Abstract
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 EpCAM-Exos 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.
Highlights
From the ‡Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia; §Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria, Australia
The abbreviations used are: A33-Exos, exosomes isolated using anti-A33 immunoaffinity beads; CCM, concentrated culture medium; CLN7, claudin-7; Culture medium (CM), culture medium; CRC, colorectal cancer; EEA1, early endosome antigen 1; EM, electron microscopy; eMV, extracellular membrane vesicle; EpCAM, epithelial cell adhesion molecule; EpCAM-Exos, exosomes isolated using anti-EpCAM immunoaffinity beads; ESCRT, endosomal sorting complex required for transport; intraluminal vesicles (ILVs), intraluminal vesicle; MUC13, mucin 13; multivesicular bodies (MVBs), multivesicular body; PDCD6IP/Alix, programmed cell death 6 interacting protein; PM, plasma membrane; Rsc, ratio of normalized spectral counts; shed microvesicles (sMVs), shed microvesicle; SSM, solid support magnet; TSG101, tumor susceptibility gene 101
A comparative proteome profiling of A33positive LIM1215 exosomes with previously published murine mast cell [21] and human-urine-derived exosomes [22] revealed a subset of proteins common to the three exosome types and, for the first time, a human colon cancer exosomal proteome “signature”. As this signature might reflect the CRC exosomal profile of a restricted CRC subtype—LIM1215 cells were originally derived from a patient with inherited nonpolyposis colorectal cancer [23]—we have extended these studies to another CRC cell subtype and report here a robust proteome study of exosomes isolated from the CRC cell line LIM1863, which grows as organoids with spontaneous differentiation into crypt-like structures in vitro [24]
Summary
Cell Culture and Preparation of Concentrated Culture Medium— Human colon carcinoma LIM1863 cells grow as free-floating multicellular spheres (organoids) in which highly polarized cells localize around a central lumen. Gels were removed from the tank and fixed in 50 ml fixing solution (40% (v/v) methanol, 10% (v/v) acetic acid in water) for 30 min on an orbital shaker and stained with 30 ml SYPRO® Ruby (Invitrogen) for 30 min This was followed by destaining twice in 50 ml of 10% (v/v) methanol with 6% (v/v) acetic acid in water for 1 h. Western Blot Analysis—Exosome samples (ϳ10 g protein) were lysed in SDS sample buffer, reduced with 50 mM DTT (when required), heated for 5 min at 95 °C, and subjected to electrophoresis using precast NuPAGETM 4 –12% (w/v) Bis-Tris Precast gels (Invitrogen) in MES running buffer at a constant 150 V for 1 h. Cells were washed three times with washing buffer and imaged using a Nikon ECLIPSE TE2000-E confocal microscope equipped with a Nikon plan APO VC 60x/1.20 WI water-immersion lens
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