Title: Proteomics Analysis of Bladder Cancer Exosomes
Abstract: Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. Bladder cancer is one of the eight most frequent cancers in the Western world, and the frequency of transitional cell carcinoma (TCC), 1The abbreviations used are:TCCtransitional cell carcinoma5T4trophoblast glycoproteinBCAbicinchoninic acidFDRfalse discovery rateHLAhuman leukocyte antigenhsp90heat shock protein 90LAMPlysosome-associated membrane proteinMHCmajor histocompatability complexTEABtriethylammonium bicarbonateTSG101tumor susceptibility gene 101NHSNational Health ServiceFBSfetal bovine serumRTroom temperatureBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolhnRNPheterogeneous nuclear ribonucleoprotein2DEtwo-dimensional electrophoresisIDidentifierORAoverrepresentation analysis.1The abbreviations used are:TCCtransitional cell carcinoma5T4trophoblast glycoproteinBCAbicinchoninic acidFDRfalse discovery rateHLAhuman leukocyte antigenhsp90heat shock protein 90LAMPlysosome-associated membrane proteinMHCmajor histocompatability complexTEABtriethylammonium bicarbonateTSG101tumor susceptibility gene 101NHSNational Health ServiceFBSfetal bovine serumRTroom temperatureBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolhnRNPheterogeneous nuclear ribonucleoprotein2DEtwo-dimensional electrophoresisIDidentifierORAoverrepresentation analysis. which accounts for 90% of bladder cancers, is second only to prostate cancer as a malignancy of the genitourinary tract. Urine cytology and cystoscopy remain the predominant clinical tools for diagnosing and monitoring the disease, but cytology is poorly sensitive, particularly for low grade tumors, and does not serve as a prognostic tool. Cystoscopy is an invasive procedure, and there is pressing need to identify informative molecular markers that can be used to replace it. transitional cell carcinoma trophoblast glycoprotein bicinchoninic acid false discovery rate human leukocyte antigen heat shock protein 90 lysosome-associated membrane protein major histocompatability complex triethylammonium bicarbonate tumor susceptibility gene 101 National Health Service fetal bovine serum room temperature 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol heterogeneous nuclear ribonucleoprotein two-dimensional electrophoresis identifier overrepresentation analysis. transitional cell carcinoma trophoblast glycoprotein bicinchoninic acid false discovery rate human leukocyte antigen heat shock protein 90 lysosome-associated membrane protein major histocompatability complex triethylammonium bicarbonate tumor susceptibility gene 101 National Health Service fetal bovine serum room temperature 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol heterogeneous nuclear ribonucleoprotein two-dimensional electrophoresis identifier overrepresentation analysis. Recently, small cell-derived vesicles termed exosomes that are present in body fluids (1Caby M.P. Lankar D. Vincendeau-Scherrer C. Raposo G. Bonnerot C. Exosomal-like vesicles are present in human blood plasma.Int. Immunol. 2005; 17: 879-887Crossref PubMed Scopus (925) Google Scholar, 2Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13368-13373Crossref PubMed Scopus (1559) Google Scholar, 3Admyre C. Johansson S.M. Qazi K.R. Filén J.J. Lahesmaa R. Norman M. Neve E.P. Scheynius A. Gabrielsson S. Exosomes with immune modulatory features are present in human breast milk.J. Immunol. 2007; 179: 1969-1978Crossref PubMed Scopus (762) Google Scholar, 4Gatti J.L. Métayer S. Belghazi M. Dacheux F. Dacheux J.L. Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles.Biol. Reprod. 2005; 72: 1452-1465Crossref PubMed Scopus (144) Google Scholar, 5Bard M.P. Hegmans J.P. 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Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.Kidney Int. 2006; 70: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 8Nilsson J. Skog J. Nordstrand A. Baranov V. Mincheva-Nilsson L. Breakefield X.O. Widmark A. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer.Br. J. Cancer. 2009; 100: 1603-1607Crossref PubMed Scopus (558) Google Scholar). These nanometer-sized vesicles, which are secreted by most cell types, originate from multivesicular bodies of the endocytic tract and reflect a subproteome of the cell. Exosomes are enriched in membrane and cytosolic proteins, and this molecular repertoire appears to be of particular functional importance to the immune system (9Théry C. Ostrowski M. Segura E. Membrane vesicles as conveyors of immune responses.Nat. Rev. Immunol. 