Single-cell and spatial transcriptomic profiling reveal fibrosis-related tumor states in lung squamous cell carcinoma associated with idiopathic pulmonary fibrosis

Background: Patients with idiopathic pulmonary fibrosis (IPF) have an elevated risk of lung cancer, particularly lung squamous cell carcinoma (LUSC), which is associated with limited treatment options and poor outcomes. Although LUSC occurring in IPF patients is collectively referred to as LUSC associated with IPF regardless of location, tumors arising within usual interstitial pneumonia (UIP) lesions may harbor distinct molecular traits shaped by the local fibrotic microenvironment compared with those arising outside UIP. Methods: We performed integrated single-cell RNA sequencing (scRNA-seq) and Digital Spatial Profiling (GeoMx DSP) to characterize tumors and adjacent lung tissue from LUSC patients with UIP patterns. scRNA-seq was performed on paired tumor and adjacent lung tissue from an In-UIP case and an Out-UIP case, while GeoMx profiling was conducted in an independent cohort (In-UIP n=3, Out-UIP n=3). scRNA-seq data were processed using Scanpy (v1.10.4) and spatial transcriptomics data were processed using GeomxTools (v3.8.0). Cellular trajectories were analyzed using scTour (v1.0.0). Spatial deconvolution was performed using Cell2location (v0.9.6), with validation in an external fibrosing ILD spatial dataset. Results: To infer lineage relationships from the scRNA-seq data, we applied partition-based graph abstraction (PAGA), which demonstrated a connection between basal cells and malignant cells, suggesting basal cells as a cellular origin of LUSC. Tumor cells were further resolved into four transcriptionally distinct malignant states, one of which was characterized by upregulation of oxidative stress-response and detoxification programs, including induction of multiple WNT ligand genes. scTour vector-field and pseudotime modeling showed expansion of this state at late phase exclusively in the In-UIP tumor. Spatial profiling by GeoMx DSP followed by Cell2location deconvolution confirmed significant enrichment of this stress-tolerant malignant state in In-UIP tumors compared with Out-UIP tumors. In adjacent non-malignant regions, UIP lungs exhibited marked expansion of basal and club populations, reflecting UIP-specific epithelial remodeling. Analysis of an independent spatial ILD cohort validated this epithelial shift specifically in IPF. Conclusions: Single-cell and spatial analyses demonstrate that UIP lesions create a selective niche that reshapes local epithelial composition and promotes regional enrichment of a stress-tolerant malignant cell state. These data indicate that the fibrotic lung microenvironment of IPF conditions malignant cell states and their spatial organization, nominating microenvironment-driven programs as potential therapeutic targets in LUSC associated with IPF.