Comparison of Early- and Late-Stage EGFR-Mutated Lung Adenocarcinomas Using Spatial Transcriptomics

Background: The epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma (LUAD) exhibits significant stage-dependent molecular and spatial heterogeneity, which remains incompletely characterized. Elucidating stage-associated differences in gene expression, signaling pathways, and immune landscapes is critical for developing stage-stratified precision therapy for EGFR-mutated LUAD.Methods: This study integrated bulk transcriptome profiling and digital spatial profiling (DSP) to investigate pathological stage-associated differences. DSP was applied to 23 EGFR-mutated LUAD patients (18 early-stage and 5 late-stage) to elucidate spatial heterogeneity across three tissue compartments: tumor cells, immune cells, and macrophages. Public bulk RNA sequencing data from 38 patients with EGFR-mutated LUAD, including 29 early-stage (I+II) and 9 late-stage (III+IV) cases, were analyzed.Results: Bulk RNA sequencing identified 580 differentially expressed genes between early- and late-stage EGFR-mutated LUAD. Late-stage tumors exhibited enhanced proliferation, marked by elevated E2F, G2M checkpoint, and MYC activities, alongside activated cell cycle, DNA replication, and DNA repair pathways. Notable metabolic reprogramming was also observed, including upregulated serine and purine metabolism. Spatial transcriptomic analysis confirmed increased proliferative, oncogenic, metastatic, and glycolytic signatures in late-stage tumor cells, consistent with findings from bulk RNA sequencing. Pathway enrichment analysis across distinct compartments revealed six stage and compartment specific modules, with modules related to cell cycle, DNA repair, invasion, and metabolism significantly upregulated in the late-stage tumor cell segment. Moreover, late-stage tumors displayed an increase in exhausted CD8+ T cells and elevated expression of immune checkpoints (PD-L2, PVR in tumor cells; B7-H3, LAG3 in immune cells), contributing to an immunosuppressive microenvironment.Conclusions: These findings delineate stage-specific transcriptional, metabolic, and spatial immune reprogramming during the progression of EGFR-mutated LUAD. These results may provide new insights into the molecular mechanisms underlying tumor progression and identify potential targets for stage-stratified precision therapy in EGFR-mutated LUAD.