2009; 9: 581-593Crossref PubMed Scopus (2817) Google Scholar). Exosomes also comprise an array of lipids, mRNA, and microRNA, which are likely involved in conveying intercellular communication processes (10Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall 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 (8399) Google Scholar). Importantly, many exosomal components are simply not present as free soluble molecules in body fluids, such as certain microRNA species, which are encapsulated within the exosome lumen (6Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol. Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1856) Google Scholar, 10Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall 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 (8399) Google Scholar). Therefore, the ability to isolate exosomes from urine (2Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13368-13373Crossref PubMed Scopus (1559) Google Scholar), plasma (1Caby M.P. Lankar D. Vincendeau-Scherrer C. Raposo G. Bonnerot C. Exosomal-like vesicles are present in human blood plasma.Int. Immunol. 2005; 17: 879-887Crossref PubMed Scopus (925) Google Scholar), saliva (11Ogawa Y. Kanai-Azuma M. Akimoto Y. Kawakami H. Yanoshita R. Exosome-like vesicles with dipeptidyl peptidase IV in human saliva.Biol. Pharm. Bull. 2008; 31: 1059-1062Crossref PubMed Scopus (180) Google Scholar), or other physiological sources (3Admyre C. Johansson S.M. Qazi K.R. Filén J.J. Lahesmaa R. Norman M. Neve E.P. Scheynius A. Gabrielsson S. Exosomes with immune modulatory features are present in human breast milk.J. Immunol. 2007; 179: 1969-1978Crossref PubMed Scopus (762) Google Scholar) holds significant potential for obtaining novel and complex sets of biomarkers in a non-invasive manner. Exosome analysis may therefore be of value in disease diagnosis and monitoring in a variety of settings (6Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol. Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1856) Google Scholar, 7Zhou H. Pisitkun T. Aponte A. Yuen P.S. Hoffert J.D. Yasuda H. Hu X. Chawla L. Shen R.F. Knepper M.A. Star R.A. Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.Kidney Int. 2006; 70: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 12Rabinowits G. Gerçel-Taylor C. Day J.M. Taylor D.D. Kloecker G.H. Exosomal microRNA: a diagnostic marker for lung cancer.Clin. Lung Cancer. 2009; 10: 42-46Abstract Full Text PDF PubMed Scopus (1019) Google Scholar, 13Taylor D.D. Akyol S. Gercel-Taylor C. Pregnancy-associated exosomes and their modulation of T cell signaling.J. Immunol. 2006; 176: 1534-1542Crossref PubMed Scopus (242) Google Scholar, 14Skriner K. Adolph K. Jungblut P.R. Burmester G.R. Association of citrullinated proteins with synovial exosomes.Arthritis Rheum. 2006; 54: 3809-3814Crossref PubMed Scopus (189) Google Scholar). Exosomes as indicators of pathology were first documented in the context of renal injury where a differential proteomics approach revealed changes in urinary exosome phenotype following renal injury (7Zhou H. Pisitkun T. Aponte A. Yuen P.S. Hoffert J.D. Yasuda H. Hu X. Chawla L. Shen R.F. Knepper M.A. Star R.A. Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.Kidney Int. 2006; 70: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). The researchers identified exosomally expressed Fetuin-A as a marker that became elevated 50-fold within hours following nephrotoxin exposure in rodents. Exosomal Fetuin-A elevation was also apparent in patients with acute renal injury before changes in urinary creatinine were observed (7Zhou H. Pisitkun T. Aponte A. Yuen P.S. Hoffert J.D. Yasuda H. Hu X. Chawla L. Shen R.F. Knepper M.A. Star R.A. Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.Kidney Int. 2006; 70: 1847-1857Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Clinical exosome analysis may also prove useful for solid cancers, such as ovarian or lung cancer, where the quantity of epithelial cell adhesion molecule-positive serum exosomes may correlate with tumor stage/grade. Such disease-associated exosomes express microRNA species not detected in healthy subjects (6Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol. Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1856) Google Scholar, 12Rabinowits G. Gerçel-Taylor C. Day J.M. Taylor D.D. Kloecker G.H. Exosomal microRNA: a diagnostic marker for lung cancer.Clin. Lung Cancer. 2009; 10: 42-46Abstract Full Text PDF PubMed Scopus (1019) Google Scholar), although in this respect, there is little correlation between microRNA and disease bulk (6Taylor D.D. Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol. Oncol. 