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

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.

KEYWORDS:

• Extracellular vesicles
• exosomes
• melanoma
• microvesicles
• subpopulations
• vesicle isolation
• tissue-derived vesicles
• tandem mass tag
• mass spectrometry

Acknowledgments

We thank Bengt R. Johansson for helpful discussions regarding electron microscopy and cell anatomy. We also thank the Electron Microscopy Unit (EMU), and later the Centre for Cellular Imaging (CCI), at the University of Gothenburg (and the National Microscopy Infrastructure, NMI (VR-RFI 2016-00968)) for microscopy support. We thank the VBG Group Herman Krefting Foundation for Asthma and Allergy Research for supporting the Krefting Research Centre at the University of Gothenburg. The Proteomics Core Facility is grateful to the Inga-Britt and Arne Lundbergs Forskningsstiftlese for the donation of the Orbitrap Fusion Tribrid MS instrument. We thank Gunnar Nilsson at the Karolinska Institute, Stockholm, Sweden, for the kind gift of the human mast cell line HMC-1. The authors acknowledge Roberto Cattaneo for his assistance with graphic design.

Authors’ contribution

RC, CL and JL designed the study. RC performed most of the experiments and analyzed the data. CL and CM performed the flow cytometry experiments and flow cytometry analysis. CL performed proteomics data analysis. SCJ performed the ELISA experiments. JLH helped perform the electron microscopy experiments. AC helped with particle measurement experiments. ROB provided metastatic melanoma tissues. NK performed ExoView^TM experiments. IJ performed the immunohistochemistry analysis. JF and AT performed the mass spectrometry experiments. YSG gave conceptual advice. The data were interpreted collectively, and the manuscript was written by CL with the support of RC, SC and JL. All authors have read, commented on, and given approval of the final version of the manuscript.

Disclosure statement

SCJ is currently employed at Codiak Biosciences in Cambridge, MA, developing exosomes as therapeutics. RC, CL, AC and JL are developing multiple EV-associated patents for putative clinical utilization.

Supplementary Material

Supplemental data for this article can be accessed here

Additional information

Funding

This work was funded by the Swedish Research Council [K2014-85X-22504-01-3], the Swedish Heart and Lung Foundation [20120528], the Swedish Cancer Foundation [CAN2014/844], and the Knut och Alice Wallenberg Foundation (Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden).