Isolation of extracellular vesicles from pleural effusion of patients with lung cancer for downstream application in the clinical setting

Malignant pleural effusion (MPE) is a common clinical manifestation of advanced non-small cell lung cancer (NSCLC) and represents a valuable source of tumor-derived components, including extracellular vesicles (EVs). The aim of this study was to compare three different EV enrichment methods with potential applicability in clinical practice: a commercial Norgen kit (NOR), immunoaffinity capture (IA), and ultracentrifugation (UC). Following EV characterization, IA demonstrated the best overall performance in terms of EV yield and purity, NOR showed intermediate efficiency, while UC was the least effective method. The IA method exhibited characteristics suitable for potential clinical implementation, whereas NOR and UC may require combination with additional enrichment approaches. Advancing pleural effusion-based liquid biopsy toward clinical application will depend on the development of robust, scalable, and user-friendly EV enrichment workflows, along with harmonized guidelines for sample collection, preprocessing, and data reporting. Establishing such standards will enhance reproducibility, enable meaningful cross-study comparisons, and accelerate the integration of EV-based biomarkers into precision oncology.