Abstract 5115: Comparative proteomics of exosomes secreted by a panel of cancer cell lines
Authors: Lavaei Mehrdad , Fijneman Remond Ja , Fluitsma Donna , Knol Jaco C. , Piersma Sander R. , Pham Thang V. , Steenbergen Renske , Meijer Gerrit A. , Verheul Henk MW , Pegtel Michiel , Jimenez Connie R. 
Published: June 2012

Abstract
BACKGROUND Exosomes are 40-100 nm membrane vesicles that are released by cancer and normal cells, after the fusion of multivesicular bodies with the plasma membrane. Multiple functions have been attributed to exosomes including antigen presentation and intercellular communication. Importantly, exosomes carry tumor-specific antigens and have been identified in various biofluids. Therefore, exosomes may provide an attractive platform for biomarker discovery. Mass spectrometry-based proteomics has contributed significantly to our understanding of the molecular composition of exosomes. Yet a comprehensive quantitative analysis of exosomes and corresponding cell lysates derived from a panel of different cancer cell lines and primary cells is missing.
AIM Identification of exosome core proteins and cancer-type specific proteins to obtain insight into aberrant exosomal functions in cancer and for candidate biomarker discovery
APPROACH Quantitative proteomics of exosomes released by a panel of 9 cancer cell lines and 2 normal cell types and their corresponding total cell lysates.
METHODS Exosomes were harvested by differential centrifugation from the following human cancer cell lines: HT29, HCT116, SW480, SW1398, CaCo2; H460; MCF-7, PC3 and LNCaP. The primary human cells included are endothelial cells and keratinocytes. The proteomics workflow is based on protein fractionation by 1DGE, in-gel digestion, nano-liquid chromatography coupled to tandem mass spectrometry on an LTQ-FTMS. Spectral counting was used for protein quantitation. Dedicated statistics and bioinformatics tools were used for significance analysis of altered protein abundances and coupling to biological functions as described (Pham et al., Bioinformatics 2010; Albrethsen et al., Mol.Cell.Prot. 2010).
RESULTS Exosome isolation by differential centrifugation was reproducible as deduced from replicate analyses. The purity of the exosome preparation was verified using analysis of specific exosome markers in our dataset, electron microscopy and western blot and was shown to be good.
The total dataset of exosomes and cell lysates contains ∼4500 proteins with 1300-1400 exosome proteins per cell line. The core exosome proteome shared by all cells analyzed was highly enriched for the terms protein synthesis, RNA binding and lipid raft proteins among others. We are currently comparing the exosome protein profile of each cell type with the protein profile of the cell lysate, as well as with the exosomes from other cell types and coupling the data to gene ontology and pathway analysis tools.
CONCLUSIONS & OUTLOOK Exosomes may be considered a relevant new platform for biomarker discovery. In cancer exosomes, we identified established tumor type-specific antigens and proteins belonging to oncogenic pathways. For the validation of potential biomarkers in biofluids, we will employ targeted mass spectrometry and antibodies against the candidate biomarkers.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5115. doi:10.1158/1538-7445.AM2011-5115