Exosomal Secretion of Cytoplasmic Prostate Cancer Xenograft-derived Proteins
2009; Elsevier BV; Volume: 8; Issue: 6 Linguagem: Inglês
10.1074/mcp.m800443-mcp200
ISSN1535-9484
AutoresFlip H. Jansen, Jeroen Krijgsveld, Angelique van Rijswijk, Gert-Jan van den Bemd, Mirella S. van den Berg, Wytske M. van Weerden, Rob Willemsen, Lennard J. M. Dekker, Theo M. Luider, Guido Jenster,
Tópico(s)Prostate Cancer Treatment and Research
ResumoNovel markers for prostate cancer (PCa) are needed because current established markers such as prostate-specific antigen lack diagnostic specificity and prognostic value. Proteomics analysis of serum from mice grafted with human PCa xenografts resulted in the identification of 44 tumor-derived proteins. Besides secreted proteins we identified several cytoplasmic proteins, among which were most subunits of the proteasome. Native gel electrophoresis and sandwich ELISA showed that these subunits are present as proteasome complexes in the serum from xenograft-bearing mice. We hypothesized that the presence of proteasome subunits and other cytoplasmic proteins in serum of xenografted mice could be explained by the secretion of small vesicles by cancer cells, so-called exosomes. Therefore, mass spectrometry and Western blotting analyses of the protein content of exosomes isolated from PCa cell lines was performed. This resulted in the identification of mainly cytoplasmic proteins of which several had previously been identified in the serum of xenografted mice, including proteasome subunits. The isolated exosomes also contained RNA, including the gene fusion TMPRSS2-ERG product. These observations suggest that although their function is not clearly defined cancer-derived exosomes offer possibilities for the identification of novel biomarkers for PCa. Novel markers for prostate cancer (PCa) are needed because current established markers such as prostate-specific antigen lack diagnostic specificity and prognostic value. Proteomics analysis of serum from mice grafted with human PCa xenografts resulted in the identification of 44 tumor-derived proteins. Besides secreted proteins we identified several cytoplasmic proteins, among which were most subunits of the proteasome. Native gel electrophoresis and sandwich ELISA showed that these subunits are present as proteasome complexes in the serum from xenograft-bearing mice. We hypothesized that the presence of proteasome subunits and other cytoplasmic proteins in serum of xenografted mice could be explained by the secretion of small vesicles by cancer cells, so-called exosomes. Therefore, mass spectrometry and Western blotting analyses of the protein content of exosomes isolated from PCa cell lines was performed. This resulted in the identification of mainly cytoplasmic proteins of which several had previously been identified in the serum of xenografted mice, including proteasome subunits. The isolated exosomes also contained RNA, including the gene fusion TMPRSS2-ERG product. These observations suggest that although their function is not clearly defined cancer-derived exosomes offer possibilities for the identification of novel biomarkers for PCa. For several decades now, prostate-specific antigen (PSA) 1The abbreviations used are:PSAprostate-specific antigenPCaprostate cancerADAMa disintegrin and metalloproteaseIPIInternational Protein IndexGAPDHglyceraldehyde-3-phosphate dehydrogenaseLTQlinear trap quadrupoleHRShepatocyte growth factor-regulated tyrosine kinase substrate (also known as HGS)ERGETS-related gene1Done-dimensionalVCaPVertebral Cancer of the Prostate. has been utilized as the "gold standard" biomarker for the detection of prostate cancer (PCa) (1Stamey T.A. Yang N. Hay A.R. McNeal J.E. Freiha F.S. Redwine E. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate.N. Engl. J. Med. 1987; 317: 909-916Crossref PubMed Scopus (2025) Google Scholar). Its introduction caused a dramatic decrease in the prevalence of advanced stages of PCa (2McDavid K. Lee J. Fulton J.P. Tonita J. Thompson T.D. Prostate cancer incidence and mortality rates and trends in the United States and Canada.Public Health Rep. 2004; 119: 174-186Crossref PubMed Scopus (97) Google Scholar). However, ongoing efforts are being made to discover new biomarkers for PCa because it became clear that PSA has limited diagnostic specificity and prognostic value, leading to an enormous increase in unnecessary biopsies and overtreatment of low risk PCa patients (3Thompson I.M. Pauler D.K. Goodman P.J. Tangen C.M. Lucia M.S. Parnes H.L. Minasian L.M. Ford L.G. Lippman S.M. Crawford E.D. Crowley J.J. Coltman Jr., C.A. