Dual size-exclusion chromatography for efficient isolation of plasma-derived extracellular vesicles and their applications for companion diagnostics of non-small cell lung cancer

Abstract

An extracellular vesicle (EV), a vesicle with a lipid bilayer membrane structure, contains comprehensive information such as nucleic acids and proteins of the cell of its origin. In particular, EVs can protect parental information from degrading enzymes in the blood and contain membrane proteins because of the property of the lipid bilayer, differentiating it from conventional biomarkers. In addition, cancer cells actively release more EVs than normal cells due to the enhanced metabolic processes. Therefore, EVs are emerging as an important biomaterial for the discovery of novel cancer biomarkers or personalized diagnoses such as companion diagnostics. To carry out EV studies, ultracentrifuge or polymer precipitation methods have been developed. However, it was difficult to obtain high-purity EVs from the blood due to the presence of contaminants such as low-density lipoproteins (LDLs) and albumin. In this dissertation, a size-exclusion chromatography (SEC)-based EV separation method was developed to increase the purity of EVs by using blood cancer plasma (acute myelogenous leukemia). The size distribution and characteristics of EVs, LDLs, and soluble proteins in the plasma were confirmed by varying the designing parameters such as resin pore size, sequential dual stacking, and the ratio of beds and the optimal parameters were selected by observing the properties of contaminants. The separated EVs showed higher purity than conventional SEC and polymer precipitation methods, and this effect was also appeared in solid cancer plasma (non-small cell lung cancer). For the application of companion diagnosis, EV-derived DNA was analyzed to detect the epidermal growth factor receptor (EGFR) mutations. As the isolated nano-sized EVs appeared to contain highly pure ctDNA, comparable diagnostic results to direct analysis of plasma were obtained. Furthermore, to overcome the limitations of the conventional analytical methods based on nucleic acid amplification, the novel method based on the affinity of EGFR-gefitinib and surface-enhanced Raman scattering (SERS) were developed for direct analysis of EGFR status. The developed protein analysis method was able to distinguish EGFR status according to WT, primary (19 deletion) and secondary mutations (19 deletion/T790M), and also to be analyzed in EV of 100 μl plasma. In conclusion, in this study, the developed dual SEC allows the isolation of highly pure EVs in a simple and rapid manner from plasma., In addition, the separated EVs reflected the cancer EGFR mutations when using non-small cell lung cancer plasma. And the developed EGFR analytical method could reflect tumor EGFR status via EV proteins. These results will aid in the investigation of EV-based novel biomarkers and at the same time, open the possibility of utilizing EV proteins as a diagnostic tool in predicting the response of target drugs in the future.