Size-Exclusion Chromatography as a Technique for the Investigation of Novel Extracellular Vesicles in Cancer.

Daniel S K Liu,Jonathan Krell,Flora M Upton,Adam E Frampton,Long R Jiao,Christopher Limb,Eleanor Rees

doi:10.3390/cancers12113156

Abstract

Simple SummaryExtracellular vesicles (EVs) are small particles that are released by cancer cells, and they may hold vital information for researchers looking for early markers for diagnosis. Size-exclusion chromatography (SEC) is a classical technique that has become increasingly popular and can be used for rapid isolation and investigation of both their cargo and functionality. This systematic review highlights its main technical aspects, the type of materials involved and by covering the findings of the identified papers hopes to demonstrate the utility of this method in cancer research to date.Cancer cells release extracellular vesicles, which are a rich target for biomarker discovery and provide a promising mechanism for liquid biopsy. Size-exclusion chromatography (SEC) is an increasingly popular technique, which has been rediscovered for the purposes of extracellular vesicle (EV) isolation and purification from diverse biofluids. A systematic review was undertaken to identify all papers that described size exclusion as their primary EV isolation method in cancer research. In all, 37 papers were identified and discussed, which showcases the breadth of applications in which EVs can be utilised, from proteomics, to RNA, and through to functionality. A range of different methods are highlighted, with Sepharose-based techniques predominating. EVs isolated using SEC are able to identify cancer cells, highlight active pathways in tumourigenesis, clinically distinguish cohorts, and remain functionally active for further experiments.

Highlights

Extracellular vesicles (EVs) are small particles defined by a lipid bilayer and an inability to replicate, and they lack organelles or a functional nucleus
By 2017, differential ultracentrifugation (dUC) accounted for 45% of all experiments with a reported 190 unique isolation methods registered on EV-TRACK, an online tool designed to improve standardisation across the field [14]
The high purity of Size-exclusion chromatography (SEC)-isolated EVs leads to a clearer “signal” than is found for many other isolation methods, with clinical samples

Summary

Introduction

Extracellular vesicles (EVs) are small particles defined by a lipid bilayer and an inability to replicate, and they lack organelles or a functional nucleus. They have been termed various names, such as “shedding membrane vesicles”, “membrane fragments” [1], “microvesicles” [2], or more recently “exosomes”. We make a broad distinction between subsets of EVs by their biogenesis, size, or density, and this is dependent on the experimental isolation method used. There remains a considerable overlap in what is isolated for experimental characterisation, and studies investigating vesicular biogenesis show that both large and small vesicles can demonstrate direct budding from the plasma membrane [5]. Exosomes are the dominant area of research, having been shown to act as a distinct intercellular signalling mechanism, shuttling messenger

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