Distinct Spatial Immune Architectures in Tumor and Tumor-Adjacent Tissues of Early-Stage Non-Small Cell Lung Cancer

Background: Lung cancer remains the leading cause of cancer-related deaths in the United States with over 124,000 estimated deaths for 2026. Previous studies have found that tumor-adjacent lung tissues may provide additional insight into the immune microenvironment of early-stage NSCLC. Methods: Multiplex immunofluorescence (mIF) imaging was performed on 192 tissues from 101 early-stage non-small cell lung cancer patients including 91 matched tumor-adjacent pairs, using three mIF panels. Spatial analyses were performed to identify distinctions between tissues and identify associations with clinical and genomic features. Results: Tumor tissue showed significantly higher densities of T cells, macrophages, B cells, and memory/regulatory populations than adjacent tissue (p<0.001). Tumors exhibited greater spatial heterogeneity, with higher prevalence of spatial patterning (47.7% vs. 31.2%) and more consistently localized organization, whereas adjacent tissue showed stronger individual-cell clustering. Pairwise colocalization (Ripley's L-cross) revealed selective spatial segregation in tumors (including B-cells from memory/regulatory cells and among cytotoxic T-cell subsets) and reduced immune proximity to malignant cells relative to the strong immune-epithelial association in adjacent tissue. Spatial features were linked to genomic features or patient outcomes: tumor neoantigen burden exhibited a positive association with CD3+ T cells in the tumor, while colocalization between CD45RO+CD57+GZMB+ cells and CD57+GZMB+ cells was negatively associated with overall survival outside the tumor. Conclusion: Tumor and adjacent tissues harbor distinct spatial immune architectures, with spatial features displaying associations with genomic features or patient outcomes. These findings highlight immune microenvironment reorganization and the importance of incorporating spatial context from both compartments into risk assessment in NSCLC.