Publications (32) View all
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Article: Proteome profiling of exosomes derived from human primary and metastatic colorectal cells reveal differential expression of key metastatic factors and signal transduction components.
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ABSTRACT: Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist pre-metastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signalling molecules fundamental to tumour progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40-100 nm in diameter, were of a buoyant density ∼1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, EGFR, JAG1, SRC, TNIK) and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalisation of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in CRC cell exosomes. The selective enrichment of metastatic factors and signalling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the crosstalk between tumour and stromal cells in the tumour microenvironment.Proteomics 04/2013; · 4.43 Impact Factor -
Article: Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids.
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ABSTRACT: Exosomes are naturally occurring biological nanomembraneous vesicles (~40-100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, miRNA, lipids and oncogenic potential. Here we describe the isolation, by sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from the highly polarized human colon carcinoma cell line LIM1863. Both exosome populations (A33- Exos and EpCAM-Exos) were indistinguishable by electron microscopy and contained stereotypical exosome markers such as TSG101, Alix and HSP70. The salient finding of this study, revealed by GeLC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 (MUC13), apical intestinal enzymes sucrase isomaltase (SI) and increased expression of dipeptidyl peptidase IV (DPP4) and the apically-restricted pentaspan membrane glycoprotein prominin 1 (PROM1). By comparison, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1 (EEA1), the golgi membrane protein ADP-ribosylation factor (ARF1) and clathrin. Our observations are consistent with EpCAM- and A33-Exos being released from the apical and basolateral surfaces, respectively, and the EpCAM-Exos proteome profile with widely-published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed to clearly distinguish A33- and EpCAM-Exos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos. Neither MHC class I or II molecules were observed by MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalisation of EpCAM, claudin-7 and CD44 in EpCAM-Exos. Given that these molecules are known to complex together to promote tumour progression, further characterisation of exosome sub-populations will enable a deeper understanding of their possible role in regulation of the tumour microenvironment.Molecular & Cellular Proteomics 12/2012; · 7.40 Impact Factor -
Article: Identifying mutated proteins secreted by colon cancer cell lines using mass spectrometry.
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ABSTRACT: Secreted proteins encoded by mutated genes (mutant proteins) are a particularly rich source of biomarkers being not only components of the cancer secretome but also actually implicated in tumorigenesis. One of the challenges of proteomics-driven biomarker discovery research is that the bulk of secreted mutant proteins cannot be identified directly and quantified by mass spectrometry due to the lack of mutated peptide information in extant proteomics databases. Here we identify, using an integrated genomics and proteomics strategy (referred to iMASp - identification of Mutated And Secreted proteins), 112 putative mutated tryptic peptides (corresponding to 57 proteins) in the collective secretomes derived from a panel of 18 human colorectal cancer (CRC) cell lines. Central to this iMASp was the creation of Human Protein Mutant Database (HPMD), against which experimentally-derived secretome peptide spectra were searched. Eight of the identified mutated tryptic peptides were confirmed by RT-PCR and cDNA sequencing of RNA extracted from those CRC cells from which the mutation was identified by mass spectrometry. The iMASp technology promises to improve the link between proteomics and genomic mutation data thereby providing an effective tool for targeting tryptic peptides with mutated amino acids as potential cancer biomarker candidates. This article is part of a Special Issue entitled: Integrated omics.Journal of proteomics 07/2012; · 5.07 Impact Factor -
Article: Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.
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ABSTRACT: Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling.Methods 01/2012; 56(2):293-304. · 4.01 Impact Factor -
Article: ExoCarta 2012: database of exosomal proteins, RNA and lipids.
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ABSTRACT: Exosomes are membraneous nanovesicles of endocytic origin released by most cell types from diverse organisms; they play a critical role in cell-cell communication. ExoCarta (http://www.exocarta.org) is a manually curated database of exosomal proteins, RNA and lipids. The database catalogs information from both published and unpublished exosomal studies. The mode of exosomal purification and characterization, the biophysical and molecular properties are listed in the database aiding biomedical scientists in assessing the quality of the exosomal preparation and the corresponding data obtained. Currently, ExoCarta (Version 3.1) contains information on 11,261 protein entries, 2375 mRNA entries and 764 miRNA entries that were obtained from 134 exosomal studies. In addition to the data update, as a new feature, lipids identified in exosomes are added to ExoCarta. We believe that this free web-based community resource will aid researchers in identifying molecular signatures (proteins/RNA/lipids) that are specific to certain tissue/cell type derived exosomes and trigger new exosomal studies.Nucleic Acids Research 01/2012; 40(Database issue):D1241-4. · 8.03 Impact Factor