2008; 110: 13-21Abstract Full Text Full Text PDF PubMed Scopus (1856) Google Scholar, 12Rabinowits G. Gerçel-Taylor C. Day J.M. Taylor D.D. Kloecker G.H. Exosomal microRNA: a diagnostic marker for lung cancer.Clin. Lung Cancer. 2009; 10: 42-46Abstract Full Text PDF PubMed Scopus (1019) Google Scholar). Other recent examples include studies of urinary exosomes in prostate cancer with exosomes expressing protein markers 5T4 (15Mitchell P.J. Welton J. Staffurth J. Court J. Mason M.D. Tabi Z. Clayton A. Can urinary exosomes act as treatment response markers in prostate cancer?.J. Transl. Med. 2009; 7: 4Crossref PubMed Scopus (227) Google Scholar), prostate cancer gene 3 (PCA-3) (8Nilsson J. Skog J. Nordstrand A. Baranov V. Mincheva-Nilsson L. Breakefield X.O. Widmark A. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer.Br. J. Cancer. 2009; 100: 1603-1607Crossref PubMed Scopus (558) Google Scholar), or mRNA (TMPRSS2-ERG) (8Nilsson J. Skog J. Nordstrand A. Baranov V. Mincheva-Nilsson L. Breakefield X.O. Widmark A. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer.Br. J. Cancer. 2009; 100: 1603-1607Crossref PubMed Scopus (558) Google Scholar, 16Jansen F.H. Krijgsveld J. van Rijswijk A. van den Bemd G.J. van den Berg M.S. van Weerden W.M. Willemsen R. Dekker L.J. Luider T.M. Jenster G. Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins.Mol. Cell. Proteomics. 2009; 8: 1192-1205Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) associated with prostate cancer. To our knowledge, exosomes have not yet been studied in the context of other urological malignancies such as renal cancer, and to date, only one report describes the urine-derived microparticles from bladder cancer patients (17Smalley D.M. Sheman N.E. Nelson K. Theodorescu D. Isolation and identification of potential urinary microparticle biomarkers of bladder cancer.J. Proteome Res. 2008; 7: 2088-2096Crossref PubMed Scopus (148) Google Scholar). In that report, they examined the proteome of a highly complex mixture of microvesicles, exosomes, and other urinary constituents that can be pelleted by high speed ultracentrifugation, identifying eight proteins that may be elevated in cancer. However, given the nature of the sample analyzed, it is unknown whether these proteins are exosomally expressed. Identification of the principal and most relevant molecular markers in these and other clinical scenarios remains a major challenge. In part, this is because exosomes present within complex body fluids originate from heterogeneous cell types. For example, plasma exosomes may be derived from platelets, lymphocytes, or endothelial cells (1Caby M.P. Lankar D. Vincendeau-Scherrer C. Raposo G. Bonnerot C. Exosomal-like vesicles are present in human blood plasma.Int. Immunol. 2005; 17: 879-887Crossref PubMed Scopus (925) Google Scholar), and a proportion may arise from well perfused organs such as the liver (18Conde-Vancells J. Rodriguez-Suarez E. Embade N. Gil D. Matthiesen R. Valle M. Elortza F. Lu S.C. Mato J.M. Falcon-Perez J.M. Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes.J. Proteome Res. 2008; 7: 5157-5166Crossref PubMed Scopus (427) Google Scholar) and likely other organs as well (16Jansen F.H. Krijgsveld J. van Rijswijk A. van den Bemd G.J. van den Berg M.S. van Weerden W.M. Willemsen R. Dekker L.J. Luider T.M. Jenster G. Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins.Mol. Cell. Proteomics. 2009; 8: 1192-1205Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Similarly, exosomes present in urine arise from urothelial cells of the kidney and downstream of the renal tract (2Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13368-13373Crossref PubMed Scopus (1559) Google Scholar, 8Nilsson J. Skog J. Nordstrand A. Baranov V. Mincheva-Nilsson L. Breakefield X.O. Widmark A. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer.Br. J. Cancer. 2009; 100: 1603-1607Crossref PubMed Scopus (558) Google Scholar, 15Mitchell P.J. Welton J. Staffurth J. Court J. Mason M.D. Tabi Z. Clayton A. Can urinary exosomes act as treatment response markers in prostate cancer?.J. Transl. Med. 2009; 7: 4Crossref PubMed Scopus (227) Google Scholar). Importantly, all proteomics studies of exosomes isolated from body fluids are unavoidably complicated by the presence of high abundance non-exosomal proteins contaminating the preparations. Examples include albumin, immunoglobulin, and complement components present in exosomes prepared from malignant effusions (5Bard M.P. Hegmans J.P. Hemmes A. Luider T.M. Willemsen R. Severijnen L.A. van Meerbeeck J.P. Burgers S.A. Hoogsteden H.C. Lambrecht B.N. Proteomic analysis of exosomes isolated from human malignant pleural effusions.Am. J. Respir. Cell Mol. Biol. 2004; 31: 114-121Crossref PubMed Scopus (315) Google Scholar) and Tamm-Horsfall protein present in exosomes purified from urine (2Pisitkun T. Shen R.F. Knepper M.A. Identification and proteomic profiling of exosomes in human urine.