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Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation

Journal article
Authors Rossella Crescitelli
Cecilia Lässer
Su Chul Jang
Aleksander Cvjetkovic
Carina Malmhäll
Nasibeh Karimi
Johanna L Höög
Iva Johansson
Johannes Fuchs
Annika Thorsell
Y. S. Gho
Roger Olofsson Bagge
Jan Lötvall
Published in Journal of Extracellular Vesicles
Volume 9
Issue 1
ISSN 2001-3078
Publication year 2020
Published at Sahlgrenska Cancer Center
Krefting Research Centre
Core Facilities, Proteomics
Department of Chemistry and Molecular Biology
Wallenberg Centre for Molecular and Translational Medicine
Institute of Medicine, Department of Internal Medicine and Clinical Nutrition
Language en
Links https://www.tandfonline.com/doi/ful...
Keywords Extracellular vesicles, exosomes, melanoma, microvesicles, subpopulations, vesicle isolation, tissue-derived vesicles, tandem mass, tag, mass spectrometry, transferrin receptor, malignant-melanoma, membrane-vesicles, exosomes, cells, markers, rme-8, visualization, endocytosis, micrornas, Cell Biology
Subject categories Clinical Medicine

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 (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.

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