Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer. Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer. Although a rare cancer worldwide (0.5 per 100,000 in the USA), intrahepatic cholangiocarcinoma (CCA) 1The abbreviations used are: CCA, cholangiocarcinoma;APF, advanced periductal fibrosis;DAB, diaminobenzidine;DPBS, Dulbecco's PBS;IARC, International Agency for Research on Cancer;IDA, information dependent acquisition;IHC, immunohistochemistry;iTRAQ, isobaric tags for relative and absolute quantitation;OGE, OFFGEL electrophoresis;Ov, Opisthorchis viverrini;PBS, phosphate buffered saline;PBST, phosphate buffered saline tween-20;PVDF, polyvinylidene difluoride;NDMA, N-nitrosodimethylamine;MMTS, methyl methanethiosulfonate;TMA, tissue microarray;TPP, Trans Proteomic Pipeline;TEAB, triethylammonium bicarbonate. 1The abbreviations used are: CCA, cholangiocarcinoma;APF, advanced periductal fibrosis;DAB, diaminobenzidine;DPBS, Dulbecco's PBS;IARC, International Agency for Research on Cancer;IDA, information dependent acquisition;IHC, immunohistochemistry;iTRAQ, isobaric tags for relative and absolute quantitation;OGE, OFFGEL electrophoresis;Ov, Opisthorchis viverrini;PBS, phosphate buffered saline;PBST, phosphate buffered saline tween-20;PVDF, polyvinylidene difluoride;NDMA, N-nitrosodimethylamine;MMTS, methyl methanethiosulfonate;TMA, tissue microarray;TPP, Trans Proteomic Pipeline;TEAB, triethylammonium bicarbonate. has the highest incidence in the world (96 per 100,000 in Northeastern Thailand (1.Sripa B. Kaewkes S. Sithithaworn P. Mairiang E. Laha T. Smout M. Pairojkul C. Bhudhisawasdi V. Tesana S. Thinkamrop B. Bethony J.M. Loukas A. Brindley P.J. Liver fluke induces cholangiocarcinoma.PLoS Med. 2007; 4: e201Crossref PubMed Scopus (556) Google Scholar)) in areas of the Greater Mekong subregion of Southeast Asia that overlap with transmission of the food-borne parasite Opisthorchis viverrini (Ov). As experimental and epidemiological evidence strongly implicate infection with this food borne pathogen in the development of CCA, Ov is one of only three eukaryotic pathogens considered Group 1 carcinogens by the International Agency for Research on Cancer (IARC) (2.IARC Schistosomes, liver flukes and Helicobacter pylori. IARC monographs on the evaluation of carcinogenic risks to humans.in: IARC monographs on the evaluation of carcinogenic risks to humans. 61. IARC, Lyon1994: 218-221Google Scholar). Ov infection occurs during the consumption of undercooked fish containing the encysted metacercarial stage of the parasite (3.Sripa B. Bethony J.M. Sithithaworn P. Kaewkes S. Mairiang E. Loukas A. Mulvenna J. Laha T. Hotez P.J. Brindley P.J. Opisthorchiasis and Opisthorchis-associated cholangiocarcinoma in Thailand and Laos.Acta Trop. 2011; 120: S158-S168Crossref PubMed Scopus (230) Google Scholar). After ingestion of metacercaria, the parasites excyst and migrate to the intrahepatic bile duct to mature and remain patent for decades, causing prolonged mechanical, toxicological and immunopathological damage to the biliary epithelium of the host. The pathological consequences of chronic Ov infection occur primarily in the intrahepatic bile ducts (4.Sripa B. Brindley P.J. Mulvenna J. Laha T. Smout M.J. Mairiang E. Bethony J.M. Loukas A. The tumorigenic liver fluke Opisthorchis viverrini–multiple pathways to cancer.Trends Parasitol. 2012; 28: 395-407Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), where CCA also arises. The location of Ov-associated CCA makes early diagnosis difficult (5.Malhi H. Gores G.J. Cholangiocarcinoma: modern advances in understanding a deadly old disease.J. Hepatol. 2006; 45: 856-867Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), with individuals presenting late and usually because of nonspecific symptoms. Thus, despite being a slow growing tumor, Ov-associated CCA is commonly diagnosed at an advanced stage, when the primary cancer is no longer amenable to surgical extirpation and has metastasized to other organs (4.Sripa B. Brindley P.J. Mulvenna J. Laha T. Smout M.J. Mairiang E. Bethony J.M. Loukas A. The tumorigenic liver fluke Opisthorchis viverrini–multiple pathways to cancer.Trends Parasitol. 2012; 28: 395-407Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The median survival rate for individuals after diagnosis is less than 24 months, highlighting the urgent need for diagnostic markers for Ov-associated CCA. Although the etiology of Ov-associated CCA is multi-factorial, it is clear that the chronic inflammation and prolonged immunopathology associated with chronic Ov infection are key underlying processes in the transition to CCA (6.Haswell-Elkins M.R. Sithithaworn P. Mairiang E. Elkins D.B. Wongratanacheewin S. Kaewkes S. Mairiang P. Immune responsiveness and parasite-specific antibody levels in human hepatobiliary disease associated with Opisthorchis viverrini infection.Clin. Exp. Immunol. 1991; 84: 213-218Crossref PubMed Scopus (72) Google Scholar). As determined from our human studies in Ov endemic areas (4.Sripa B. Brindley P.J. Mulvenna J. Laha T. Smout M.J. Mairiang E. Bethony J.M. Loukas A. The tumorigenic liver fluke Opisthorchis viverrini–multiple pathways to cancer.Trends Parasitol. 2012; 28: 395-407Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), Ov-associated CCA is the culmination of a series of clearly defined clinical and subclinical events caused by the persistent production of the growth factors and cytokines associated with chronic inflammation, would healing, and fibrogenesis (4.Sripa B. Brindley P.J. Mulvenna J. Laha T. Smout M.J. Mairiang E. Bethony J.M. Loukas A. The tumorigenic liver fluke Opisthorchis viverrini–multiple pathways to cancer.Trends Parasitol. 2012; 28: 395-407Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 7.Pinlaor S. Yongvanit P. Prakobwong S. Kaewsamut B. Khoontawad J. Pinlaor P. Hiraku Y. Curcumin reduces oxidative and nitrative DNA damage through balancing of oxidant-antioxidant status in hamsters infected with Opisthorchis viverrini.Mol. Nutr. Food Res. 2009; 53: 1316-1328Crossref PubMed Scopus (55) Google Scholar, 8.Prakobwong S. Yongvanit P. Hiraku Y. Pairojkul C. Sithithaworn P. Pinlaor P. Pinlaor S. Involvement of MMP-9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1-dependent DNA damage in a hamster model.Int. J. Cancer. 2010; 127: 2576-2587Crossref PubMed Scopus (73) Google Scholar). The continuous accumulation of desmoplastic (fibrotic) elements along the intrahepatic biliary tract leads to advanced periductal fibrosis (APF) and then, in some cases, to CCA (4.Sripa B. Brindley P.J. Mulvenna J. Laha T. Smout M.J. Mairiang E. Bethony J.M. Loukas A. The tumorigenic liver fluke Opisthorchis viverrini–multiple pathways to cancer.Trends Parasitol. 2012; 28: 395-407Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). In this regard, Ov-associated CCA is not greatly different from other infection related cancers that induce a chronic inflammation that culminates in cancer: e.g. hepatocellular fibrosis and hepatocellular carcinoma from Hepatitis B and Hepatitis C viral infection (9.Sheikh M.Y. Choi J. Qadri I. Friedman J.E. Sanyal A.J. Hepatitis C virus infection: molecular pathways to metabolic syndrome.Hepatology. 2008; 47: 2127-2133Crossref PubMed Scopus (212) Google Scholar, 10.Castello G. Scala S. Palmieri G. Curley S.A. Izzo F. HCV-related hepatocellular carcinoma: From chronic inflammation to cancer.Clin. Immunol. 2010; 134: 237-250Crossref PubMed Scopus (118) Google Scholar). The progression from Ov infection to CCA can be observed in a robust Syrian hamster model that uses sub-carcinogenic levels of dietary N-nitrosodimethylamine (NDMA) to accelerate the onset of CCA. Hamsters that are infected with Ov and fed a diet supplemented with NDMA progress to CCA whereas those that are infected with Ov but do not receive NDMA do not progress to CCA during a six month animal trial (11.Thamavit W. Bhamarapravati N. Sahaphong S. Vajrasthira S. Angsubhakorn S. Effects of dimethylnitrosamine on induction of cholangiocarcinoma in Opisthorchis viverriniinfected Syrian golden hamsters.Cancer Res. 1978; 38: 4634-4639PubMed Google Scholar). In this animal model of CCA, the biliary epithelium of the hamster is markedly inflamed and displays fibrosis advancing along its length after 12 weeks of infection (12.Bhamarapravati N. Thammavit W. Vajrasthira S. Liver changes in hamsters infected with a liver fluke of man, Opisthorchis viverrini.Am. J. Trop. Med. Hyg. 1978; 27: 787-794Crossref PubMed Scopus (125) Google Scholar), with this is fibrotic deposition routinely present at the biliary epithelium that leads to CCA (8.Prakobwong S. Yongvanit P. Hiraku Y. Pairojkul C. Sithithaworn P. Pinlaor P. Pinlaor S. Involvement of MMP-9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1-dependent DNA damage in a hamster model.Int. J. Cancer. 2010; 127: 2576-2587Crossref PubMed Scopus (73) Google Scholar, 13.Chamadol N. Pairojkul C. Khuntikeo N. Laopaiboon V. Loilome W. Sithithaworn P. Yongvanit P. Histological confirmation of periductal fibrosis from ultrasound diagnosis in cholangiocarcinoma patients.J. Hepatobiliary Pancreat. Sci. 2014; 21: 316-322Crossref PubMed Scopus (48) Google Scholar). In the current study, isobaric labeling (8-plex iTRAQ) and tandem mass spectrometry (MS/MS) were used to identify differentially expressed protein markers of CCA using animal and human models of CCA. In the hamster model of Ov-induced CCA, protein expression levels in the livers from Ov-infected hamsters that had progressed to CCA were compared with normal uninfected hamsters as well as to hamsters that were "Ov-infected" but did not receive NDMA and were thus CCA-free (Fig. 1). Specifically, we attempted to identify protein markers of CCA that were distinct from those associated with the strong inflammation caused by Ov infection; that is, differentially expressed proteins that were either (1) associated with Ov-induced CCA but not Ov-associated inflammation when compared with normal controls, or (2) proteins associated with both inflammation and CCA but which exhibited significantly different expression in Ov-induced CCA when compared with Ov-associated inflammation. To verify these observations in human Ov-associated CCA, immunoblotting experiments and immunohistochemical analysis of CCA tissue microarrays (TMAs) were also assayed for the expression levels for these dysregulated proteins. The results of this study provide protein "signatures" of both Ov-associated inflammation and Ov-associated CCA that can be further exploited for clinical application as well as insight into the relationship between Ov-associated chronic inflammation and Ov-associated CCA. Quantitative 8-plex iTRAQ experiments were conducted on whole liver protein preparations from three groups of hamsters: "Normal," "Ov-induced CCA," and "Ov-infected" groups using three biological replicates from each group. Four 8-plex iTRAQ channels were used in each experiment which included two labeled control samples as an internal control; two additional channels were used for a separate study on the effects of curcumin on CCA and Ov infection (see supplemental Table S1 for details of iTRAQ labeling). For verification in hamster liver tissue Western blotting experiments and immunohistochemical analysis were performed using four biological replicates from each of the three experimental groups. All human CCA tissue was obtained with informed consent from patients at Srinagarind Hospital, Khon Kaen University, Thailand. For Western blotting experiments in human CCA tissue, seven biological replicates were used and in each replicate adjacent nontumor tissue was used as a control. Candidate proteins were further verified using IHC analysis on a TMA containing tissue from 68 CCA patients, 48 male and 20 female. The aim of this study was to identify potential leads for further study and sample numbers were predominantly guided by (1) reagent costs; and (2) sample availability. Ov metacercariae were obtained from naturally infected cyprinoid fish in Khon Kaen province, Thailand using established methods (8.Prakobwong S. Yongvanit P. Hiraku Y. Pairojkul C. Sithithaworn P. Pinlaor P. Pinlaor S. Involvement of MMP-9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1-dependent DNA damage in a hamster model.Int. J. Cancer. 2010; 127: 2576-2587Crossref PubMed Scopus (73) Google Scholar). In brief, fish were digested with 0.25% pepsin-HCl and metacercariae isolated from the resulting slurry. Metacercariae were examined microscopically, counted and viable cysts were used to infect hamsters (Mesocricetus auratus). All animal tissue samples were taken from hamsters used in an immunohistochemical study of Ov-induced CCA described in (8.Prakobwong S. Yongvanit P. Hiraku Y. Pairojkul C. Sithithaworn P. Pinlaor P. Pinlaor S. Involvement of MMP-9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1-dependent DNA damage in a hamster model.Int. J. Cancer. 2010; 127: 2576-2587Crossref PubMed Scopus (73) Google Scholar) and approved by the Animal Ethics Committee of Khon Kaen University, Thailand (AEKKU 22/2557). The experimental design is shown in Fig. 1A. Hamsters were maintained at the animal research facility of the Faculty of Medicine, Khon Kaen University using protocols approved by the Khon Kaen University Animal Ethics Committee. Twelve male Syrian golden hamsters (Mesocricetus auratus) aged between 4–6 weeks were randomly divided into three groups of four animals: (1) the Normal group, which received a conventional murine diet (CP-SWT, Thailand); (2) the Ov-induced CCA group, which were infected with 50 Ov metacercariae by oral inoculation and fed the control diet supplemented with NDMA for the first two months of the trial (administered in water available ad libitum at 12.5 ppm); and (3) the Ov-infected group, which were infected with 50 Ov metacercariae by oral inoculation and fed the control diet. Human liver tissues were prepared as described in (14.Khoontawad J. Hongsrichan N. Chamgramol Y. Pinlaor P. Wongkham C. Yongvanit P. Pairojkul C. Khuntikeo N. Roytrakul S. Boonmars T. Pinlaor S. Increase of exostosin 1 in plasma as a potential biomarker for opisthorchiasis-associated cholangiocarcinoma.Tumour Biol. 2014; 35: 1029-1039Crossref PubMed Scopus (16) Google Scholar). Written informed consent was obtained from 68 CCA patients, 48 male and 20 female, who underwent liver resection at Srinagarind Hospital, Khon Kaen University, Thailand between 1999–2010 (HE571283). Patients had a mean age in years of 57 ± 7.7 (38–74 years). The Human Research Ethics Committee, Khon Kaen University, approved the study protocols for obtaining liver samples from the biobank of the Liver Fluke and Cholangiocarcinoma Research Center (HE571294). Frozen liver tissue from seven paired tumor cases (adjacent nontumor and tumor) were used for Western blotting and paraffin-embedded liver tissues from the same cases were used for immunohistochemistry analysis. Tissue microarrays (TMAs) were