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Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te / j p ro t

Characterization and proteomic analysis of ovariancancer-derived exosomes

Bing Lianga, Peng Penga, She Chenb, Lin Lib, Meijun Zhangb, Dongyan Caoa, Jiaxin Yanga,Haixia Lia, Ting Guia, Xialu Lib,⁎, Keng Shena,⁎aDepartment of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking UnionMedical College, Beijing 100730, ChinabNational Institute of Biological Sciences, Beijing 102206, China

A R T I C L E I N F O

⁎ Corresponding authors. Tel.: +86 10 6521250E-mail addresses: lixialu@nibs.ac.cn (X. L

1874-3919/$ – see front matter © 2013 Elseviehttp://dx.doi.org/10.1016/j.jprot.2012.12.029

A B S T R A C T

Article history:Received 18 April 2012Accepted 26 December 2012Available online 16 January 2013

Keywords:Exosomes

Ovarian cancer is themost lethal type of cancer among all frequent gynecologicmalignancies,because most patients present with advanced disease at diagnosis. Exosomes are importantintercellular communication vehicles, released by various cell types. Herewe presented firstlythe protein profile of highly purified exosomes derived from two ovarian cancer cell lines,OVCAR-3 and IGROV1. The exosomes derived from ovarian cancer cell lines were round andmostly 30–100 nm in diameter when viewed under an electron microscope. The exosomalmarker proteinsTSG101 andAlixwere detected in exosomepreparations. The range of densitywas between 1.09 g/ml and 1.15 g/ml. A total of 2230 proteinswere identified from two ovariancell-derived exosomes. Among them, 1017 proteins were identified in both exosomesincluding all of the major exosomal protein markers. There were 380 proteins that are notreported in the ExoCarta database. In addition to common proteins from exosomes of variousorigins, our results showed that ovarian cancer-derived exosomes also carried tissue specificproteins associated with tumorigenesis and metastasis, especially in ovarian carcinoma.Based on the known roles of exosomes in cellular communication, these data indicate thatexosomes released by ovarian cancer cells may play important roles in ovarian cancerprogression and provide a potential source of blood-based protein biomarkers.

© 2013 Elsevier B.V. All rights reserved.

ProteomicsOvarian cancer

1. Introduction

Ovarian cancer is the most lethal type of cancer amongall frequent gynecologic malignancies all over the world [1,2].Most patients are diagnosed with epithelial ovarian cancerin advanced stages and with subsequent platinum/taxanechemoresistance appearance, which are the two most impor-tant reasons for the highmortality associated with this cancertype [2,3]. Although serum CA-125 level evaluation andultrasonography are clinically accepted methods for thediagnosis of ovarian cancer, they are not satisfactory methodsfor the early detection of ovarian cancer because of false

7.i), shenkeng@vip.sina.com

r B.V. All rights reserved.

positive and false negative results [2]. Cytoreductive surgeryand subsequent platinum/taxane-based postoperative adju-vant chemotherapy have made some progress in improvingthe survival rate of patients with ovarian cancer, but thefinal recurrence and chemoresistance of the disease are stillunsolved. So it is very urgent to find more sensitive diagnosticbiomarkers and establish novel therapeutic strategies.

Exosomes are spherical and bilayered proteolipids with adiameter of 30–100 nm. They are enriched in various bioac-tivematerials, including proteins, lipids,miRNAs andmRNAs[4,5]. Exosomes are released from various cell types, includ-ing reticulocytes [6], immune cells (dendritic cells/T cells/B

(K. Shen).

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cells) [7–9], mast cells [10], several kinds of epithelial cells[11,12], and tumor cells [13,14]. Exosomes have also beenfound in several types of body fluids including plasma [15],malignant effusions [16,17], urine [18], saliva [12] andamniotic fluid [19]. They have been proposed as a potentialsource of diagnostic marker and demonstrated to playimportant roles in cellular intercommunication [10,13,19,20].

In addition to some common exosomal proteins such asTSG101, Alix, CD9, CD81, CD63, GTPase active proteins andcytoskeletal proteins including actin, and tubulin proteins[8], exosomes may carry some disease associated proteinsfor they are a sub-part of the originated cells [20]. Thereforeexosomes in urine, saliva, plasma and other body fluids holdsignificant potential for obtaining some novel or combiningsets of diagnostic biomarkers in a non-invasive manner [19].

Several experiments have shown that exosomes are presentin ovarian cancer cell culture supernatants and ovarian cancerpatients' plasma/serum or ascites [15,16,21,22]. Douglas D.Taylor et al. found diagnostic microRNAs from EpCAM-positiveexosomes in ovarian cancer patients' serum [21]. Li et al.detected claudin-containing exosomes in plasma from patientswith ovarian cancer [15]. Peng et al. demonstrated exosomesin the ascites of patients with ovarian cancer and revealedthe origin and effects of these exosomes on anti-tumorimmunity [16]. Therefore analysis of biological characteris-tics of ovarian cancer-derived exosomes will be of value inthe diagnosis of ovarian cancer, monitoring the therapeuticefficacy of exosomes, exploring their roles in tumor progres-sion and so on. These studies about ovarian cancer-derivedexosomes only highlight the expression of individualmoleculeson exosomes. To our knowledge, the systematic proteomicanalysis of ovarian cancer-derived exosomes, whether from thecancer cell conditioned medium, plasma or ascites of patients,has never been reported.

Exosomes in body fluidmay originate fromheterogeneouscell types. For example, plasma exosomes may be derivedfrom immune cells, platelets, endothelial cells, hepatocytesand other cells [23,24]. Similarly, exosomes present in theascites of ovarian cancer patients may originate from differentcell types [20,23]. More importantly, proteomic analyses ofexosomes from plasma or ascites reveal that the exosomes areunavoidably contaminated by highly abundant non-exosomalproteins such as albumin, immunoglobulin and complementcomponents. A global proteomic analysis of exosomes froma single ovarian cancer cell line cultured in conditionedmedium may be one approach to resolve the problem. In thisarticle, we purified exosomes from the conditioned mediumof two ovarian cancer cell lines, OVCAR-3 and IGROV1, andperformed the first systematic proteomic analysis of humanovarian cancer-derived exosomes.

2. Experimental procedures

2.1. Cell culture

OVCAR-3 is a cell line originating from the malignant ascitesof a patient with papillary adenocarcinoma of the ovary[25]. IGROV1 is a cell line derived from the tumor tissue of

a 47-year-old woman suffering from stage III ovarian adeno-carcinoma [26]. Well characterized ovarian cancer cell linesOVCAR-3 and IGROV1 were obtained from the NIH cellbank. In vitro cultured OVCAR-3 and IGROV1 cells were usedas the exosome source for this study because they have beenextensively characterized previously, and are representativeof the behavior and phenotype of epithelial ovarian adenocar-cinoma. The cells were maintained in RMPI-1640 (HyClone),supplemented with 10% dFBS (which had been depleted ofbovine-derived exosomes by ultracentrifugation for 16 hat 120,000 g, followed by filtration through a 0.2 μm filter(Millipore)). The cells were seeded into 15 cm dishes (Corning),and incubated in humidified air in 5% CO2 at 37 °C.

2.2. Exosome isolation and purification

Ovarian cancer cells were grown up to 70%–80% in theirstandard medium with 10% dFBS. The medium was removed.Cells were rinsed three times with phosphate-buffered saline(PBS) and then grown in serum-free medium (20 ml for a15 cm dish). After 24 h–48 h of incubation, the conditionedmedium was collected, centrifuged once at 500 g for 10 min,and 2000 g for 20 min to eliminate cell and cell debriscontamination [10]. The supernatant was further centrifugedat 16,500g for 30 min at 4 °C. Then the exosomes werepelleted by ultracentrifugation at 120,000g for 80 min at 4 °C.The exosome pellets were washed once in PBS, pelleted byultracentrifugation at 120,000g for 80 min at 4 °C again. Thefinal exosome pellets were resuspended in PBS. To get pureovarian cancer-derived exosomes, a further purification stepwas performed as previously described with little modification[27]. Briefly, the PBS-suspended exosome preparations werediluted in 3.5 ml PBS and underlayered on top of a densitycushion composed of 20 mMTris/30% sucrose/deuterium oxide(D2O)/HCL pH 7.35 (0.5 ml) forming a visible interphase. Thesamples were ultracentrifuged at 120,000 g, for 70 min at 4 °Cin a SW60Ti swinging bucket rotor (Beckman Coulter, Fullerton,CA). Exosomes contained in the 30% sucrose/D2O/Tris cushionand interphase were diluted 8 times with PBS and centrifugedat 120,000 g, for 70 min at 4 °C. The final exosome pellets ofhigher purity were resuspended in PBS. After measuring thetotal protein concentration of the purified exosomes usingBradford dye assays (Bio-Rad Laboratories, Hercules, CA), theexosome preparations were stored at −80 °C until use.

2.3. Sucrose density gradient centrifugation

PBS-washed exosomes were resuspended in 2 ml 2.5 Msucrose, 20 mMHEPES/NaOH pH 7.2. A linear sucrose gradient(0.25 M–2.5 M sucrose, 20 mM HEPES/NaOH pH 7.2) was lay-ered on top of the exosome suspension of 2.5 M sucrose in aSW32Ti tube (Beckman Instruments, Inc.) and centrifuged at170,000 g for 16 h at 4 °C. After centrifugation, the gradientswere removed from the top in 12 fractions, and the density ofeach fraction was estimated by weighing a fixed volume ofeach fraction. Then each fraction was diluted 10 times withPBS and centrifuged at 120,000 g for 80 min at 4 °C. Finally thepellet of each fraction was resuspended in 80 μl PBS, and usedfor Western-blotting.

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2.4. Electron microscopy

2.4.1. Cryo-electron microscopy of OVCAR-3-derivedexosomesFor cryo-electron microscopy, a droplet of exosome suspen-sion (3 μl) was directly adsorbed onto glow-discharged holeycarbon-coated grids (Quantifoil, Germany). After removingexcess fluid, grids were rapidly plunged into liquid ethanewith the aid of Vitrobot™ (FEI). Then the samples were storedin liquid nitrogen and transferred into a transmission electronmicroscope using a Gatan 626 cryo-holder. The samples wereimaged on a FEI Tecnai G2 Spirit TWIN electron microscopewith source LaB6, operated at 120 kV.

2.4.2. Transmission electron microscopy of IGROV1-derivedexosomesExosomes obtained after differential centrifugation of condi-tioned cell-culture medium were loaded onto Formvarcarbon-coated 200 mesh copper grids and allowed to absorbat RT for 10 min. Excess fluid was drained with filter paperslightly. Adsorbed exosomes were negatively stained with3% phosphotungstic acid at RT for 5 min. The exosome-containing grids were air-dried and observed by means of aHitachi H-7600 transmission electron microscope operating at80.0 kV and images were captured by an AMT capture system.

2.5. Western-blot analysis

Exosomes were prepared as described above. 5× SDS-loadingbuffer was added to dissolve the proteins from exosomes,diluted to 1× SDS-loading buffer, and then heated at 95 °C for5 min. The samples were centrifuged at 13,000 rpm for 5 minto remove insoluble materials. Supernatants were subse-quently loaded onto SDS-PAGE (3% stacking gel, 12%–15%running gel), running in a Mini Protean 2 electrophoresissystem (Bio-Rad, Munich, Germany). The protein was trans-ferred to a polyvinylidene fluoridemembrane (PVDF) in transferbuffer. After being blocked with 5% non-fat milk in PBST for1 h at RT, the membrane was incubated with the primaryantibody overnight at 4 °C. Immunocomplexes were labeledwith HRP-conjugated secondary antibody and detected usingan enhanced chemiluminescence (ECL) system. Between eachincubation step, the membrane was washed three timeswith 0.5% PBST (PBS with 0.5% Tween-20). Cell lysates werecompared to exosome lysates by immuno-blotting. The follow-ing primary antibodies were used: TSG101 (Proteintech group),Alix (Proteintech group), GM130 (Proteintech group), TFRC(Proteintech group), EpCAM (Proteintech group), hnRNPA1(Sigma Aldrich Co., clone 9H10), β-actin (Santa Cruz Biotech-nology) and cytochrome C (BD Pharmingen).

2.6. 1D SDS-PAGE and in-gel digestion

The purified exosomes (40 μg) were electrophoresed on a 12%1D SDS-PAGE. After bromophenol blue ran 2–3 cm into theresolving gel, the gel was stained with Coomassie brilliantblue and cut into 4 slices. Each gel slice was destainedwith 200 μl of 50% acetonitrile (ACN) in 50 mM NH4HCO3,dehydrated with 50 μl of ACN, and dried. In-gel trypticdigestion was carried out overnight at 37 °C using a 10 ng/μl

solution of sequencing-grade trypsin (Promega, Madison, WI)in 50 mM ammonium bicarbonate, pH 8.0. Tryptic peptidesresulting from the digestion were extracted with 5% formicacid/50% acetonitrile and 0.1% formic acid/75% acetonitrilesequentially and then concentrated to ~20 μl.

2.7. Nano-LC–ESI-MS/MS

The extracted peptides were separated by an analyticalcapillary column (50 μm×10 cm) packed with 5 μm sphericalC18 reversed phase material (YMC, Kyoto, Japan). A WatersnanoACQUITY UPLC system (Waters, Milford, USA) was usedto generate the following HPLC gradient: 0–30% B in 60 min,and 30–70% B in 15 min (A=0.1% formic acid in water, B=0.1%formic acid in acetonitrile). The eluted peptides were sprayedinto an LTQ ORBITRAP Velosmass spectrometer (ThermoFisherScientific, San Jose, CA, USA) equipped with a nano-ESI ionsource. Themass spectrometerwas operated in data-dependentmode with one MS scan followed by ten HCD (high-energycollisional dissociation) MS/MS scans for each cycle.

2.8. Database searches

Database searches were performed on an in-house Mascotserver (Matrix Science Ltd., London, UK) against the IPI(International Protein Index) human protein database. Thesearch parameters were: 7 ppm mass tolerance for precursorions; 0.02 Da mass tolerance for product ions; and two missedcleavage sites were allowed for trypsin digestion. Methionineoxidation was set as a variable modification. The search resultswere filtered with both peptide significance threshold andexpectation value to be below0.05. TheMascot Percolator scoreswere used for all peptides.

2.9. Gene ontology annotation

To obtain the subcellular localization of identified proteins,we searched the LocDB (http://www.rostlab.org/services/locDB)[28] database. Biological processes, molecular functions andthe pathways of the proteins involved were analyzed byPANTHER (http://www.pantherdb.org/) consortiumdatabases[29]. We also performed the detailed enrichment GO analysisof different exosomal protein subsets including the overlappedproteins from the two ovarian cancer cell lines and ovariancancer-specific proteins absent in the ExoCarta repository.

3. Results

3.1. Characterization of ovarian cancer-derived exosomes

The exosomes secreted by OVCAR-3 and IGROV1 cells wereisolated from serum-free culture supernatants by a combina-tion of differential centrifugation to remove cell and cell debris.Then further purification step was performed by ultracentrifu-gation on cushion of 30% sucrose/D2O/Tris pH 7.35 to removenon-membranous proteins, protein aggregates and other largervesicles. Western blots were performed to detect the exosomalmarker proteins. The published exosomal marker molecules

(1)

(2)

TSG101

OVCAR3-exo IGROV1-exo

96 kDaAlix

45kDa

A

B

C

D OVCAR-3

WCL EXO WCL EXO

IGROV1

Cytochrome C

GM130 130 kDa

15kDa

Density: 1.228 1.168 1.158 1.154 1.143 1.112 1.097 1.080 1.086 1.073 1.069 1.075 g/ml

12 11 10 9 8 7 6 5 4 3 2 1Fraction:

TSG101

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OVCAR-3-exosome

1772

IGROV1-exosome

1475

755

458

1017

Ovcar3 -exosome

IGROV1-exosome

Exocarta database

494

682380

184

152233

1514

A

B

Fig. 2 – A—Venn diagrams depicting overlap in proteinsidentified in OVCAR-3- and IGROV1-derived exosomes. Atotal of 2230 proteins were identified from the ovarian cancercell line-derived exosomes, and 1017 proteins werecommonly identified in the two cell lines. B—Three-wayVenn diagram depicting the overlap between the genes ofencoding exosomal proteins derived from OVCAR-3 andIGROV1 and previous exosomal studies published in theExoCarta database. Here, 682 proteins were found to becommonly identified in both ovarian cancer cell-derivedexosomes and previous studies published in the ExoCartadatabase.

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TSG101 and Alix were detected in exosome preparationsderived from OVCAR-3 and IGROV1 cell lines (Fig. 1A). Thepreparations were examined by cryo- or transmission electronmicroscopy and were shown to be mostly 30–100 nm indiameter (Fig. 1B). The exosome preparations were layered ona continuous sucrose and ultracentrifuged to separate floatingexosomes from other non-exosomal secreted proteins andprotein aggregates. Twelve fractions were collected from thetop of the gradients. The known exosomal marker proteinTSG101 was found to be present between fractions 6–9 with thedensity range of 1.09–1.15 g/ml (Fig. 1C). Such analysis con-firmed that ovarian cancer cell-derived exosomes are similar tothose from other cell types in density [13,14].

The cis-Golgi protein GM130 was detected in whole celllysates but was not detectable in exosomes, which mayindicate that the exosome preparations were not contami-nated by cellular debris. Similarly, cytochrome C was notdetected in exosomes, but was obviously positive in thewhole cell lysate, which indicated that almost no contami-nating apoptotic body was present in the exosome prepara-tions (Fig. 1D). The overall viability of OVCAR-3 and IGROV1was all more than 96% judged by trypan blue staining ofdead cells.

3.2. Proteomic analysis of exosomes derived from twoovarian cancer cell lines

To determine the protein profile of ovarian cancer derivedexosomes, total exosomal proteins were separated by 1DSDS-PAGE on 12% gel and then sliced into 4 bands. After everyband was cut into pieces and subjected to trypsin digestion in1.5 ml Eppendorf tubes respectively overnight, the extractedpeptides were analyzed by nano-LC–MS/MS. Protein databasesearching of MS/MS data resulted in identifying 1772 proteinsin OVCAR-3 derived exosomes and 1475 proteins in IGROV1derived exosomes respectively. Furthermore, only those pro-teins identified with 2 or more peptides, an expected valueof less than 0.05, and a false discovery rate (FDR) of 1% wereincluded. 1017 proteins were identified in the exosomes fromboth cell lines. The high overlap of proteins between exosomesfrom two ovarian cancer cell lines suggests that the proceduresused for exosome isolation and purification are reproducible,and the methods for protein identification are reliable.

Western-blot confirmed that OVCAR-3 and IGROV1-derivedexosomes contained β-actin, EpCAM, hnRNPA1, hnRNPK, andAlix that were identified by MS/MS analysis. TFRC was notverified in exosomes from IGROV1 by Western-blot although

Fig. 1 – A—Western-blotting analysis of exosomes isolated fromlysate proteins were separated on 12% SDS-PAGE and transferreagainst Alix and TSG101. B—(1) Cryo-electronmicroscopy imagesshow that exosomes are round and mostly 30–100 nm in diametare shown to be almost uncontaminated by cellular debris or proIGROV1-derived exosomes negatively stained with phosphotunground and 30–100 nm in diameter. C—Fractions of linear sucroseExosome marker protein, TSG101 was strongly detected in fractiand 1.15 g/ml, which is consistent with the typical exosome denprotein positive in apoptotic bodies and GM130, a cis-Golgi appathe cultured medium of the two ovarian cancer cell lines, but we

it was identified by MS/MS. The exosomal marker proteinTSG101 was also detected in exosomes derived from another 4ovarian cancer cell lines (not shown). The relative expression ofexpected published exosomal proteins was evaluated between

conditioned medium of OVCAR-3 and IGROV1. Exosomald to PVDF membrane. Blots were probed with antibodiesof exosomes from OVCAR-3 ovarian cancer cells. The imageser except for a few that are larger than 100 nm. The samplestein aggregates. (2) Transmission electron micrographs ofstic acid. The images show that small vesicles are almostdensity gradients were analyzed by Western blotting.

ons 6, 7, and 8. The density range is around between 1.09sity of 1.09–1.19 g/ml. D—Cytochrome C, a mitochondrialratus protein were not detected in prepared exosomes fromre strongly positive in the whole cellular lysates (WCL).

Fig. 3 – Validation of the MS/MS results by Western blotting.To validate several exosomal proteins identified in ourstudy, Western blotting was performed for both whole celllysates (WCL) and exosomes. The proteins were separatedon a 12% SDS-PAGE gel, transferred to PVDF membrane,and probed with specific antibodies against Alix, EpCAM,β-actin, TFRC, hnRNPA1, hnRNPK and TSG101.

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whole cell lysates (WCL) and exosomes (EXO) by Westernblotting. The total amount of loading protein in WCL laneswas about 10 times more than in EXO lanes. The staining

Subcellular Loc

0.00% 5.00% 10.00% 15.00% 20.0

mitochondrion

vesicle

proteinaceous extracellular matrix

nucleus

plasma membrane

endosome

Golgi apparatus

endoplasmic reticulum

peroxisome

cytoplasm

extracellular region

Fig. 4 – Classification of identified proteins by subcellular localizwas determined by the LocDB database. Distributions of OVCAR-localization. There are more frequent extracellular region proteinexosomes.

intensities of some exosome-specific proteins in EXO laneswere approximate to or even higher than those in WCL lanes,although with different loading protein amounts, such as Alixin both cells, and TSG101 and EpCAM in OVCAR-3 cells (Fig. 3).The absolute integrated OD of each band (IOD) was calculatedwith the Gel-Pro Analyzer 4.0 software (listed in SupplementalTable 3). These indicated that the reported exosomal markerproteins such as TSG101, Alix, and EpCAM, may be enrichedin exosome preparations compared with cell lysates, whilehnRNPA2 and hnRNPKwere not shown to be enrichedwhetherin OVCAR-3 or IGROV1 exosome preparations. Additionally, itseems that several molecules, including TSG101, EpCAM andβ-actin, were more enriched in OVCAR-3-derived exosomepreparations than in the exosomes of IGROV1 cells. Enrichingsome exosomal marker proteins in exosomes is also character-istic of exosomes produced by other cell types such as bladdercancer cells, colorectal cancer cells and hepatocytes [13,14,30].

By comparing the identified exosomal protein gene sym-bols with the ExoCarta database of exosomal proteins, theresults showed that there were about 1398 proteins identifiedin at least two trials. Among them, about 380 proteins havenot been previously identified in human originated exosomes(ExoCarta repository), while the published top 25 proteins thatwere often identified in exosomal studies as exosomal markerswere all identified in our two experiments except for HLA-DRA[31] (http://exocarta.org/exosome_markers).

3.3. Annotation of identified proteins

The identified proteins were classified by subcellular local-ization, biological process and molecular functions. We usedthe LocDB and PANTHER database to annotate the identifiedproteins in the two ovarian cancer-derived exosomes andExoCarta repository proteins respectively. Fig. 4 shows thesubcellular localization of the identified proteins in the twoexperiments and the ExoCarta repository proteins. Proteinsubcellular localization distributions are highly similar be-tween the two ovarian cancer cell lines. More frequentextracellular region proteins present in the ExoCarta database

ation

0% 25.00% 30.00% 35.00%

Exocarta database IGROV1-EXOOVCAR-3-EXO

ation. The subcellular localization of the identified proteins3 and IGROV1 in our study are highly similar in subcellulars in the ExoCarta database than in the ovarian cancer-derived

Biological Process

0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00%

cell communication (GO:0007154)

cellular process (GO:0009987)

localization (GO:0051179)

transport (GO:0006810)

cellular component organization (GO:0016043)

apoptosis (GO:0006915)

system process (GO:0003008)

reproduction (GO:0000003)

response to stimulus (GO:0050896)

regulation of biological process(GO:0050789)

homeostatic process (GO:0042592)

developmental process (GO:0032502)

generation of precursor metabolites andenergy (GO:0006091)

metabolic process (GO:0008152)

cell cycle (GO:0007049)

immune system process (GO:0002376)

cell adhesion (GO:0007155)

Exocarta database

IGROV1-EXO

OVCAR-3-EXO

Fig. 5 – Biological processes in which exosomal proteins are involved were determined by PANTHER analysis. OVCAR-3, IGROV1and ExoCarta database exosomal proteins were classified by biological process and compared.

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maybe due to different protein identificationmethods. In termsof the biological processes as shown in Fig. 5, the exosomalproteins are mainly involved in metabolic process, cell com-munication, cellular process, transport, developmental process,cell cycle, immune system process and so on. Compared withthe ExoCarta database, the ovarian cancer-derived exosomessignificantly enrich some proteins involved in the processes ofmetabolism, cell cycle, reproduction, systemprocess, apoptosis,and transport. The molecular functions of these proteinsare shown in Fig. 6, ovarian cancer-derived exosomes enrichsome proteins having binding and catalytic activities. Detailed

Molecu

0.00% 5.00% 10.00% 15.00% 20.00%

ion channel activity (GO:0005216)

transporter activity (GO:0005215)

translation regulator activity(GO:0045182)

transcription regulator activity(GO:0030528)

enzyme regulator activity (GO:0030234)

catalytic activity (GO:0003824)

motor activity (GO:0003774)

receptor activity (GO:0004872)

antioxidant activity (GO:0016209)

structural molecule activity (GO:0005198)

binding (GO:0005488)

Fig. 6 –Molecular functions of theseproteinsweredeterminedbyPAand ExoCarta exosomal proteins were compared.

exosome protein information for the two ovarian cancer cellline-derived exosomes is provided in Supplemental Tables 1and 2.

We further performed detailed GO enrichment analysisin biological process for the two ovarian cancer shared 1017proteins. As shown in Fig. 7, the exosomes derived from the twoovarian cancer lines enriched some of the proteins involvedin the following biological processes: protein ubiquitination,RNA catabolic process, viral reproduction, cell cycle, antigenprocessing and presentation, cellular protein complex disas-sembly and protein targeting transport. Some of these enriched

lar Function

25.00% 30.00% 35.00% 40.00% 45.00% 50.00%

Exocarta databaseIGROV1-EXOOVCAR-3-EXO

NTHER analysis. Themolecular functions ofOVCAR-3, IGROV1

Fig. 7 – The result of detailed GO enrichment analysis of biological processes among the overlapped 1017 proteins between OVCAR-3- and IGROV1-derived exosomes. Thecolor saturation degrees of boxes positively correlate with the significance of enrichment in biological processes.

178JO

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LO

FPR

OT

EO

MIC

S80

(2013)

171–182

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processes are associated with exosome biogenesis such asprotein ubiquitination, viral reproduction and protein targetingtransport, while other processes may be associated withspecific functions of the exosome such as antigen processingand presentation, and cell cycle.

Previous exosomal proteomic studies revealed that exosomescontain a conserved set of common proteins from various celltypes and cell type specific proteins. The common proteinsinclude MV (microvesicle) biogenesis associated proteins (suchas Alix, TSG101 and clathrin), small GTPases (Rab proteins),annexin proteins, tetraspanins, heat shock proteins and 14-3-3proteins. Most of these proteins have been known as exosomalmarker molecules: CD9, CD63, CD81, Tsg101, Alix, HSP70 andHSP90. All of these proteins were identified in our studies(Supplemental Tables 1 and2). There are also somemicrovesiclebiogenesis associated proteins only present in our study such asrab12, 18, and 23; vacuolar protein sorting-associated pro-teins (VPS26A and VPS29); VTI1B; membrane budding pro-teins (ARCN1, COPE, COPG, PICALM) and so on.

Table 1 shows the top 25 pathways that involve theseexosomal proteins. Pathways of cytoskeletal regulation byRho GTPase, the heterotrimeric G-protein signaling pathwayand the ubiquitin proteasome pathway may be associatedwith exosome formation, secretion and intracellular traffick-ing [4,5,8,32–34]. Most other pathways may be involved intumor growth and progression including the integrin signal-ing pathway, EGF receptor signaling pathway, Wnt signalingpathway, PI3 kinase pathway, FGF receptor signaling path-way, angiogenesis pathway, Ras pathway, P53 pathway andso on [2,35,36].

4. Discussion

Exosomes are nanometer sized vesicles secreted by various celltypes. Recently, more and more studies prove that exosomesplay important roles in cellular communications. There havebeen some studies about exosomes in colorectal cancer [14,20],bladder cancer [13], prostate cancer [37], pancreatic cancer [38],cardiovascular diseases [39] and malignancies of the centralnervous system [40]. These studies highlight the roles of severalexosomal molecules and the proteomic analysis of exosomes.So far, there was no report about the systematic proteomicanalysis of exosomes, whether found in malignant effusion,blood or cell culture medium, in the context of ovarian cancer.However, it is so important to know the proteomic profile ofthe ovarian cancer-derived exosomes, when developing somenovel diagnostic biomarkers and therapeutic targets based onexosomes. Furthermore, analyzing the protein composition ofexosomes is very helpful to further understand themechanismof their biogenesis and the functional roles in this most lethalgynecologic malignancy.

We have examined exosomes isolated from ovarian cancercell lines, OVCAR-3 and IGROV1. They are round, mostly30–100 nm in diameter and have a density of 1.09–1.15 g/mlon sucrose gradients. The latter property is a very usefulcharacteristic, which can be employed to separate exosomesfrom other vesicles or protein aggregates. We also performedWestern blots for detecting those proteins, which were notputatively expressed in exosomes such as cytochrome C

and GM130. Based on the size, density and marker moleculeexpression of the vesicles, our experimental proceduresobtained a good yield of exosomes with high purity, whichwas essential for downstream proteomic analysis.

We successfully identified a total of 2230 exosomalproteins from two ovarian cancer cell lines by performingan LC–MS/MS workflow. The number of identified proteinsin this study is larger than the previous exosome proteomicstudies of high quality [13,14,20]. In order to avoid thecontaminants from bovine serum, we cultured cells in amedium with 10% dFBS (which had been depleted of bovine-derived exosomes by ultracentrifugation for 16 h at 120,000 g,followed by filtration through a 0.2 μm filter (Millipore))before cells reached 70%–80% confluency. Then, after wash-ing the cells three times with PBS, the medium was changedto serum-free medium and cultured for 24–48 h. The prepa-rations were also purified by sucrose/D2O cushion centrifu-gation. By combining the observation of exosomes undera cryo-electron microscope and the Western-blotting resultsof cytochrome C and GM130, we think we got highly purifiedovarian cancer cell-derived exosomes. In addition, we foundthat the number of identified proteins from exosomes wasincreasing with improvement in technology by reviewingrecent published articles about exosomal proteomic analysis[14,18,20,23,41]. Recently, Wang et al. reported that 2179 pro-teinswere identified fromurine exosomes bymultidimensionalprotein identification (MudPIT) [18]. Therefore we think thelarger number of identified proteins in our study was mainlydue to the high sensitivity of our MS protein identificationworkflow for detecting the low abundant exosomal proteins.However the lack of tools for accurately estimating the levelsof sample contamination is a major difficulty in the field. Webelieve that more and more low abundant proteins will beidentified from exosomes in future studies.

An inspection of proteome data set for exosomes derivedfrom OVCAR-3 and IGROV1 revealed the presence of manyproteins common to exosomes from various origins, includingTSG101, Alix, heat shock proteins, tetraspanins, rabs, annexins,cytoskeletal proteins and some enzymes, which may be associ-ated with biogenesis, structure and trafficking of exosomes. Bycomparing the gene symbols of the identified exosomal proteinsfrom the two ovarian cell lines with ExoCarta, a gene symboldatabase of published proteins identified from exosomes, wefound that 1018 proteins (47.9%) among all our identified proteingene symbols were present in the database and 1107 proteinswere unreported as revealed in the Venn diagram (Fig. 2B). 682(64.2%) proteins among the common proteins in exosomesfrom the two ovarian cancer cell lines were present in thedatabase, while the remaining 380 proteins were absent.Multilevel GO enrichment analysis in biological process re-vealed that exosomes derived from ovarian cancer cellsenriched some proteins associated with protein ubiquitination,RNA catabolic process, viral reproduction, cell cycle, antigenprocessing and presentation, cellular protein complex disas-sembly and protein targeting transport (shown in Fig. 7). Proteinubiquitination is required for exosome formation [8,42,43].MacDonald et al. found that protein ubiquitination is essentialfor the recruitment of endosomal sorting complexes requiredfor transport (ESCRT) machinery onto endosomal membranesandmultivesicular body intralumenal vesicle (ILV) formation

Table 1 – The top 25 pathways of the overlapped proteinsinvolved by PANTHER analysis.

No. Pathways Proteinidentified

1 Integrin signaling pathway (P00034) 442 Huntington disease (P00029) 373 Parkinson disease (P00049) 364 Inflammation mediated by chemokine and

cytokine signaling pathway (P00031)33

5 EGF receptor signaling pathway (P00018) 316 Cytoskeletal regulation by Rho GTPase (P00016) 247 Wnt signaling pathway (P00057) 218 PI3 kinase pathway (P00048) 209 FGF signaling pathway (P00021) 2010 Angiogenesis (P00005) 1811 Ubiquitin proteasome pathway (P00060) 1612 PDGF signaling pathway (P00047) 1613 Heterotrimeric G-protein signaling pathway-Gi

alpha and Gs alpha mediated pathway(P00026)

16

14 Ras pathway (P04393) 1515 Cadherin signaling pathway (P00012) 1316 Apoptosis signaling pathway (P00006) 1217 p53 pathway (P00059) 1218 De novo purine biosynthesis (P02738) 1219 Nicotinic acetylcholine receptor signaling

pathway (P00044)12

20 T cell activation (P00053) 1121 Heterotrimeric G-protein signaling

pathway-Gq alpha and Go alpha mediatedpathway (P00027)

