Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation
Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation
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DOI：
10.1080/20013078.2020.1722433
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发表时间：
2020-01-01
影响因子：
16
通讯作者：
Lotvall, Jan
中科院分区：
文献类型：
--
作者：
Crescitelli, Rossella;Lasser, Cecilia;Lotvall, Jan
迄今为止进行的大多数细胞外囊泡（EV）研究都是在细胞系上进行的，对这些细胞系如何代表体内EV的特征知之甚少。本研究的目的是建立一种方法来分离和分类直接从肿瘤组织中分离的EV亚群。首先，我们建立了从转移性黑色素瘤组织中分离EV亚群的方案，其包括酶处理（胶原酶D和DNA酶）。用微分超离心分离小的和大的EV，并将其进一步分离成高密度和低密度（HD和LD）级分。然后使用电子显微镜、Bioanalyzer（R）、纳米颗粒跟踪分析和定量质谱分析深入分析所有EV亚群。具有不同大小、形态以及RNA和蛋白质货物的EV亚群可以从转移性黑素瘤组织中分离。LD EV表现出RNA谱，存在18 S和28 S核糖体亚基。相比之下，HD EV具有核糖体RNA亚基的小峰或无峰的RNA谱。定量蛋白质组学显示，几种蛋白质，如flotillin-1富集在大型和小型LD EV中，而ADAM 10仅富集在小型LD EV中。相比之下，丝裂霉素仅在大EV中富集。我们的结论是，酶处理提高EV隔离致密纤维化组织没有任何明显的影响，分子或形态特征。通过提供直接从肿瘤组织中分离的EV的几个亚群的详细分类，我们可以更好地了解EV在肿瘤生物学中的功能及其在生物标志物发现中的可能用途。
The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer (R), nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery.