Proteomic Analysis of Exosomes Isolated from Human Malignant Pleural Effusions

Abstract

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.

Proteomic Analysis of Exosomes Secreted by Human Mesothelioma Cells

“…Initial exosome isolations from pleural effusion turned out to be troublesome, caused by high amounts of immunoglobulins and complement factors present in the fluid. 13 Exosomes in pleural effusions will not only be secreted by mesothelioma cells, but also by mesothelial cells or from cells of hematopoietic origin present in the fluid. Therefore, mesothelioma cell line-derived exosomes were studied for their protein content.…”

Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

“…Electron Microscopy-Melanoma cells, cultured in buffered and acidic conditions, were fixed with 2.5% glutaraldehyde, post-fixed in 1% OsO 4 , and then dehydrated in alcohol and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and finally examined with a Philips 208 transmission electron microscope (FEI Co.).…”

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

“…Their physiological functions are diverse, generally related to the functions of the parent cell (28). Exosomes are produced by tumors in vivo and they are often abundant in malignant effusions for example in advanced ovarian carcinoma (29) and pleural malignant mesothelioma (30). Exosomes released from malignified epithelial cells that no longer respect their basal membranes but grow invasively may be useful biomarkers in blood plasma (31)(32)(33)(34) and urine (35) in some cancer types.…”