11

22 Endothelin signaling pathway (P00019) 1123 Alzheimer disease-presenilin pathway

(P00004)10

24 Muscarinic acetylcholine receptor 2 and 4signaling pathway (P00043)

10

25 Interleukin signaling pathway (P00036) 10

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[44]. The mechanism of multivesicular body intralumenalvesicle formation is similar to the budding of virus out ofcytosol. Some of the protein machineries that control MVBformation are also similar with the production of variousenveloped viruses such as HIV and Ebola [43,45]. The biologicalprocesses of cellular protein complex disassembly and proteintargeting transport are also essential for exosome biogenesis.

In addition to some common proteins involved in exosomebiogenesis, recent evidences have demonstrated that exosomesalso harbor some cell/tissue-specific protein signatures [4,20,46]. In our study, the detailed GO enrichment analysis resultrevealed that ovarian cancer-derived exosomes enriched thesignal transduction proteins of cell cycle checkpoint, P53class mediators, and DNA damage response (shown in Fig. 7).As we all know, cell cycle checkpoint and DNA damage areimportant or even decisive factors for tumorigenesis [47,48].The majority of epithelial ovarian cancer is characterizedby frequent mutations in p53 [35]. Our results showed thatovarian cancer-derived exosomes also enriched a set ofproteins involved in antigen processing and presentation ofpeptide antigen. It was consistent with previous reports thattumor-derived exosomes could induce anti-tumor immuneresponse and be used as a source of tumor antigens [49–51].

Although the BioVenn analysis found that there were 380proteins shared by two ovarian cancer cells derived exosomesthat are absent in the ExoCarta database, the number maybe smaller in fact because some of them have been identifiedin previous studies but are not included in the ExoCartadatabase. In addition, some of them belonged to families ofproteins described in the ExoCarta database. We performed adetailed enrichment GO analysis of the 682 proteins (commonto the two ovarian cancer-derived exosomes and the ExoCartadatabase) and the 380 proteins (data not shown). The specific380 proteins were also significantly enriched proteins in-volved in cell cycle checkpoint, DNA damage response, andP53 classmediators, while the common 682 proteins had no suchenrichment. All these data demonstrated that ovarian cancercell-derived exosomes carried some ovarian cancer-specificproteins.

Table 1 showed that ovarian cancer-derived exosomeproteins were highly enriched in carcinogenesis-associatedsignal pathways. Some pathways are associated with migra-tion, invasion, immune modulation and angiogenesis, andthese processes arenecessary for tumorigenesis andmetastasis.Based on the known functions of exosomes in cellular commu-nications [4,5,10], exosomes carrying tumorigenesis-associatedmolecules released by ovarian cancer cells might play someimportant roles in tumor growth and metastasis by providinga conducive tumor micro-environment. The pathway analysisalso found that the exosomes contained Huntington disease-and Parkinson disease-associated proteins. Both are neurode-generative diseases as a result of neurodegenerative processes.Previous studies indicated that exosomes may act as vehiclesof proteins associated with neurodegenerative diseases [52–54].By analysis, we also found that a subset of proteins that wasoverexpressed in ovarian cancer tissue was present in theexosomal protein lists. These molecules include epithelialcell surface antigen (EpCAM), proliferation cell nuclear antigen(PCNA), tubulin beta-3 chain (TUBB3), epidermal growth factorreceptor (EGFR), apolipoprotein E (APOE), claudin 3 (CLDN3),

fatty acid synthase (FASN), ERBB2, and L1CAM (CD171) [55]. Inview of the high challenge of metastatic and chemo-resistantcharacteristics of ovarian cancer, our exosomal proteomic datamay provide some new information and directions for futureresearch. Exosomes containing cell-type and disease associatedproteinsmay also be a source of diagnosticmarkers and a targetof new therapeutic methods.

5. Conclusion

We have successfully isolated and purified exosomes fromconditioned culture supernatants of two human ovarian cancercell lines.Wehaveperformed thehighquality proteomic analysisof ovarian cancer-derived exosomes and identified someproteins that had not been identified in exosomes before. Thegene ontology (GO) analysis results revealed that ovarian cancercell-derived exosomes may carry cell/tissue specific proteins,which may provide some new biomarkers for diagnosis andindicate exosomal functions in ovarian cancer. The commonproteins associatedwith exosome biogenesismay provide somenew information on understanding themechanism of exosomesecretion in ovarian cancer. Further studies shouldbe conducted

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to validate some candidate diagnostic markers and confirmthe functional roles of exosomes in the malignant disease.

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.jprot.2012.12.029.

Acknowledgments

We thank the Youth Fund of National Natural Science Founda-tion of China (program no.: 81101976) for the financial supportof this investigation. We thank Wanzhong He and Sha Zhangfor technical assistance with electron microscopy experiments.

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