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Proteomic Analysis of Exosomes Isolated from Human Malignant Pleural Effusions

Department of Pulmonary Medicine, Department of Neurology, and Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands

Address correspondence to: M. P. L. Bard, Department of Pulmonary Medicine, Erasmus Medical Centre H-Ee2253a, P.O. Box 1738, 3000 DR, Rotterdam, The Netherlands. E-mail: m.bard{at}erasmusmc.nl

Exosomes are membrane vesicles from endosomal origin secreted by various cells such as hematopoietic, epithelial, and tumor cells. Exosomes secreted by tumor cells contain specific antigens potentially useful for immunotherapeutic purposes. Our aim was to determine if exosomes are present in human cancerous pleural effusions and to identify their proteomic content. Exosomes were purified by sucrose gradient ultracentrifugation, and electron microscopy was used to check both concentration and purity of exosomes. Proteins were separated by one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and protein bands were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blotting. Exosomes were present in pleural fluid obtained from patients suffering from mesothelioma (n = 4), lung cancer (n = 2), breast cancer (n = 2), and ovarian cancer (n = 1). As previously reported by others, antigen-presenting molecules, cytoskeletal proteins, and signal transduction–involved proteins were present. Proteins not previously reported were identified (SNX25, BTG1, PEDF, thrombospondin 2). Different types of immunoglobulins and complement factors were abundantly present in the sucrose fractions containing exosomes. Exosome-directed specificity of these immunoglobulins was not observed. In conclusion, sucrose gradient ultracentrifugation allows isolation of exosomes from malignant pleural effusions. However, pleural fluid proteins and especially immunoglobulins are coisolated and may hamper the use of exosomes isolated from malignant effusion for immunotherapy programs.

Abbreviations: B-cell translocation gene, BTG • dendritic cell, DC • electron microscopy, EM • immunoglobulin, Ig • heat shock protein, HSP • matrix-assisted laser desorption ionisation time-of-flight, MALDI-TOF • major histocompatibility complex, MHC • polyacrylamide gel electrophoresis, PAGE • pigment epithelium-derived factor, PEDF • sodium dodecyl sulfate, SDS • sorting nexin, SNX

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