Molecular lipidomics of exosomes released by PC-3 prostate cancer cells Alicia Llorente a, b, ⁎, Tore Skotland a, b , Tuulia Sylvänne c , Dimple Kauhanen c , Tomasz Róg d , Adam Orlowski d , Ilpo Vattulainen d, e , Kim Ekroos c , Kirsten Sandvig a, b, f a Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital,—The Norwegian Radium Hospital, 0379 Oslo, Norway b Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, 0379 Oslo, Norway c Zora Biosciences Oy, 02150 Espoo, Finland d Department of Physics, Tampere University of Technology, 33101 Tampere, Finland e MEMPHYS,—Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark f Department of Biosciences, University of Oslo, 0316 Oslo, Norway abstract article info Article history: Received 24 February 2013 Received in revised form 12 April 2013 Accepted 17 April 2013 Available online 24 April 2013 Keywords: Biomarkers Exosomes High-throughput quantitative lipidomics Microvesicles Prostate cancer The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differ- ences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potential- ly be used as cancer biomarkers. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Cells are able to release exosomes to the extracellular environment [1,2]. Exosome release occurs upon fusion of multivesicular bodies with the plasma membrane [3]. The molecular mechanisms of this pro- cess are still unclear, but there are several studies that have provided in- formation about some of the implicated molecules. For example, p53 [4], diacylglycerol kinase [5], phospholipase D2 [6], sphingomyelinase [7], cholesterol [8] and several small GTPases [9–11] have been pro- posed to play a role in exosome release. It is considered today that exosomes function in cell–cell communication [1,12,13]. Exosomes are able to affect neighboring cells in various ways, for example by inducing intracellular signaling or by transferring different molecules such as proteins, mRNAs or microRNAs [14]. Furthermore, it has been suggested that microvesicles play a role in several diseases [15,16]. Concerning cancer, microvesicles have been suggested to contribute to cancer cell survival, invasiveness and metastases [2]. Exosomes show an amazingly diverse mixture of proteins, lipids and nucleic acids [17]. Databases such as Vesiclepedia, Exocarta and EVpedia, designed to collect information related to the composition of extracellular vesicles, contain several thousands of protein and mRNA entries [18]. In contrast to the extensive information about the protein and mRNA com- position of exosomes, current knowledge about the molecular lipid com- position of these extracellular vesicles is unknown. To this point exosome lipidomes have predominantly been delineated at the lipid class and sum species level [7,19]. Although slightly deviating lipid contents have been observed between various populations of exosomes, it has been shown that exosomes are often enriched in cholesterol, sphingomyelin and satu- rated phospholipids, suggesting that exosome membranes contain lipid raft-like domains [7,19,20]. The structure and function of membranes and domains are determined by the assembled molecular lipids and membrane-bound proteins. Thus, the precise protein and lipid makeup of the exosome membrane is required to gain knowledge about their structures and functions, which is essential for understanding the mecha- nism of exosome formation, release and function. Furthermore, exosomes contain several molecules that are specific to the parent cells from which they are released [21]. This, together with the fact that exosomes can reach biological fluids, has led to the idea of using exosomes as a source for noninvasive diagnostic biomarkers for Biochimica et Biophysica Acta 1831 (2013) 1302–1309 ⁎ Corresponding author at: Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital—The Norwegian Radium Hospital, 0379 Oslo, Norway. Tel.: +47 90892782. E-mail address: alillo@rr-research.no (A. Llorente). 1388-1981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbalip.2013.04.011 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip