Proteomic Analysis of Exosomes from Mutant KRAS Colon Cancer Cells Identifies Intercellular Transfer of Mutant KRAS
2012; Elsevier BV; Volume: 12; Issue: 2 Linguagem: Inglês
10.1074/mcp.m112.022806
ISSN1535-9484
AutoresMichelle Demory Beckler, James N. Higginbotham, Jeffrey L. Franklin, Amy‐Joan L. Ham, Patrick J. Halvey, Imade Imasuen, Corbin A. Whitwell, Ming Li, D.C. Liebler, Robert J. Coffey,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoActivating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression. Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression. K-RAS (KRAS) is a small, monomeric GTPase whose biological activity is specified by its nucleotide binding state. Multiple lines of evidence highlight the importance of KRAS in colorectal cancer (CRC). 1The abbreviations used are:CRCcolorectal cancerDiD1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanineEGFRepidermal growth factor receptorEXOexosomeFDRfalse discovery rateMRMmultiple reaction monitoringWCLwhole cell lysateWTwild-type. 1The abbreviations used are:CRCcolorectal cancerDiD1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanineEGFRepidermal growth factor receptorEXOexosomeFDRfalse discovery rateMRMmultiple reaction monitoringWCLwhole cell lysateWTwild-type.For example, activating missense mutations in KRAS, which lock the protein into the GTP-bound state, occur in 30% to 40% of CRCs and are strongly associated with poor prognosis (1Pylayeva-Gupta Y. Grabocka E. Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web.Nat. Rev. Cancer. 2011; 11: 761-774Crossref PubMed Scopus (1215) Google Scholar, 2Cox A.D. Der C.J. Ras history: the saga continues.Small GTPases. 2010; 1: 2-27Crossref PubMed Scopus (472) Google Scholar). Also, mutant KRAS negatively predicts responsiveness to anti-EGF receptor (EGFR) therapy (3De Roock W. Piessevaux H. De Schutter J. Janssens M. De Hertogh G. Personeni N. Biesmans B. Van Laethem J.L. Peeters M. Humblet Y. Van Cutsem E. Tejpar S. KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab.Ann. Oncol. 2008; 19: 508-515Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar). colorectal cancer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine epidermal growth factor receptor exosome false discovery rate multiple reaction monitoring whole cell lysate wild-type. colorectal cancer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine epidermal growth factor receptor exosome false discovery rate multiple reaction monitoring whole cell lysate wild-type. Early attempts to decipher the neoplastic consequences of mutant KRAS relied on overexpression studies. A drawback of these studies is their failure to simulate the genetic conditions present in human tumors, where there is often one wild-type (WT) and one mutant KRAS allele (1Pylayeva-Gupta Y. Grabocka E. Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web.Nat. Rev. Cancer. 2011; 11: 761-774Crossref PubMed Scopus (1215) Google Scholar). More recently, KRAS mutant CRC cell lines have been engineered to selectively contain either the wild-type or the mutant KRAS allele (4Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras.Science. 1993; 260: 85-88Crossref PubMed Scopus (600) Google Scholar), and a single mutant Kras allele has been activated in the intestine using genetically engineered mice (5Haigis K.M. Kendall K.R. Wang Y. Cheung A. Haigis M.C. Glickman J.N. Niwa-Kawakita M. Sweet-Cordero A. Sebolt-Leopold J. Shannon K.M. Settleman J. Giovannini M. Jacks T. Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon.Nat. Genet. 2008; 40: 600-608Crossref PubMed Scopus (469) Google Scholar). Detailed studies using these complementary approaches demonstrate a wide range of tumor-promoting effects of mutant KRAS (reviewed in Ref. 6Velho S. Haigis K.M. Regulation of homeostasis and oncogenesis in the intestinal epithelium by Ras.Exp. Cell Res. 2011; 317: 2732-2739Crossref PubMed Scopus (13) Google Scholar). Much of what is known about mutant KRAS pertains to its ability to alter the behavior of a transformed cell in a cell autonomous manner. With the exception of increased tumor vascularity via increased tumor-derived VEGF expression (7Okada F. Rak J.W. Croix B.S. Lieubeau B. Kaya M. Roncari L. Shirasawa S. Sasazuki T. Kerbel R.S. Impact of oncogenes in tumor angiogenesis: mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells.Proc. Natl. Acad. Sci. 1998; 95: 3609-3614Crossref PubMed Scopus (228) Google Scholar, 8Mazure N.M. Chen E.Y. Yeh P. Laderoute K.R. Giaccia A.J. Oncogenic transformation and hypoxia synergistically act to modulate vascular endothelial growth factor expression.Cancer Res. 1996; 56: 3436-3440PubMed Google Scholar), non-cell autonomous effects of mutant KRAS have been much less studied. Exosomes are 30- to 100-nm secreted vesicles that have emerged as a novel mode of intercellular communication (9Schorey J.S. Bhatnagar S. Exosome function: from tumor immunology to pathogen biology.Traffic. 2008; 9: 871-881Crossref PubMed Scopus (606) Google Scholar). We recently reported that exosomes purified from conditioned medium of mutant KRAS CRC cells contained higher levels of the EGFR ligand amphiregulin (AREG) and enhanced invasiveness of recipient cancer cells relative to exosomes from isogenically matched wild-type KRAS cells (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). These results prompted us to perform a comprehensive analysis of exosomes purified from these cells. Herein, we show that mutant KRAS induces many changes in exosomal protein composition. Notably, we show that (i) KRAS is contained within exosomes, (ii) exosomes can transfer mutant KRAS to cells expressing only wild-type KRAS, and (iii) mutant KRAS-containing exosomes enhance wild-type KRAS cell growth in collagen matrix and soft agar. These results have important implications for the progression of CRC tumors by providing a mechanism by which the tumor microenvironment may be influenced by non-cell autonomous signals released by mutant KRAS-expressing tumor cells. DKs-8, DLD-1, DKO-1 (4Shirasawa S. Furuse M. Yokoyama N. Sasazuki T. Altered growth of human colon cancer cell lines disrupted at activated Ki-ras.Science. 1993; 260: 85-88Crossref PubMed Scopus (600) Google Scholar), and RIE-1 cells were cultured as described elsewhere (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 11Barnard J.A. Graves-Deal R. Pittelkow M.R. DuBois R. Cook P. Ramsey G.W. Bishop P.R. Damstrup L. Coffey R.J. Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family.J. Biol. Chem. 1994; 269: 22817-22822Abstract Full Text PDF PubMed Google Scholar). Cells were maintained in serum-containing DMEM (Mediatech, Manassas, VA). Bovine growth serum was purchased from HyClone (Logan, UT), and all other cell culture reagents were purchased from Mediatech unless otherwise stated. Triscarboxyethylphosphine was purchased from Pierce (Rockford, IL), sequencing grade trypsin was obtained from Promega (Madison, WI), and trifluoroethanol and dithiothreitol were acquired from Acros (Geel, Belgium). Trifluoroacetic acid, ammonium bicarbonate, and urea were purchased from Fisher Scientific (Pittsburgh, PA). All other reagents were purchased from Sigma (St. Louis, MO). For a list of other reagents, see the supplemental "Experimental Procedures" section. Exosomes were isolated from conditioned medium of DKs-8, DLD-1, and DKO-1 cells as previously described, with slight modification (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Briefly, cells were cultured in DMEM supplemented with 10% bovine growth serum until 80% confluent. The cells were then washed three times with PBS and cultured for 48 h in serum-free medium. The serum-free conditioned medium was removed and centrifuged for 10 min at 300 × g to remove cellular debris, and the resulting supernatant was then filtered through a 0.22-μm polyethersulfone filter (Nalgene, Rochester, NY) to reduce microparticle contamination. The filtrate was concentrated ∼300-fold with a 100,000 molecular-weight cutoff centrifugal concentrator (Millipore). The concentrate was then subjected to high-speed centrifugation at 150,000 × g for 2 h. The resulting exosome-enriched