Two Distinct Populations of Exosomes Are Released from LIM1863 Colon Carcinoma Cell-derived Organoids
Two Distinct Populations of Exosomes Are Released from LIM1863 Colon Carcinoma Cell-derived Organoids
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DOI：
10.1074/mcp.m112.021303
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发表时间：
2013-03-01
影响因子：
7
通讯作者：
Simpson, Richard J.
中科院分区：
文献类型：
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作者：
Tauro, Bow J.;Greening, David W.;Simpson, Richard J.
外泌体是内吞来源的天然存在的生物纳米膜囊泡（类似于40至100 nm），其从不同细胞类型释放到细胞外空间。它们具有多效性功能，例如抗原呈递和蛋白质货物、mRNA、microRNA、脂质和致癌潜力的细胞间转移。在这里，我们描述了通过使用抗A33-和抗EpCAM-偶联的磁珠的连续免疫捕获分离从源自人结肠癌细胞系LIM 1863的类器官释放的两个不同的外来体群体。外泌体群体（A33-Exos和EpCAM-Exos）不能通过电子显微镜区分，并且含有常规外泌体标记物，例如TSG 101、阿利克斯和HSP 70。通过基于凝胶的LC-MS/MS揭示的该研究的显著发现是在EpCAM-Exos中唯一鉴定出经典顶端运输分子CD 63（LAMP 3）、粘蛋白13和顶端肠酶蔗糖酶异麦芽糖酶，以及二肽基肽酶IV和顶端限制性五跨膜糖蛋白p53 in 1的表达增加。相反，A33-Exos制备物富含基底外侧运输分子，例如早期内体抗原1、高尔基体膜蛋白ADP-核糖基化因子和网格蛋白。我们的观察结果与EpCAM-和A33-Exos分别从顶侧和基底侧表面释放，以及EpCAM-Exos蛋白质组谱与广泛发表的定型外泌体一致。还进行了LIM 1863衍生的脱落微泡（sMV）的蛋白质组分析，以清楚地区分A33-和EpCAM-Exos与sMV。有趣的是，抗原呈递分子的MHC I类家族的几个成员仅在A33-Exos中观察到，而在EpCAM-Exos中通过MS既没有观察到MHC I类分子也没有观察到MHC II类分子。此外，我们在任何细胞外囊泡研究中首次报告了EpCAM、密蛋白-7和CD 44在EpCAM-Exos中的共定位。鉴于已知这些分子复合在一起以促进肿瘤进展，外泌体亚群的进一步表征将使人们能够更深入地了解它们在调节肿瘤微环境中的可能作用。Molecular & Cellular Proteomics 12：10.1074/mcp. M112.021303，587-598，2013.
Exosomes are naturally occurring biological nanomembranous vesicles (similar to 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. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.021303, 587-598, 2013.