A single-nucleus transcriptomic characterization of EGFR G719X/S768I double-mutant lung adenocarcinoma identifies an immunosuppressive niche defined by COL4A3-CD44 interaction

Abstract

EGFR double mutations (G719X/S768I) represent a clinically recalcitrant subtype of lung adenocarcinoma (LUAD) characterized by diminished tyrosine kinase inhibitor (TKI) sensitivity. However, the specific tumor microenvironment (TME) features and intercelluar crosstalk driving this therapeutic resistance poorly defined. We performed single-nucleus RNA sequencing (snRNA-seq) on formalin-fixed paraffin-embedded (FFPE) tissues from six EGFR G719X/S768I double-mutant (EGFR_double) and five no EGFR mutation (Non_EGFR) LUAD patients. To provide a comprehensive reference, we integrated our FFPE data with publicly fresh-tissue single cell RNA sequencing (scRNA-seq) data with seven EGFR single-mutant LUAD (EGFR_single) and five adjacent normal (Adjacent), generated a comprehensive atlas of 246,086 cells. Findings were further validated using multiplex immunohistochemistry (mIHC) and pharmacogenomic profiling. We identified a specialized EMT-like malignant subpopulation (MP4) uniquely enriched in EGFR_double, which exhibits a distinct lipid-glycosylation metabolic profile that stabilizes CD44 expression. Cell-cell communication analysis revealed enhanced COL4A3-CD44 interactions between EMT-like malignant MP4 cells and metabolically reprogrammed, immunosuppressive alveolar macrophages in EGFR_double compared with Non_EGFR. MIHC confirmed in situ proximity of this significantly increased COL4A3-CD44 co-localization in EGFR_double. Furthermore, drug sensitivity analysis identified specific vulnerabilities of this subtype. Our study identifies a previously unrecognized malignant-myeloid niche and TME in EGFR_double. These findings provide a translational framework for identifying a potential novel therapeutic targets and suggest that combinatorial strategies targeting the COL4A3-CD44 interaction may overcome therapeutic recalcitrance in this challenging clinical population.