pellet was resuspended in PBS containing 25 mm HEPES (pH 7.2) and washed by centrifuging again at 150,000 × g for 3 h. The wash steps were repeated a minimum of three times until no trace of phenol-red was detected. The resulting pellet was resuspended in PBS containing 25 mm HEPES (pH 7.2) as described previously (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), and the protein concentrations of the exosome preparations were determined with a MicroBCA kit (Pierce). The number of exosomes per microgram of protein was determined by means of nanoparticle tracking analysis (NanoSight, Wiltshire, UK) according to the manufacturer's recommendations. Analysis was performed on three independent preparations of exosomes. Three separate preparations of exosomes were purified, and the same protein concentration of each sample was analyzed. The samples were digested with trypsin using a trifluoroethanol (TFE) digestion procedure as described elsewhere, with minor modifications (12Wang Y. Wang H. Gao L. Liu H. Lu Z. He N. Polyacrylamide gel film immobilized molecular beacon array for single nucleotide mismatch detection.J. Nanosci. Nanotechnol. 2005; 5: 653-658Crossref PubMed Scopus (21) Google Scholar), and isoelectric focusing was adapted from Cargile et al. (13Cargile B.J. Sevinsky J.R. Essader A.S. Stephenson Jr., J.L. Bundy J.L. Immobilized pH gradient isoelectric focusing as a first-dimension separation in shotgun proteomics.J. Biomol. Tech. 2005; 16: 181-189PubMed Google Scholar). For detailed methods of TFE digestion and isoelectric focusing of tryptic peptides, see the supplemental "Experimental Procedures" section. LC-MS/MS analyses were performed on an LTQ-Orbitrap hybrid mass spectrometer (Thermo Electron, San Jose, CA) equipped with an Eksigent nanoLC and autosampler (Dublin, CA). Peptides were resolved on a 100 μm × 11 cm fused silica capillary column (Polymicro Technologies, LLC, Phoenix, AZ) packed with 5 μm, 300 Å Jupiter C18 (Phenomenex, Torrance, CA) using an inline 100 mm × 4 cm solid phase extraction column packed with the same C18 resin as that previously described (14Licklider L.J. Thoreen C.C. Peng J. Gygi S.P. Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column.Anal. Chem. 2002; 74: 3076-3083Crossref PubMed Scopus (186) Google Scholar). Liquid chromatography was carried out at room temperature at a flow rate of 0.6 μl min> using a gradient mixture of 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B). For additional details, see the supplemental "Experimental Procedures" section. The "ScanSifter" algorithm read the tandem mass spectra stored as centroided peak lists from Thermo RAW files and transcoded them to mzML files (15Ma Z.Q. Tabb D.L. Burden J. Chambers M.C. Cox M.B. Cantrell M.J. Ham A.J. Litton M.D. Oreto M.R. Schultz W.C. Sobecki S.M. Tsui T.Y. Wernke G.R. Liebler D.C. Supporting tool suite for production proteomics.Bioinformatics. 2011; 27: 3214-3215Crossref PubMed Scopus (28) Google Scholar). For detailed analysis, see the supplemental "Experimental Procedures" section. Protein groups identified were submitted to Webgestalt for GOSlim analysis and to the Ingenuity Pathways analysis package. In order to classify protein groups, the data were sorted based on relative levels, and proteins were identified that differed by greater than 3-fold between groups and had a false discovery rate (FDR) of less than 0.05. Classifications of proteins that had significantly different levels were made based on the DAVID (http://david.abcc.ncifcrf.gov/) and Uniprot databases. To identify any potential proteins that might differentiate cellular mutant KRAS status, we statistically evaluated the differences using our previously published approach, with some modifications (16Li M. Gray W. Zhang H. Chung C.H. Billheimer D. Yarbrough W.G. Liebler D.C. Shyr Y. Slebos R.J. Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.J. Proteome Res. 2010; 9: 4295-4305Crossref PubMed Scopus (86) Google Scholar). To calculate the rate ratio, reported spectral counts are reverse calculated using model-generated rates and provided offset numbers. The rate ratio is the ratio of the group rates expressed as the base-2 log of the ratio of the rates (16Li M. Gray W. Zhang H. Chung C.H. Billheimer D. Yarbrough W.G. Liebler D.C. Shyr Y. Slebos R.J. Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.J. Proteome Res. 2010; 9: 4295-4305Crossref PubMed Scopus (86) Google Scholar). This ratio represents the quantitative difference between the compared groups. A generalized linear mixed effect model (17Faraway J.J. Extending the Linear Model with R: Generalized Linear, Mixed Effects and Nonparametric Regression Models. Chapman & Hall/CRC, Boca Raton, FL2006Google Scholar) was fitted to handle exosome values expressed as count data with repeated measurements for each cell line sample; p values were obtained for each comparison based on a likelihood ratio test. Rate ratios were determined by comparing the expected values of the two mutant states. An FDR controlling procedure was applied to handle the multiple comparisons when testing thousands of proteins simultaneously. To test whether there was a monotone increasing trend in exosomal levels for DKs-8, DLD-1, and DKO-1 cells, a Jonckheere–Terpstra trend test was applied (18Jonckheere A.R. A distribution-free k-sample test against ordered alternatives.Biometrika. 1954; 41: 133-145Crossref Google Scholar). See the supplemental "Experimental Procedures" section for additional statistical considerations. DKs-8 and DKO-1 cells were grown to 80% confluence and serum starved overnight. One million cells were incubated with 100 μg of the indicated exosomes or mock treated for 1 h under constant rotation at 37 °C. Cells were then pelleted and washed three times with ice-cold PBS, and the detection of peptides was performed as described in the supplemental "Experimental Procedures" section. Peptide samples were analyzed in triplicate (2-μl injection volume) on a TSQ Vantage triple quadrupole mass spectrometer (Thermo-Fisher, San Jose, CA) equipped with an Eksigent nanoLC solvent delivery system (Eksigent, Dublin, CA), an autosampler, and a nanospray source. For details, see the supplemental "Experimental Procedures" section (19Halvey P.J. Ferrone C.R. Liebler D.C. GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples.J. Proteome Res. 2012; 11: 3908-3913Crossref PubMed Scopus (30) Google Scholar). Concentrations for KRAS peptides were normalized to protein input and reported as fmol/μg protein. For the relative quantification of exosomal marker proteins, the labeled reference peptide method was used (20Zhang H. Liu Q. Zimmerman L.J. Ham A.J. Slebos R.J. Rahman J. Kikuchi T. Massion P.P. Carbone D.P. Billheimer D. Liebler D.C. Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry.Mol. Cell. Proteomics. 2011; 10M110.006593Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Cell samples were lysed and digested prior to LC-multiple reaction monitoring (LC-MRM) analysis as described elsewhere (21Halvey P.J. Zhang B. Coffey R.J. Liebler D.C. Slebos R.J. Proteomic consequences of a single gene mutation in a colorectal cancer model.J. Proteome Res. 2012; 11: 1184-1195Crossref PubMed Scopus (27) Google Scholar). A stable isotope labeled version of the β-actin peptide GYSFTTTAER was used as an internal standard (25 fmol/μl). The integrated chromatographic peak areas for the transitions of each targeted peptide were obtained from Skyline (22MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2963) Google Scholar), summed, and normalized to summed peak areas for the β-actin internal standard, as we have described elsewhere (20Zhang H. Liu Q. Zimmerman L.J. Ham A.J. Slebos R.J. Rahman J. Kikuchi T. Massion P.P. Carbone D.P. Billheimer D. Liebler D.C. Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry.Mol. Cell. Proteomics. 