Dynamic remodeling of the extracellular vesicle proteome during systemic therapy in advanced non–small cell lung cancer.

Abstract

e15059 Background: Non–small cell lung cancer (NSCLC) remains associated with poor prognosis in advanced stages, and reliable biomarkers for monitoring treatment response are still limited. While liquid biopsy applications have largely focused on circulating nucleic acids, extracellular vesicles (EVs) represent a stable and biologically informative source of tumor- and microenvironment-derived proteins. EV proteomic profiling may provide dynamic insights into therapy-induced molecular changes and improve patient stratification. Methods: Plasma samples were collected from patients with advanced NSCLC at baseline (P0, pre-treatment) and at first radiological evaluation (P1), prior informed consent was obtained. Patients were stratified according to treatment regimen (chemotherapy, immunotherapy, or chemo-immunotherapy). EVs were isolated from plasma using an affinity-based enrichment method optimized for low sample volumes and characterized by nanoparticle tracking analysis and Western blotting. EV protein cargo was analyzed by data-independent acquisition (DIA) LC–MS/MS with label-free quantification. Multivariate analyses, functional enrichment (GO/KEGG), and protein–protein interaction network analyses were performed to identify treatment- and time-dependent proteomic signatures. Results: EV proteomic profiling identified 418 EV-associated proteins across all samples. Comparison between baseline (P0) and post-treatment (P1) revealed 108 differentially expressed proteins (43 enriched at P0, 65 at P1). In immunotherapy-treated patients, baseline EVs displayed heterogeneous profiles enriched in epithelial organization, lipid metabolism, complement activation, and coagulation pathways. Post-treatment EVs exhibited marked proteomic remodeling, with enrichment of immune modulation, oxidative stress response, metabolic adaptation, and cytoskeletal remodeling pathways. Functional analyses indicated increased complement regulation, protease inhibition, and immune–stromal interactions, consistent with therapy-induced tumor microenvironment reprogramming. Post-treatment EV profiles partially converged with other treatment groups while retaining immunotherapy-specific signatures. Conclusions: These findings support further study of plasma-derived EVs as they capture dynamic proteomic changes associated with immune checkpoint inhibition in NSCLC and represent a promising non-invasive tool for monitoring immunotherapy-induced molecular remodeling and for biomarker-driven patient stratification and follow-up.