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13368-13373Crossref PubMed Scopus (1559) Google Scholar). As such, great care must be taken in the interpretation of the large data sets produced by proteomics studies, requiring careful validation of the proteins of interest. The protein composition of exosomes using a single homogenous cell type is one approach that may be used to uncover the protein components of exosomes produced by various cell types. There remain two major issues in the realm of exosome proteomics that complicate our interpretation of lists of identified proteins. Foremost are the diverse methods chosen for exosome purification that in some studies have involved attempts to remove contaminants through a key biophysical property of the vesicles, i.e. their capacity to float on sucrose (19Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze H.J. B Lymphocytes secrete antigen-presenting vesicles.J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2323) Google Scholar, 20Lamparski H.G. Metha-Damani A. Yao J.Y. Patel S. Hsu D.H. Ruegg C. Le Pecq J.B. Production and characterization of clinical grade exosomes derived from dendritic cells.J. Immunol. Methods. 2002; 270: 211-226Crossref PubMed Scopus (503) Google Scholar) or other dense media (21Graner M.W. Alzate O. Dechkovskaia A.M. Keene J.D. Sampson J.H. Mitchell D.A. Bigner D.D. Proteomic and immunologic analyses of brain tumor exosomes.FASEB J. 2009; 23: 1541-1557Crossref PubMed Scopus (309) Google Scholar). Not all published studies, however, have taken such steps, preferring a far simpler pellet (or pellet and wash) approach. These latter preparations may be significantly contaminated by components of the cellular secretome, cell fragments, and other components. All of these factors could lead to false positive identifications of exosome proteins. The second key issue centers on the MS approaches utilized in various exosome proteomics studies. Many early examples relied only on a peptide mass fingerprinting approach, lacking robust peptide sequence data (22Théry C. Boussac M. Véron P. Ricciardi-Castagnoli P. Raposo G. Garin J. Amigorena S. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.J. Immunol. 2001; 166: 7309-7318Crossref PubMed Scopus (1157) Google Scholar, 23Wubbolts R. Leckie R.S. Veenhuizen P.T. Schwarzmann G. Möbius W. Hoernschemeyer J. Slot J.W. Geuze H.J. Stoorvogel W. Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation.J. Biol. Chem. 2003; 278: 10963-10972Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar), and more recently, search criteria that are generally recommended for MS-derived sequence data have not been specified in all studies. In this study, we have listed only those proteins identified by good quality MS/MS data for two or more peptides. Variability in the robustness and bias in bioinformatics analysis of data sets and in the steps taken to validate identified proteins is an additional factor that impacts the confidence in the identification lists produced. In this study, we aimed to perform the first proteomics analysis of human bladder cancer exosomes. We took extensive steps to produce high purity and quality-assured exosome preparations prior to beginning proteomics workflows. Solubilizing the sample with SDS and a reducing agent (DTT) was a critical step that allowed for global protein identification using nanoscale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. In this study, we present the identification of a significant number of exosomally expressed proteins (353 in total) of unrivaled quality. Critical manual examination of these identifications revealed issues with multiple (physiologically impossible) MHC Class I identifications that were attributed to a misdesignation of nomenclature by MASCOT due to peptide (and target protein) homology. The data were subjected to unbiased overrepresentation analysis (examining ExoCarta and Gene Ontology databases) to reveal a proteome consistent with exosomes, particularly of carcinoma origin. Validation of several identified proteins, by combining ultracentrifugation on a linear sucrose gradient with Western blotting and/or analysis of exosome-coated latex beads, demonstrated