue) versus high-risk (red) group in EGFR-mutant patients (left), in non-EGFR–mutant patients (medium), and all EGFR-mutant patients(right) highlighting ACVR1 gene expression is correlated with the OS of EGFR-mutant patients but not non-EGFR–mutant patients. (F) Proportion of ACVR1⁺CD68⁺ cells among total ACVR1⁺ cells in paired normal and tumor tissues. Each line represents an individual sample. (G) Kaplan–Meier plots for overall survival (OS), with log-rank tests between cases with high and low macrophage polarization signatures in the LUAD database. (Left) Higher expression of the M1-associated ATM signature was significantly associated with improved overall survival (log-rank p = 0.021). (Right) Expression of the M2-associated PTM signature showed no significant association with survival (log-rank p = 0.42). (H) Kaplan–Meier plots for overall survival (OS), with log-rank tests between cases with high and low ACVR1 gene expression in the LUAD TCGA database. The Kaplan–Meier curves were used to visualize and compare the OS of the low-risk (blue) versus high-risk (red) group in KRAS-mutant patients (left) and in non-EGFR–mutant patients (right). (I) Kaplan–Meier plots for overall survival (OS), with log-rank tests between cases with high and low ACVR1 gene expression in other cancer types of TCGA database. The Kaplan–Meier curves were used to visualize and compare the OS of the low-risk versus high-risk group in indicated cancer types. (J) ACVR1 expressed Macrophage is enriched in non-responders. Both mean expression of ACVR1 and ratio of ACVR1 expressed macrophages were higher in non-responders. Public Data were used27–32. (K) Heatmap showing correlation between cytotoxicity T cell scores and ACVR1 in indicated cancer types assessed by TIDE33.