Isolation and characterization of extracellular vesicles from pleural effusion samples of patients with advanced non-small cell lung cancer

Introduction: The incidence of lung cancer (LC) in Serbia has increased over the last three decades. Up to 40%of patients with advanced non-small cell lung cancer (NSCLC) develop pleural effusion (PE). The aim of thisstudy was to evaluate the usability of existing methods for isolation of extracellular vesicles from PE samplesof patients with advanced non-small cell lung cancer and to characterize them for further use in the clinical/diagnostic/research setting. Material & Methods: PE samples diluted in PBS (1:1) from patients with advanced NSCLC were used. In-house spherical porous methacrylate-based copolymer coupled with VHH antibodies was used for the isolation of extracellular vesicles (EV) from PE samples. Flow-cytometry was performed for detection of exosomal markers. Results: A pool of PE was prepared from 5 patients with advanced NSCLC. Flow cytometry confirmed that the isolation of EVs was successful using the in-house affinity chromatography method. The presence of CD9 antigen was detected, as well as a decrease in the signal after the addition of Triton X-100. Further plans include the analysis of CD63 and CD81 antigens using flow cytometry, NTA analysis to determine the number and diameter of obtained vesicles, as well as TEM and SEM to determine their morphology. Discussion: We aim to investigate for the first time whether this method is applicable to pleural effusion samples as a new cancer liquid biopsy sample type. We also plan to evaluate the method in comparison with a commercial isolation kit.