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

Abstract

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.

Keywords: SEC; biomarkers; cancer; exosomes; extracellular vesicles; size-exclusion chromatography.

Figure 1

Larger particles take a more direct path through the gel and elute first. (A) Sample is layered onto column prepared with gel matrix. (B) With continuous addition of wash buffer, separation of the sample according to size occurs along the column.

Figure 2

Schematic graph showing make up of consecutive fractions. EVs elute first but there is often an overlap between similarly sized particles e.g., small EVs and lipoproteins. Assessing these potential contaminants should be a mandatory feature of size-exclusion chromatography (SEC) EV studies. NTA, Nanoparticle Tracking Analysis; DLS, Dynamic Light Scattering; HDL, High-Density Lipoproteins; WB, Western Blot; ELISA, Enzyme-Linked ImmunoSorbent Assay; BCA, BicinChoninic Acid.