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Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation
Journal of Extracellular Vesicles ( IF 15.5 ) Pub Date : 2020-02-11
Rossella Crescitelli, Cecilia Lässer, Su Chul Jang, Aleksander Cvjetkovic, Carina Malmhäll, Nasibeh Karimi, Johanna L. Höög, Iva Johansson, Johannes Fuchs, Annika Thorsell, Yong Song Gho, R. Olofsson Bagge, Jan Lötvall

ABSTRACT

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®, 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.



中文翻译:

优化分离后通过定量蛋白质组学鉴定的人类转移性黑色素瘤组织细胞外囊泡亚群

摘要

迄今为止,大多数细胞外囊泡(EV)研究都是在细胞系上进行的,但对这些细胞在体内如何表现出EV的特性知之甚少 。这项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出的电动汽车的亚群。首先,我们为转移性黑素瘤组织的EV亚群建立了隔离方案,其中包括酶处理(胶原酶D和DNase)。通过差异超速离心分离大小的电动汽车,然后将其进一步分为高密度和低密度(HD和LD)部分。然后使用电子显微镜,Bioanalyzer®,纳米颗粒跟踪分析和定量质谱分析对所有EV亚种群进行深入分析。可以从转移性黑色素瘤组织中分离出具有不同大小,形态,RNA和蛋白质货物的电动汽车亚群。LD EVs显示存在18S和28S核糖体亚基的RNA谱。相反,HD EV的RNA谱具有核糖体RNA亚基的小峰或无峰。定量蛋白质组学显示,大,小型LD EV均富集了几种蛋白质(例如flotillin-1),而小型LD EV仅富集了ADAM10。相反,米托菲林仅在大型电动汽车中富集。我们得出的结论是,酶处理可改善从致密纤维化组织中分离出的EV,而对分子或形态特征没有任何明显影响。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,我们可能会更好地了解EV在肿瘤生物学中的功能及其在生物标记物发现中的可能用途。定量蛋白质组学显示,大,小型LD EV均富集了几种蛋白质(例如flotillin-1),而小型LD EV仅富集了ADAM10。相反,米托菲林仅在大型电动汽车中富集。我们得出的结论是,酶处理可改善从致密纤维化组织中分离出的EV,而对分子或形态特征没有任何明显影响。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,我们可能会更好地了解EV在肿瘤生物学中的功能及其在生物标记物发现中的可能用途。定量蛋白质组学显示,大,小型LD EV均富集了几种蛋白质(例如flotillin-1),而小型LD EV仅富集了ADAM10。相反,米托菲林仅在大型电动汽车中富集。我们得出的结论是,酶处理可改善从致密纤维化组织中分离出的EV,而对分子或形态特征没有任何明显影响。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,我们可能会更好地了解EV在肿瘤生物学中的功能及其在生物标记物发现中的可能用途。我们得出的结论是,酶处理可改善从致密纤维化组织中分离出的EV,而对分子或形态特征没有任何明显影响。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,我们可能会更好地了解EV在肿瘤生物学中的功能及其在生物标记物发现中的可能用途。我们得出的结论是,酶处理可改善从致密纤维化组织中分离出的EV,而对分子或形态特征没有任何明显影响。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,我们可能会更好地了解EV在肿瘤生物学中的功能及其在生物标记物发现中的可能用途。

更新日期:2020-04-20
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