correct surface orientation of several MS-identified membrane proteins at densities consistent with exosomes. The robust approaches taken emphasize our confidence in the validity of the identifications generated and highlight that 72 (of 353) proteins have not been previously shown to be exosomally expressed by other human proteomics studies. The data will be useful for future studies in this underinvestigated disease and will form a platform not only for future clinical validation of some of these putative markers but also to aid further investigations into novel aspects of exosome function and manufacture. HT1376 is a cell line originating from a primary TCC of the bladder (Stage T2, Grade G4) (24Rasheed S. Gardner M.B. Rongey R.W. Nelson-Rees W.A. Arnstein P. Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses.J. Natl. Cancer Inst. 1977; 58: 881-890Crossref PubMed Scopus (116) Google Scholar). In vitro cultured HT1376 cells were used as the exosome source for this study because they have been extensively characterized previously and are representative of the behavior and phenotype of TCC (24Rasheed S. Gardner M.B. Rongey R.W. Nelson-Rees W.A. Arnstein P. Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses.J. Natl. Cancer Inst. 1977; 58: 881-890Crossref PubMed Scopus (116) Google Scholar, 25Masters J.R. Hepburn P.J. Walker L. Highman W.J. Trejdosiewicz L.K. Povey S. Parkar M. Hill B.T. Riddle P.R. Franks L.M. Tissue culture model of transitional cell carcinoma: characterization of twenty-two human urothelial cell lines.Cancer Res. 1986; 46: 3630-3636PubMed Google Scholar). The cells were maintained in Dulbecco's modified Eagle's medium (Lonza) supplemented with penicillin/streptomycin and 5% FBS (which had been depleted of exosomes by overnight ultracentrifugation at 100,000 × g followed by filtration through 0.2-µm and then 0.1-µm vacuum filters (Millipore)). The cells were seeded into bioreactor flasks (from Integra) and maintained at high density culture for exosome production as described (26Mitchell J.P. Court J. Mason M.D. Tabi Z. Clayton A. Increased exosome production from tumour cell cultures using the Integra CELLine Culture System.J. Immunol. Methods. 2008; 335: 98-105Crossref PubMed Scopus (89) Google Scholar). Cells were confirmed negative for mycoplasma contamination by monthly screening (Mycoalert, Lonza). Additional well characterized bladder cancer cell lines (HT1197, RT4, RT112, and T24) (25Masters J.R. Hepburn P.J. Walker L. Highman W.J. Trejdosiewicz L.K. Povey S. Parkar M. Hill B.T. Riddle P.R. Franks L.M. Tissue culture model of transitional cell carcinoma: characterization of twenty-two human urothelial cell lines.Cancer Res. 1986; 46: 3630-3636PubMed Google Scholar, 27Southgate J. Harnden P. Trejdosiewicz L.K. Cytokeratin expression patterns in normal and malignant urothelium: a review of the biological and diagnostic implications.Histol. Histopathol. 1999; 14: 657-664PubMed Google Scholar) were obtained from ATCC or from Cancer Research UK cell bank and cultured similarly. The culture medium of HT1376 cells (typically 15–30 ml) was subjected to serial centrifugation to remove cells (400 × g for 10 min) and cellular debris (2000 × g for 15 min). The supernatant was then centrifuged at 10,000 × g for 30 min, and the supernatant was further purified by underlaying with a 30% sucrose, D2O cushion and subjected to ultracentrifugation at 100,000 × g for 2 h. The cushion was collected, and exosomes were washed in PBS as described previously (20Lamparski H.G. Metha-Damani A. Yao J.Y. Patel S. Hsu D.H. Ruegg C. Le Pecq J.B. Production and characterization of clinical grade exosomes derived from dendritic cells.J. Immunol. Methods. 2002; 270: 211-226Crossref PubMed Scopus (503) Google Scholar, 28Andre F. Schartz N.E. Movassagh M. Flament C. Pautier P. Morice P. Pomel C. Lhomme C. Escudier B. Le Chevalier T. Tursz T. Amigorena S. Raposo G. Angevin E. Zitvogel L. Malignant effusions and immunogenic tumour-derived exosomes.Lancet. 2002; 360: 295-305Abstract Full Text Full Text PDF PubMed Scopus (737) Google Scholar, 29Clayton A. Mitchell J.P. Court J. Mason M.D. Tabi Z. Human tumour-derived exosomes selectively impair lymphocyte responses to interleukin-2.Cancer Res. 2007; 67: 7458-7466Crossref PubMed Scopus (408) Google Scholar). Exosome pellets were resuspended in 100–150 µl of PBS and frozen at −80 °C. The quantity of exosomes was determined by the micro-BCA protein assay (Pierce/Thermo Scientific), and this gave an average of 12 µg (±2.2 S.E., n = 5) exosomes/ml of culture medium for the HT1376 c