Background/Objectives
Biomarkers for predicting immunotherapeutic efficacy in non-small cell lung cancer (NSCLC) are limited, with PD-L1 expression on tumor tissue being the only approved biomarker. Blood-derived extracellular vesicles (EVs) may serve as a minimally invasive alternative, better reflecting the tumor immune microenvironment. This study aimed to profile surface markers of circulating immune cell-derived EVs in NSCLC patients and evaluate their potential as predictive biomarkers for immunotherapy response.
Methods
Large extracellular vesicles (lEVs) were isolated from plasma samples of NSCLC patients and non-cancerous donors via differential centrifugation, and subsequently characterized by nanoparticle tracking analysis, immunoblotting, and flow cytometry. Immune marker expression profiles were analyzed across cohorts and correlated with tumor PD-L1 expression, blood counts, and clinical response to immunotherapy.
Results
A comprehensive flow cytometry-based screen identified 126 out of 351 detectable surface antigens on plasma-derived lEVs, including 21 immune checkpoint molecules. Notably, CD28, CD226, CD160, and CD47 exhibited high expression levels. Comparative analysis demonstrated quantitative differences in marker expression between lEVs and their cellular counterparts, indicating a selective marker enrichment on lEVs. In a discovery cohort (n=5 healthy, n=5 NSCLC), 21 antigens were significantly differentially expressed between both groups, with elevated levels observed in NSCLC patients. These results were validated in an independent cohort (n=14 non-cancer; n=26 NSCLC), confirming increased levels of T cell- and NK cell-associated markers on lEVs from NSCLC patients. Notably, CTLA-4 was significantly enriched on lEVs from immunotherapy responders compared to non-responders and was able to discriminate between the two groups with an area under the curve (AUC) of 0.80, suggesting its potential as a novel predictive biomarker.
Conclusion
This study offers a comprehensive characterization of the immune vesiculome in NSCLC patients and identifies novel candidate biomarkers for predicting immunotherapy response. The results highlight the potential of blood-derived EVs to guide personalized treatment strategies in NSCLC.
Keywords
NSCLC, large EVs, biomarker, immunotherapy, immune cells
Funding/Acknowledgments
The project was funded by Novartis (INCA prize) and Maria-Möller-Stiftung.