2011; 10M110.006593Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). For Western blotting, DKs-8, DLD-1, and DKO-1 cells were lysed, and proteins were resolved via SDS-PAGE and Western blotted as described in the supplemental "Experimental Procedures" section. For Western blotting, 100 μg whole cell lysate (WCL) and 10 μg exosome protein (EXO) were used for CTNND1, ITGAV, ITGB1, RAP1, and SRC; 100 μg of WCL and 30 μg of EXO were used for CTNNA, ITGA2, LYN, and KRAS; 200 μg WCL and 30 μg EXO were used for CTTN and EPHA2; 50 μg WCL and 20 μg EXO were used for ITGA6; and 50 μg WCL was used for TUBA. Exosomes were purified from conditioned medium of DKs-8 or DKO-1 cells, and imaging was performed as described elsewhere (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The diameter of 100 exosomes from each of two independent exosome preparations (200 total) was determined using ImageJ software. To determine whether exosomes are internalized, exosomes isolated from DKO-1 or DKs-8 cells were labeled with 100 μg 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD)/mg EXO and washed to remove unincorporated DiD. DKO-1 or DKs-8 recipient cells were grown to 50% confluence and cultured in serum-free medium overnight. DiD-labeled exosome internalization by recipient cells was performed as previously described (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Briefly, cells were removed from culture dishes, washed, and incubated with DiD-stained exosomes under constant rotation for the indicated times. Cells were immediately diluted into 100 vol ice-cold PBS supplemented with 10% BSA, followed by washing. The cells were analyzed via flow cytometry to determine the total fluorescence (surface-associated exosomes + internalized exosomes) and the percentage of labeled cells. The cells were then exposed to 200 mm Sudan Black in PBS supplemented with 0.5% DMSO and 5% bovine serum for 5 min at 4 °C to quench DiD fluorescence on the surface of recipient cells. Flow cytometry was repeated to determine the fluorescence of internalized exosomes. DKs-8 and DKO-1 exosomes were stained with the lipophilic membrane dye DiD (Invitrogen, Grand Island, NY) as described elsewhere (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Recipient DKs-8, DKO-1, or RIE-1 cells were plated on coverslips in 24-well tissue culture dishes at a density of 5.0 × 104 cells/well. After the cells had been maintained in serum-containing medium for 24 h, the cells were washed and incubated with serum-free medium for an additional 24 h. The adherent cells were then incubated with 100 μg of DiD-stained exosomes for 30 min at 37 °C on an orbital shaker. Subsequently, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.2% Triton-X100, incubated with AlexaFluor 488-Phalloidin (Invitrogen), and mounted on slides. Internalization was visualized on a Zeiss LSM 510 Meta confocal microscope with a 63× objective. For growth in a three-dimensional collagen gel matrix (23Chung E. Graves-Deal R. Franklin J.L. Coffey R.J. Differential effects of amphiregulin and TGF-alpha on the morphology of MDCK cells.Exp. Cell Res. 2005; 309: 149-160Crossref PubMed Scopus (35) Google Scholar), three layers of collagen were used in 48-well tissue culture dishes. Prior to being plated in collagen, DKs-8 and DKO-1 cells were trypsinized, syringed, and resuspended in serum-free DMEM at a concentration of 5 × 105 cells/ml. The top and bottom layers contained 150 μl/well of PureCol collagen (Advanced Biomatrix, San Diego, CA) diluted to 2 mg/ml in serum-free DMEM. The middle layer consisted of 150 μl/well of 2 mg/ml collagen in serum-free DMEM and 5 × 103 cells. Serum-free medium or serum-free medium supplemented with 50 μg of DKs-8 or DKO-1 exosomes was added. Medium was replaced twice weekly for 2 weeks. Colonies were detected by using a Gel Count imager (Oxford Optronix, Oxford, UK). Three technical replicates were performed for each of three experiments. The mean colony number for each sample was plotted ± the S.E., and statistical significance was reported as p < 0.05. The results for colony diameters are reported as a boxplot through their five summary statistics: sample minimum (lowest bar), lower quartile (Q1, the lower hinge), median (Q2), upper quartile (Q3, the upper hinge), and the sample maximum (highest bar). A two-sample t test was applied to assess the