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter.N. Engl. J. 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Prev. 2000; 9: 1133-1147PubMed Google Scholar). On the RNA level, the PCA3 test and especially the recently discovered fusion of TMPRSS2 with ETS transcription factors may hold promise for PCa detection and potentially prognosis in the near future (9de Kok J.B. Verhaegh G.W. Roelofs R.W. Hessels D. Kiemeney L.A. Aalders T.W. Swinkels D.W. Schalken J.A. DD3(PCA3), a very sensitive and specific marker to detect prostate tumors.Cancer Res. 2002; 62: 2695-2698PubMed Google Scholar, 10Kumar-Sinha C. Tomlins S.A. Chinnaiyan A.M. Recurrent gene fusions in prostate cancer.Nat. Rev. Cancer. 2008; 8: 497-511Crossref PubMed Scopus (575) Google Scholar). One of the drawbacks of the latter two as markers for PCa is the fact that they are detected in urine, after a standardized prostatic massage, instead of in serum or plasma. This will hamper retrospective validation as most historical biorepositories do not contain urine. Although several validation studies of promising candidates have been performed in the past or are currently underway, no single marker has yet outperformed PSA, justifying ongoing efforts in searching for PCa biomarkers. One approach is the screening of large series of serum samples from men with and without PCa. However, given the large sample variability, the high complexity, and dynamic range of proteins in serum samples, large numbers of human serum samples have to be analyzed to achieve any statistical significance. Also identified proteins may be related to secondary body defense mechanisms rather than being directly derived from the tumor cells as are most tumor markers applied in the clinic today. To circumvent these problems, we have exploited the xenograft model system as a platform for the discovery of new biomarkers for PCa (11van Weerden W.M. de Ridder C.M. Verdaasdonk C.L. Romijn J.C. van der Kwast T.H. Schroder F.H. van Steenbrugge G.J. Development of seven new human prostate tumor xenograft models and their histopathological characterization.Am. J. Pathol. 1996; 149: 1055-1062PubMed Google Scholar). As has recently been reported, this model system is indeed capable of identifying human proteins that are shed into the circulation by human prostate cancer cells (12van den Bemd G.J. Krijgsveld J. Luider T.M. van Rijswijk A.L. Demmers J.A. Jenster G. Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.Mol. Cell. Proteomics. 2006; 5: 1830-1839Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In the present study we further exploited this approach and performed an in-depth proteomics analysis of serum of mice carrying androgen-sensitive (PC346) or androgen-independent prostate cancer xenografts (PC339). Among the discovered human proteins were numerous cytoplasmic proteins, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenases A and B, and various subunits of the proteolytic proteasome complex (12van den Bemd G.J. Krijgsveld J. Luider T.M. van Rijswijk A.L. Demmers J.A. Jenster G. Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.Mol. Cell. Proteomics. 2006; 5: 1830-1839Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Many of these cytoplasmic proteins are also present in the human plasma proteome as retrieved from the database of the Human Proteome Organisation Plasma Proteome Project (13Omenn G.S. States D.J. Adamski M. Blackwell T.W. Menon R. Hermjakob H. Apweiler R. Haab B.B. Simpson R.J. Eddes J.S. Kapp E.A. Moritz R.L. Chan D.W. Rai A.J. Admon A. Aebersold R. Eng J. Hancock W.S. Hefta S.A. Meyer H. Paik Y.K. Yoo J.S. Ping P. Pounds J. Adkins J. Qian X. Wang R. Wasinger V. Wu C.Y. Zhao X. Zeng R. Archakov A. Tsugita A. Beer I. Pandey A. Pisano M. Andrews P. Tammen H. Speicher D.W. Hanash S.M. Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database.Proteomics. 2005; 5: 3226-3245Crossref PubMed Scopus (691) Google Scholar). We hypothesized that the presence of cytoplasmic tumor-derived proteins in the xenograft sera could be explained by the secretion of exosomes. Exosomes are small membrane vesicles secreted by virtually every cell type, including tumor cells (14Thery C. Zitvogel L. Amigorena S. Exosomes: composition, biogenesis and function.Nat. Rev. Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3834) Google Scholar). Exosomes are formed in multivesicular bodies by inward budding, thereby encapsulating cytoplasmic components (14Thery C. Zitvogel L. Amigorena S. Exosomes: composition, biogenesis and function.Nat. Rev. Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3834) Google Scholar, 15Valenti R. Huber V. Iero M. Filipazzi P. Parmiani G. Rivoltini L. Tumor-released microvesicles as vehicles of immunosuppression.Cancer Res. 2007; 67: 2912-2915Crossref PubMed Scopus (339) Google Scholar). The exact function of exosomes in tumor cells has yet to be elucidated but is expected to relate to roles in cell-to-cell contact, tumor-stroma interaction, protein degradation, and antigen presentation (14Thery C. Zitvogel L. Amigorena S. Exosomes: composition, biogenesis and function.Nat. Rev. Immunol. 2002; 2: 569-579Crossref PubMed Scopus (3834) Google Scholar, 15Valenti R. Huber V. Iero M. Filipazzi P. Parmiani G. Rivoltini L. Tumor-released microvesicles as vehicles of immunosuppression.Cancer Res. 2007; 67: 2912-2915Crossref PubMed Scopus (339) Google Scholar). In addition to containing proteins, it was recently discovered that exosomes also contain functional RNA, proposed as "exosomal shuttle RNA" (16Valadi H. Ekstrom K. Bossios A. Sjostrand M. Lee J.J. Lotvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (9148) Google Scholar). To confirm our hypothesis that the cytoplasmic tumor-derived proteins in the serum of xenograft-bearing mice were the result of exosomal secretion, we isolated exosomes from the PC346C cell line and analyzed their protein content. To further explore the contents of exosomes we isolated and analyzed exosomal RNA from both the PC346C and VCaP cell lines. Human prostate cancer xenografts were grown on immune-incompetent mice athymic male nude (nu/nu) BALB/c mice (n = 9 for each xenograft; Taconic, Ry, Denmark) (11van Weerden W.M. de Ridder C.M. Verdaasdonk C.L. Romijn J.C. van der Kwast T.H. Schroder F.H. van Steenbrugge G.J. Development of seven new human prostate tumor xenograft models and their histopathological characterization.Am. J. Pathol. 1996; 149: 1055-1062PubMed Google Scholar, 12van den Bemd G.J. Krijgsveld J. Luider T.M. van Rijswijk A.L. Demmers J.A. Jenster G. Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.Mol. Cell. Proteomics. 2006; 5: 1830-1839Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We used the human prostate cancer cell lines PC346 (androgen-sensitive) and PC339 (androgen-independent). Specific characteristics have been described previously (17Marques R.B. van Weerden W.M. Erkens-Schulze S. de Ridder C.M. Bangma C.H. Trapman J. Jenster G. The human PC346 xenograft and cell line panel: a model system for prostate cancer progression.Eur. Urol. 2006; 49: 245-257Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Prior control serum was collected by retro-orbital punction. Tumor-bearing mice were sacrificed after 4–5 weeks, and blood was collected. Samples were stored at −80 °C. The protocol was approved by the Animal Experiments Committee under the national Experiments on Animals Act and adhered to the rules laid down in this national law that serves the implementation of "Guidelines on the protection of experimental animals" by the council of Europe under Directive 86/609/EC. After filtration using a 0.22-µm spin filter, high abundance proteins were removed utilizing Multi Affinity Removal Spin cartridges (Agilent Technologies, Wilmington, DE) according to the manufacturer's instructions. Depleted samples were concentrated on 5-kDa-cutoff ultracentrifugation columns (Agilent Technologies). Total protein concentration was determined by the Bradford method (Bio-Rad). Precast 4–20% polyacrylamide linear gradient gels (Bio-Rad) were utilized to separate 10 µg of protein of depleted mouse serum (pooled from nine individual control mice, nine PC339 xenograft-bearing mice, or nine PC346 xenograft-bearing mice) by SDS-PAGE (Mini-Protean III, Bio-Rad). Prestained high range molecular weight markers (SeeBlue, Invitrogen) were loaded on each gel. After running, gels were stained by Coomassie Brilliant Blue (Merck). Gel lanes (range, 5–200 kDa) were excised and divided into 3-mm sections. Gel slices were washed, destained twice (50% (v/v) acetonitrile in 50 mm ammonium bicarbonate), dehydrated (100% acetonitrile), and reduced with 6.5 mm DTT in 50 mm ammonium bicarbonate for 1 h at 37 °C. After alkylation with 54 mm iodoacetamide in 50 mm ammonium bicarbonate, proteins were dehydrated in 100% acetonitrile and then rehydrated with the digestion solution containing 10 ng/µl ultra grade sequencing trypsin (Promega, Madison, WI) for 30 min at room temperature. After addition of 30 µl of 50 mm ammonium bicarbonate solution, gel particles were incubated overnight at 37 °C. The peptides were extracted using 0.5% formic acid in 50% acetonitrile, dried completely in a vacuum centrifuge, and stored at −80 °C until analysis. Nanoflow LC-tandem mass spectrometry was performed for samples by coupling an Agilent 1100 HPLC system (Agilent Technologies), operated as described previously (12van den Bemd G.J. Krijgsveld J. Luider T.M. van Rijswijk A.L. Demmers J.A. Jenster G. Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.Mol. Cell. Proteomics. 2006; 5: 1830-1839Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), to a 7-tesla LTQ-FT mass spectrometer (FT-ICR-MS, Thermo Electron, Bremen, Germany). For protein identification, database searches were performed using Mascot version 2.0 (Matrix Science, London, UK) allowing 5-ppm mass deviation for the precursor ion, a 0.6-Da tolerance on the fragment ions, and trypsin as the digestion enzyme. A maximum number of one missed cleavage was allowed, and carbamidomethylated cysteine and oxidized methionine were set as fixed and optional modifications, respectively. Only peptides with Mascot scores >30 were accepted. Scaffold (version 01_05_06, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 90.0% probability as specified by the Peptide Prophet algorithm (18Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3912) Google Scholar). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (19Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3655) Google Scholar). Before we annotated a certain peptide derived from the xenograft-bearing mice as human, a stringent selection procedure was followed (see Fig. 1). First all peptide mass values identified in the serum from control mice and PC339 or PC346 xenograft-bearing mice were searched against both the Internation Protein Index (IPI) mouse and IPI human databases (version 3.18, containing 53,788 and 60,090 proteins, respectively). Then a selection was made of peptides uniquely present in the serum of PC346 or PC339 xenograft-bearing mice. These peptides were subsequently divided into a group of human-specific peptides (identified only in the IPI human database) and a group of homologous peptides (present in both the IPI human and IPI mouse databases). Homologous peptides were annotated as tumor-derived if four or more times higher abundant in the serum of PC339 or PC346 xenografted mice in comparison with control serum as listed in Scaffold. Additionally to double check human specificity, the identified human-specific peptides were blasted against the Swiss-Prot database of the National Center for Biotechnology Information (NCBI) database. To clean up samples from contaminants, for each xenograft-derived serum sample (50 µg of protein) the 2-D Clean-Up kit (Amersham Biosciences) was utilized according to the manufacturer's instructions. Next samples were solubilized in 125 µl of rehydration buffer (8 m urea, 2% CHAPS, 0.5% IPG buffer, 0.2% DTT, trace of bromphenol blue, all dissolved in H2O). The samples were loaded onto Immobiline dry strip gels (pH 3–10, non-linear, 7 cm; Amersham Biosciences). Isoelectric focusing was carried out as follows: 30 V for 10 h, 300 V for 2 h, 1000 V for 30 min, 5000 V for 90 min, 5000 V for 30 min, and 20 V for 20 h. Before starting the second dimension, strips were reduced and alkylated for 15 min in DTT equilibration buffer (6 m urea, 50 mm Tris, pH 8.8, 20% glycerol, 2% SDS, 1% DTT) and iodoacetamide equilibration buffer (6 m urea, 50 mm Tris, pH 8.8, 20% glycerol, 2% SDS, 2.5% iodoacetamide). Next the IPG strips were placed upon a Criterion XT bis-Tris gel (12%; Bio-Rad). The second dimension was run at 100 V for ±2 h with XT MOPS buffer (Bio-Rad). After running the second dimension, gels were blotted onto Protran nitrocellulose membrane in Tris-glycine-SDS buffer (Bio-Rad). The immunoblot was blocked for 1 h and after washing twice incubated overnight at 4 °C with a monoclonal antibody (1:2000) against proteasome α subunits 6, 2, 4, 5, 1, and 3 (clone MCP231, Biomol International, Exeter, UK). This corresponds with the α subunits 1, 2, 3, 5, 6, and 7 according to the nomenclature of Baumeister et al. (20Baumeister W. Walz J. Zuhl F. Seemuller E. The proteasome: paradigm of a self-compartmentalizing protease.Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar). In addition, monoclonal antibodies specifically directed against the proteasome α1 subunit (PSMA1; α6 according to the Baumeister et al. (20Baumeister W. Walz J. Zuhl F. Seemuller E. The proteasome: paradigm of a self-compartmentalizing protease.Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar) nomenclature) (clone MCP20, Biomol International) or α3 subunits (PSMA3; α7 according to the Baumeister et al. (20Baumeister W. Walz J. Zuhl F. Seemuller E. The proteasome: paradigm of a self-compartmentalizing protease.Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar) nomenclature) (clone MCP72, Biomol International) were utilized. The immunoblot was washed and incubated for 1 h with a 1:1000 solution of a goat anti-mouse horseradish peroxidase-conjugated antibody (DakoCytomation, Glostrup, Denmark). The secondary antibody was visualized with a chemiluminescence detection kit (Roche Applied Science). For reprobing, blots were immersed in a 0.04 m Tris-HCl, 0.06 m Tris base, 0.07 m SDS, 0.10 m β-mercaptoethanol solution for 20 min at 50 °C. The protocol for characterization of the proteasome by native gel electrophoresis was followed as previously described by Elsasser et al. (21Elsasser S. Schmidt M. Finley D. Characterization of the proteasome using native gel electrophoresis.Methods Enzymol. 2005; 398: 353-363Crossref PubMed Scopus (135) Google Scholar). Depleted xenograft and control serum samples were mixed with 5× sample buffer containing 250 mm Tris-HCl, pH 7.4, 50% glycerol, 60 ng/ml xylene cyanol. Samples were either directly loaded or denatured by heating at 96 °C for 5 min. Gels were run for 3–4 h at 4 °C. Gels were transferred onto Protran nitrocellulose membranes at 250 mA for 1.5 h. Serum proteasome concentrations were measured as previously described by Dutaud et al. (22Dutaud D. Aubry L. Henry L. Levieux D. Hendil K.B. Kuehn L. Bureau J.P. Ouali A. Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma.J. Immunol. Methods. 2002; 260: 183-193Crossref PubMed Scopus (75) Google Scholar) with some minor modifications. Briefly serum from control (n = 3) and PC339 (n = 3) or PC346 (n = 3) xenograft-bearing mice (1:20 diluted) was incubated for 1 h on a plate coated with a 1:4500 dilution of a monoclonal antibody against PSMA1 (clone MCP20, Biomol International). After addition of a 1:1500 solution of a rabbit anti-proteasome antibody (directed against β subunits of the proteasome; PW 8155, Biomol International) cells were extensively washed with PBS-Tween 20 buffer. Then a 1:4000 solution of goat anti-rabbit horseradish peroxidase-conjugated antibody (DakoCytomation) was added, and the plate was incubated for 1 h in the dark. To reveal horseradish peroxidase activity, 50 mm phosphate, 25 mm citrate buffer, pH 5.0 was added to the cells. After 15 min, the reaction was stopped with 2.5 m sulfuric acid. Absorbance values were measured at 492 nm. All analyses were performed in triplicate. The human prostate cancer cell line PC346C was cultured in Dulbecco's modified Eagle's medium-Ham's F-12 medium (Cambrex Bio Science, Verviers, Belgium) supplemented with 0.1 nm R1881, 2% FCS (PAN Biotech, Aidenbach, Germany), 1% insulin-transferrin-selenium (Invitrogen), 0.01% BSA (Roche Applied Science), 10 ng/ml epidermal growth factor (Sigma-Aldrich), 100 units/ml penicillin and 100 µg/ml streptomycin antibiotics (Cambrex Bio Science), 100 ng/ml fibronectin (Harbor Bio-Products, Tebu-bio, the Netherlands), 20 µg/ml fetuin (ICN Biomedicals, Zoetermeer, The Netherlands), 50 ng/ml cholera toxin (Sigma-Aldrich), 0.1 mm phosphoethanolamine (Sigma-Aldrich), and 0.6 ng/ml triiodothyronine (Sigma-Aldrich) (23Marques R.B. Erkens-Schulze S. de Ridder C.M. Hermans K.G. Waltering K. Visakorpi T. Trapman J. Romijn J.C. van Weerden W.M. Jenster G. Androgen receptor modifications in prostate cancer cells upon long-term androgen ablation and antiandrogen treatment.Int. J. Cancer. 2005; 117: 221-229Crossref PubMed Scopus (65) Google Scholar). The human PCa cell line VCaP was cultured in RPMI 1640 medium (Cambrex Bio Science) supplemented with 10% dextran-coated charcoal-treated FCS (PAN Biotech) and 100 units/ml penicillin and 100 µg/ml streptomycin antibiotics (Cambrex Bio Science). Exosomes were isolated according to the protocol described previously by Hegmans et al. (24Hegmans J.P. Bard M.P. Hemmes A. Luider T.M. Kleijmeer M.J. Prins J.B. Zitvogel L. Burgers S.A. Hoogsteden H.C. Lambrecht B.N. Proteomic analysis of exosomes secreted by human mesothelioma cells.Am. J. Pathol. 2004; 164: 1807-1815Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Briefly PC346C and VCaP were cultured in their respective medium to 80% confluency. Cultures were washed twice with PBS and incubated for 48 h in a humidified atmosphere of 5% CO2, 95% air with serum-free medium consisting of Dulbecco's modified Eagle's medium-Ham's F-12 or RPMI 1640 medium (Cambrex Bio Science) supplemented with 0.1 nm R1881. After incubation cell culture supernatants were subjected to successive centrifugations of 400 × g (10 min), 3000 × g (20 min), and 10,000 × g (30 min). Exosomes were then pelleted at 64,000 × g for 110 min using an SW28 rotor (Beckman Coulter Instruments, Fullerton, CA). Exosome pellets were resuspended in 0.32 m sucrose and centrifuged at 100,000 × g for 1 h (SW60 rotor, Beckman Coulter Instruments). For several experiments, the isolated exosomes from PC346C were further purified by immobilization onto magnetic beads. In short, 25 µl of Dynabeads, precoated with goat anti-mouse immune globulin G (Invitrogen Dynal AS, Oslo, Norway) were incubated for 1 h with 30 µl of an anti-CD9 monoclonal antibody (clone MM2/57, Chemicon International, London, UK). Thereafter beads were incubated by rotation top end over with 20 µg of exosomes for 1 h at 4 °C. After washing four times, beads and exosomes were resuspended in PBS for further experiments. Exosomes from PC346C obtained after ultracentrifugation of cell culture supernatants were resuspended in 10 µl of Milli-Q and spotted onto Formvar-coated grids (200 mesh). Adsorbed exosomes were fixed in 2% paraformaldehyde for 5 min at room temperature. After fixation the exosomes were either directly negatively stained using uranyl acetate or immunolabeled with antibodies against CD9 (clone MM2/57, Chemicon International). Antigen-antibody complexes were visualized with protein A conjugated with 10-nm colloidal gold particles (1:20 dilution; Aurion, Wageningen, The Netherlands) followed by negative staining (see above). The specificity of the labeling procedure was tested by omitting the primary antibody. Grids were examined by a Philips CM100 electron microscope at 80 kV. After resuspending the exosome pellet in PBS, 10 µg of isolated exosomes and 10 µg of supernatant fraction were applied onto two 10% SDS-polyacrylamide gels. After running, one of the gels was silver-stained as described previously by Mortz et al. (25Mortz E. Krogh T.N. Vorum H. Gorg A. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis.Proteomics. 2001; 1: 1359-1363Crossref PubMed Scopus (462) Google Scholar). This gel was used to identify distinct bands present in the exosome fraction (see Fig. 4b). Subsequently these bands were excised from a Coomassie Brilliant Blue (Merck)-stained gel and cut in 3-mm sections. Preparation for mass spectrometry was performed using the protocol described under "Preparation of Xenograft Sera for Mass Spectrometry". Peptide separation was performed on a nanoscale liquid chromatography system (nanoLC Ultimate 3000) (Dionex, Sunnyvale, CA) with a 50-min gradient (5–40% acetonitrile, H2O, 0.1% formic acid). The injection volume was 5 µl of the tryptically digested sample. Peptides were separated on a C18 PepMap column (150 mm × 75 µm inner diameter) (Dionex) at 200 nl/min after preconcentration on a trap column (1 mm × 300 µm inner diameter). Separated peptides were detected by a linear ion trap Orbitrap (LTQ-Orbitrap) mass spectrometer (Finnigan LTQ Orbitrap XL, Thermo Electron). Samples were measured in a data-dependent acquisition mode. In the measurement method used, the peptide masses are measured in a survey scan at a maximum resolution of 60,000. To obtain a maximum mass accuracy a prescan is used to keep the ion population in the Orbitrap for each scan approximately the same. During the high resolution scan in the Orbitrap the five most intense monoisotopic peaks in the spectra were fragmented and measured in the LTQ. The fragment ion masses were measured in the LTQ to have a maximum sensitivity and a maximum amount of MS/MS data. For a full analysis of the exosomal proteome, 10 µg of the isolated exosome fraction was applied onto a 10% SDS-polyacrylamide gel and run for ∼1.5 cm inside the running gel. Thereafter this gel section was excised and divided into 3-mm sections, washed, destained (100% acetonitrile followed b
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