constructed by the Department of Pathology, Faculty of Medicine, Khon Kaen University as described previously (15.Fedor H.L. De Marzo A.M. Practical methods for tissue microarray construction.in: Pancreatic Cancer. Springer, 2005: 89-101Google Scholar). The TMAs contained 68 human CCA cases. To confirm the presence of intact tumor tissue, an H&E stained section of the TMA block was prepared and reviewed by two independent pathologists. Diagnosis of CCA patients was evaluated by clinical data, imaging analysis, tumor markers, and pathology. Hamster liver tissue (100 mg) from each hamster in each group was suspended in 600 μl of lysis buffer (7 m urea, 2 m thiourea, 4% (w/v) CHAPS and 40 mm Tris-Base) and homogenized with a Microtube Bead Homogenizer at 4 °C for 5 min. The sample was sonicated for 1 min and incubated on ice for 30 min. The solubilized samples were centrifuged at 12,000 × g for 20 min at 4 °C. Protein samples were precipitated with 6 volumes of cold acetone at −20 °C overnight. Proteins were then pelleted using centrifugation at 8000 × g at 4 °C for 10 min and the pellets resuspended in 0.5 m triethylammonium bicarbonate (TEAB) pH 8.5 (Sigma-Aldrich, Castle Hill, Australia) and 0.1% SDS. Proteins were quantified by Bradford protein assay (Bio-Rad, Gladesville, Australia) using the manufacturer's recommendations. Total liver proteins from three biological replicates were analyzed in three 8-plex iTRAQ experiments. Liver proteins (100 μg) from three hamsters in each group were reduced, alkylated, and labeled using the iTRAQ 8PLEX Multiplex Kit (AB SCIEX, Mt Waverley, Australia). Briefly, protein samples were reduced with 10 mm dithiothreitol at 60 °C for 1 h and alkylated in 50 mm iodoacetamide or methyl methanethiosulfonate (MMTS) at 37 °C for 30 min. Proteins were then digested with 2μg of trypsin at 37 °C for 16 h. iTRAQ labeling reagents were prepared by adding 50 μl of isopropanol to each vial and these were then used to label the peptide samples for 2 h at room temperature. Labeled peptides were combined into three mixtures, representing three biological replicates, each containing four peptide samples (Ov-induced CCA, Ov-infected and two control channels). After labeling, peptide mixtures were cleaned using HiTrap ion exchange columns (GE Healthcare, Little Chalfont, UK) and desalted using a Sep-Pak Vac C18 cartridge (Waters, Milford, MA). Cleaned fractions were then lyophilized prior to OFFGELTM. The 3100 OFFGEL Fractionator and OFFGEL kit pH 3–10 (Agilent Technologies, Santa Clara, CA) with a 24-well setup were prepared using the manufacturer's recommendations. Lyophilized peptide mixtures were diluted to a final volume of 3.6 ml using the OFFGEL peptide sample solution. IPG gel strips (24 cm) with a 3–10 linear pH range (GE Healthcare) were rehydrated with the Peptide IPG Strip Rehydration Solution using the manufacturer's recommendations and 150 μl of sample was loaded in each well. Peptides were isoelectrically focused with a maximum current of 50 μA until 50 kV-h were achieved. Twenty-four fractions were recovered from each well and the wells were rinsed with 150μl of water/methanol/formic acid (49/50/1) for 15 min. Each fraction was lyophilized and then resuspended in 15μl of H2O with 5% (v/v) formic acid prior to LC-MS/MS analysis. The human CCA cell line KKU055 (moderately differentiated) was cultured in RPMI 1640 (GIBCO, Life Technologies, Waltham, MA) media containing 1% Penicillin-Streptomycin (GIBCO, Life Technologies) and 10% Fetal Calf Serum (GIBCO, Life Technologies) in a 25 cm2 culture flask (Greiner Bio One, Kremsmünster, Austria) for the first generation. For the second generation heat treated 10% FCS was used. Cell growth was carried out at 37 °C under 5% CO2 and 