Single-cell RNA sequencing reveals ancestry-associated molecular and cellular immunosuppressive features in non-small cell lung cancer

Background: Racial disparities in non-small cell lung cancer (NSCLC) outcomes persist, with Black American (BA) patients having inferior survival compared to White American (WA) patients, even after adjusting for stage, treatment, and socioeconomic factors. We hypothesize that distinct molecular and cellular features in the tumor microenvironment (TME) contribute to ancestry-specific heterogeneity and these disparities. Methods: We performed 10x scRNA-seq on treatment-naïve, early-stage NSCLC from 42 patients: 29 adenocarcinomas (15 WA, 14 BA) and 13 squamous (10 WA, 3 BA). Unsupervised clustering and annotation of 220,591 cells identified six major cell types: epithelial, endothelial, fibroblasts, myeloid, mast, and lymphocytes-each further subdivided into lineage-specific phenotypes. Analyses stratified by ancestry include: differential gene expression and cell abundance, gene set enrichment analysis, trajectory inference, and ligand-receptor analysis. Results: Malignant epithelial cells showed significant pathway enrichment of OXPHOS, fatty acid metabolism, and G2/M checkpoint in BA tumors (q≤0.01) and TNF-alpha signaling via NF-κB, IFN-gamma response, and heme metabolism in WA tumors (q=0.004, 0.008, and 0.02, respectively). Endothelial cells showed enrichment in IL-2/STAT5 signaling and fatty acid metabolism in BA tumors (q=0.01 and 0.03, respectively) and in proliferation pathways in WA tumors (Myc Targets V1, q=0.005).BA and WA tumors exhibited distinctive immunosuppressive features. BA tumors showed higher numbers of FAP- cancer associated fibroblasts (CAF; q=0.07) and M2 macrophages (q=0.004) but low effector CD8+ (p=0.02) and gammadelta T cells (p=0.018). BA tumor associated macrophages (TAM) showed preserved activation (CD40, CD80) but impaired antigen presentation (HLA-DM/DQ/DR, all q<0.05) and were enriched for hypoxia and TGF-beta signaling gene signatures (q<0.01). In contrast, WA tumors contained more FAP+ CAFs and macrophages displayed M0/M1-like features with prominent antigen presentation and IFN-I/II responses (q<0.01). Also, BA tumors showed active innate immune networks, particularly SPP1-CD44 signaling (p<0.001), whereas WA tumors exhibited stronger adaptive stimulatory (MHCII-TCR) and inhibitory (CD86-CTLA4) signaling (p<0.001). Furthermore, CD8⁺ T cells in BA tumors adopted an early exhaustion phenotype associated with TGF-beta signaling (q<0.01), while in WA tumors they showed progressive exhaustion (high PDCD1, HAVCR2, LAG3) in an IFN-alpha/gamma-driven environment (q<1E-5). Conclusions: This first large-scale, ancestry-stratified scRNA-seq atlas of NSCLC reveals molecular mechanisms underlying distinctive immunosuppressive programs in BA and WA patients and highlights molecular and cellular TME features that may contribute to racial disparities in NSCLC.