mean colony diameter difference between the samples, and statistical significance is reported as p < 0.001. Prior to being plated in agarose, RIE-1 cells were rotated end over end with the indicated exosomes (50 μg exosomes/5.0 × 105 cells) or mock treated for 2 h at 37 °C. The cells were then plated in six-well dishes in triplicate at a density of 6.25 × 104 cells/well in 0.4% Type VII agarose (Sigma) over a hardened layer of 0.8% agarose. The cells were incubated at 37 °C in 5% CO2 for 7 days, and colonies were counted using a Gel Count imager. Three technical replicates were performed for each of three experiments. The mean colony number for each sample was plotted ± the S.E., and statistical significance was reported as p < 0.01. Our prior study showed that exosomes released by mutant KRAS-expressing CRC cells contained markedly higher levels of AREG than exosomes from their isogenically matched WT KRAS derivatives (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). We hypothesized that there might be global changes in the protein composition of exosomes based on the mutant KRAS status of the producing cell. Exosomes were purified from the serum-free conditioned medium of parental DLD-1 cells and their isogenically matched derivatives: DKO-1 (mutant KRAS allele only) and DKs-8 (WT allele only) cells. Serum-containing medium was not used during collection because of the known presence of exosomes in bovine serum (24Thery C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.in: Bonifacino J.S. Current Protocols in Cell Biology. 2006Google Scholar). To ensure that the exosomal preparations derived from these cells were both enriched in exosomes and relatively devoid of larger extracellular vesicles, vesicles were subjected to exosomal marker and size analysis (Fig. 1). These vesicles contained the exosome-specific markers HSP70 (25Zhan R. Leng X. Liu X. Wang X. Gong J. Yan L. Wang L. Wang Y. Wang X. Qian L.J. Heat shock protein 70 is secreted from endothelial cells by a non-classical pathway involving exosomes.Biochem. Biophys. Res. Commun. 2009; 387: 229-233Crossref PubMed Scopus (92) Google Scholar), TSG101 (26Thery C. Boussac M. Veron 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 (1188) Google Scholar), and Flotillin-1 (10Higginbotham J.N. Demory Beckler M. Gephart J.D. Franklin J.L. Bogatcheva G. Kremers G.J. Piston D.W. Ayers G.D. McConnell R.E. Tyska M.J. Coffey R.J. Amphiregulin exosomes increase cancer cell invasion.Curr. Biol. 2011; 21: 779-786Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), but they did not contain voltage-dependent anion channel (VDAC), a mitochondrial protein (Fig. 1A). Size analysis by means of transmission electron microscopy (TEM) showed that DKs-8 vesicles had a mean diameter of 59.2 nm ± 14.2 nm and DKO-1 vesicles had a mean diameter of 56.3 nm ± 17.9 nm (Fig. 1B). Importantly, no vesicles were larger than 140 nm. Although we cannot exclude the possibility that other types of vesicles are contained in our preparations, these results are consistent with the reported size of exosomes (9Schorey J.S. Bhatnagar S. Exosome function: from tumor immunology to pathogen biology.Traffic. 2008; 9: 871-881Crossref PubMed Scopus (606) Google Scholar) and smaller than the reported size of microvesicles, which range in diameter from 200 nm to 1 μm (27Cocucci E. Racchetti G. Meldolesi J. Shedding microvesicles: artefacts no more.Trends Cell Biol. 2009; 19: 43-51Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar). Combined, these results strongly support the purity of these exosome preparations. To determine the concentration of exosomes produced by each of the cell lines, nanoparticle tracking analysis was performed. The results show relatively similar numbers of vesicles per microgram of protein for DKs-8, DLD-1, and DKO-1 exosome preparations (supplemental Fig. S1A), suggesting that the three cells lines secrete equivalent levels of exosomes under serum-free conditions. To determine whether protein composition was altered in ex
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