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Published ahead of print on February 19, 2004, doi:10.1165/rcmb.2003-0238OC

Am. J. Respir. Cell Mol. Biol., Volume 31, Number 1, July 2004, 114-121

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Submitted on June 23, 2003
Revised on February 19, 2004

PROTEOMIC ANALYSIS OF EXOSOMES ISOLATED FROM HUMAN MALIGNANT PLEURAL EFFUSIONS

Martin P.L. Bard1*, Joost P Hegmans1, Annabrita Hemmes1, Theo M Luider2, Rob Willemsen3, Lise-Anne A Severijnen3, Jan P van Meerbeeck1, Sjaak A Burgers1, Henk C Hoogsteden1, and Bart N Lambrecht1

1 Pulmonary Medicine, Erasmus Medical Centre, Rotterdam, The Netherlands, 2 Neurology, Erasmus Medical Centre, Rotterdam, The Netherlands, 3 Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands

* To whom correspondence should be addressed. E-mail: m.bard{at}erasmusmc.nl.

Exosomes are membrane vesicles from endosomal origin secreted by various cells such as haematopoietic, 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 SDS-PAGE 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 ovary cancer (n=1). As previously reported by others, antigen-presenting molecules, cytoskeletal proteins, and signal transduction-involved proteins were present. Not previously reported proteins 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.




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