Complementary single-nucleus and single-cell RNA sequencing reveal tumor state evolution and microenvironmental rewiring in NSCLC

Single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) provide complementary insights into the cellular architecture of complex tissues, yet their biological resolution in non-small cell lung cancer (NSCLC) remains incompletely defined. Here we directly compared matched tumor and normal lung tissues from patients with NSCLC to determine how each platform captures distinct cellular and biological programs within the tumor ecosystem. scRNA-seq enabled finer delineation of immune cell subpopulations and cytoplasmic metabolic programs, whereas snRNA-seq more effectively resolved epithelial lineages and nuclear regulatory pathways. Regulon analysis identified transcription factors governing alveolar type 2 (AT2) to type 1 (AT1) differentiation in a platform-dependent manner. We further defined four reproducible transcriptional modules along the AT2-to-malignant trajectory, with early modules enriched for epithelial homeostasis and late modules for oncogenic signaling. Ligand–receptor analysis revealed epithelial state–dependent microenvironmental rewiring, shifting from adhesion-related interactions in early states to extracellular matrix signaling during malignant progression. Together, these findings demonstrate that scRNA-seq and snRNA-seq provide complementary biological insights into epithelial differentiation, tumor-state evolution, and microenvironmental remodeling in NSCLC.