95% humidified air. Upon reaching 80% confluency the cells were washed with 1× PBS and trypsinized using 0.25% Trypsin-EDTA (GIBCO, Life Technologies) and pelleted at 800 × g for 5 min before they were split into 175 cm2 culture flask (Greiner Bio One) in equal volumes. Approximately 800 ml of culture supernatant was collected at 80% confluency from each generation of the cell lines. Cell culture supernatants were subjected to differential high-speed centrifugation for isolation of exosomes as follows: the supernatant was centrifuged for 30 min at 2000 × g; the supernatant was then transferred to new tubes and centrifuged for 45 min at 15,000 × g. The supernatant was then collected and centrifuged at 100,000 × g for 18 h in 3.5 25 × 89 mm thin-wall polyallomer tubes (Beckman, Grea) with a SW31Ti Ultracentrifuge rotor. The pellet was then resuspended in PBS, sterile-filtered (0.2 μm) and centrifuged for a further 2 h at 100,000 × g in a TLA100.3 Ultracentrifuge rotor. All centrifugation steps were performed at 4 °C in order to maintain exosome stability. The final pellet was resuspended in Dulbecco's PBS (DPBS, Life Technologies, Australia). Isopycnic separation was used to further purify exosomes using sequential ultracentrifugations in an OptiPrep (Sigma-Aldrich) iodixanol gradient as previously described (16.Kalra H. Adda C.G. Liem M. Ang C.S. Mechler A. Simpson R.J. Hulett M.D. Mathivanan S. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.Proteomics. 2013; 13: 3354-3364Crossref PubMed Scopus (425) Google Scholar). Briefly, after high-speed centrifugations exosome extracts were subjected to a 0.25 m sucrose and iodixanol density gradient (5–40%). Gradients were prepared in Beckman Coulter Polyallomer 14 × 89 mm thin-wall tubes, under sterile conditions. A volume of 200 μl of exosome sample was added to each gradient column and centrifuged for 18 h, at 4 °C and 100,000 × g in a SW41Ti Ultracentrifuge rotor. After centrifugation, 12 × 1 ml fractions were collected from each gradient column and diluted 1:3 with DPBS followed by a 2 h centrifugation at 100,000 × g in a TLA100.3 Ultracentrifuge rotor. The pellet was then resuspended in 200 μl DPBS and centrifuged for a further 30 min. Finally, the pellet was resuspended in 50 μl of Tris-HCL solution (pH 7.5). Samples were stored at −80 °C (17.Théry C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol. 2006; 3: 3-22Google Scholar). Electron microscopy was used to identify fractions containing exosomes and to confirm the purity of exosomes in exosome preparations. A 2 μl aliquot from each of the exosome samples from density gradient fractions in Tris-HCl solution (pH 7.5) was directly adsorbed onto glow-discharged formvar carbon-coated copper grids on SuperFrost slides (Agar Scientific, Stansted, UK) to obtain a monolayer of exosomes for analysis. Each slide was then negatively stained with freshly prepared 2% uranyl acetate in aqueous suspension. Grids were air-dried for 3 min and imaged using a JEM-100CX transmission electron microscope (JEOL, Akishima, Japan) equipped with a thermionic tungsten filament and operated at an acceleration voltage of 100 kV. Digital images were taken with a pixel size of 0.3 nm using an Olympus Morada camera using exposure times between 100 and 400 mS. A NanoSight instrument (Malvern Instruments, Malvern, UK) was used to assess exosome yield from representative exosome preparations, after iodixanol fractionation, using the manufacturer's recommendations. For NanoSight analysis, 5 μl of exosome preparation was diluted to 1 ml with PBS. This solution was taken from a 50 μl solution that was the result of purifying 72 ml of culture media. Isolated exosomes were resolubilised in 1 ml 1× PBS and centrifuged at 100,000 × g for 60 min at 40 °C to pellet the exosomes. The pellet was resuspended in 200 μl of ice cold Exosome Resuspension buffer (Total Exosome RNA and Protein Isolation Kit, Invitrogen, Carlsbad, CA). The sample was incubated for 5–10 min at room temperature to allow the pellet to dissolve. This was followed by gently pipetting of the sample to ensure that the pellet was completely dissolved. Acetone precipitation was carried out by adding 1:5 volume of acetone and incubating overnight at −20 °C. The samples were then centrifuged at 3000 rpm for 15 min at 40 °C. The supernatant was discarded and the pellet was washed again with acetone and centrifuged at 10,000 rpm for 15 min at 40 °C. The pellet was dissolved in 10 μl of laemmli buffer and incubated at 96 °C for 3–5 min. This solution was loaded on the gel against the ladder (Precision Plus ProteinTM Dual Color Standards) sequence and subjected to SDS-PAGE and in-gel digestion. Exosome protein samples (30 μg) were applied to 1-mm-thick 4% stacking, 12% resolving gel for SDS-PAGE. Electrophoresis was carried out at 100 V for 20 min and then 200 V for 50 min. The gels were stained using Coomassie Brilliant Blue and destained in 25:10:65 methanol/acetic acid/water (v/v/v). SDS-PAGE gel lanes were divided into ∼24 slices and each slice cut into small pieces. Each gel slice was processed independently and was firstly destained twice by incubation in 50% acetonitrile, 200 mm NH4HCO3 for 45 min at 37 °C and then dried using a vacuum centrifuge. The gel pieces were resuspended in 20 mm dithiothreitol (DTT) and reduced for 1 h at 65 °C. DTT was removed, and the samples alkylated by the addition of 50 mm iodoacetamide and incubation in darkness at 37 °C for 40 min. Gel pieces were washed twice in 25 mm NH4HCO3 for 15 min and completely dried in a vacuum centrifuge. Gel pieces were rehydrated with 20 μl of trypsin reaction buffer (40 mm NH4HCO3, 10% acetonitrile) containing 20 μg/ml trypsin (Sigma) for 20 min at room temperature. An additional 50 μl of trypsin reaction buffer was added to the samples and incubated overnight at 37 °C. The digest supernatant was removed from the gel slices, and residual peptides were washed from the gel slices by incubating three times with 0.1% formic acid for 45 min at 37 °C. The original supernatant and extracts were combined and dried in a vacuum centrifuge. The tryptic peptides were resuspended in 12 μl 5% formic acid before mass spectral analysis. Labeled peptides from iTRAQ experiments and tryptic peptides from in-gel digests of exosome proteins were analyzed by LC-MS/MS on a Shimadzu Prominance Nano HPLC (Shimadzu, Brisbane, Australia) coupled to a Triple TOF 5600 mass spectrometer (AB SCIEX) equipped with a nano electrospray ion source. Two μl of peptide sample (∼1.5 μg of protein for iTRAQ experiments) was injected onto a 50 mm × 300 μm C18 trap column (Agilent) at 20 μl/min. The samples were de-salted on the trap column for 5 min using 0.1% formic acid (aq) at 20 μl/min. The trap column was then placed in-line with the analytical nano-HPLC column (150 mm × 75 μm C18, 5 μm, Vydav, Theale, UK) for mass spectrometry analysis. A linear gradient of 1–40% solvent B (90/10 acetonitrile/0.1% formic acid (aq)) over 120 min at 800 nL/minute flow rate, followed by a steeper gradient from 40% to 80% solvent B in 5 min, was used for peptide elution. The ionspray voltage was set to 2000V, declustering potential 100V, curtain gas flow 25, nebuliser gas 1 (GS1) 10 and interface heater at 150 °C. 500 ms full scan TOF-MS data was acquired followed by 20 × 50 ms full scan product ion data in an Information Dependent Acquisition (IDA) mode. Full scan TOF-MS data was acquired over the mass range 350–1800 and for product ions 100–1800. Ions observed i