Two Distinct Populations of Exosomes Are Released from LIM1863 Colon Carcinoma Cell-derived Organoids

Abstract

Exosomes are naturally occurring biological nanomembranous vesicles (ϳ40 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 The microenvironment in which a tumor originates plays a critical role in its initiation, progression, and metastasis (1-3). Recent advances have indicated that although the microenvironment provides crucial signaling to maintain tissue architecture, inhibit cell growth, and constrain the malignant phenotype, it can also promote and induce cancer (4). In addition to cancer cells, the tumor microenvironment comprises normal cells, blood cells, secreted proteins and peptides, and constituents of the extracellular matrix that actively influence cell behavior. Secreted proteins, peptides, and physiological small molecules such as soluble cytokines and chemokines are currently recognized as the main exocrine and juxtacrine factors underlying cell-to-cell communication within the tumor microenvironment and providing the metastatic niche in distant organs (5). In addition to soluble secreted proteins and peptides, most cell types also release extracellular membrane vesicles (eMVs) 1 that transfer information between cells in the microenv...

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