Two distinct populations of exosomes released from LIM1863 colon carcinoma cell-derived organoids.pdf (593.82 kB)
Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids
journal contributionposted on 2021-08-12, 06:32 authored by BJ Tauro, David GreeningDavid Greening, RA Mathias, Suresh MathivananSuresh Mathivanan, H Ji, Richard SimpsonRichard Simpson
Exosomes are naturally occurring biological nanomembranous vesicles (̃40 to 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, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863. The exosome populations (A33-Exos and EpCAM-Exos) could not be distinguished via electron microscopy and contained stereotypical exosome markers such as TSG101, Alix, and HSP70. The salient finding of this study, revealed via gel-based LC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1, the Golgi membrane protein ADP-ribosylation factor, 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 in order to clearly distinguish A33- and EpCAMExos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos, whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalization of EpCAM, claudin-7, and CD44 in EpCAM-Exos. Given that these molecules are known to complex together to promote tumor progression, further characterization of exosome subpopulations will enable a deeper understanding of their possible role in regulation of the tumor microenvironment. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
This work was supported by the National Health & Medical Research Council of Australia program through Grant No. 487922 (R.J.S.), Fellowship No. 1016599 (S.M.), and Early Career CJ Martin Fellowship No. APP1037043 (R.A.M.). B.J.T. is supported by The University of Melbourne Research Scholarship (MRS). Analysis of proteomic data described in this work was supported using the Australian Proteomics Computational Facility funded by the National Health & Medical Research Council of Australia through Grant No. 381413. This work was supported by funds from the Operational Infrastructure Support Program provided by the Victorian Government Australia. We acknowledge the Australian Cancer Research Foundation for providing funds to purchase the Orbitrap (TM) mass spectrometer.
JournalMolecular and Cellular Proteomics
PublisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
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Science & TechnologyLife Sciences & BiomedicineBiochemical Research MethodsBiochemistry & Molecular BiologyEXTRACELLULAR MEMBRANE-PARTICLESEPITHELIAL-CELLSPROMININ-1 CD133TUMOR-STROMACANCERPROTEINSEPCAMTETRASPANINSPROTEOMICSCLAUDIN-7OrganoidsCell Line, TumorHumansColonic NeoplasmsCalcium-Binding ProteinsCell Cycle ProteinsDNA-Binding ProteinsMembrane GlycoproteinsCell Adhesion MoleculesNeoplasm ProteinsProteomeTranscription FactorsAntigens, NeoplasmHistocompatibility Antigens Class IChromatography, LiquidProteomicsLysosome-Associated Membrane GlycoproteinsTandem Mass SpectrometryCell-Derived MicroparticlesExosomesEndosomal Sorting Complexes Required for TransportClaudinsEpithelial Cell Adhesion MoleculeHyaluronan Receptors