Extracellular Vesicle Isolation and Analysis using Centrifugal Microfluidic System for Liquid Biopsy

Abstract

Liquid biopsy, an emerging method for less-invasive testing than conventional tissue biopsy, has high potential not only to give a practical diagnostic method, but also to offer a more comprehensive information of the disease since biomarkers are secreted from the overall location of the disease. Among representative biomarkers including circulating tumor cells (CTCs), extracellular vesicles (EVs) and circulating tumor DNA (ctDNA) for liquid biopsy, challenges and overcoming developments of EV applications are discussed and demonstrated in this thesis. Recently, EVs have emerged as novel biomarker with the growing evidence that EVs play an important role in intercellular communication in tumor microenvironments. Unlike the other biomarkers, EVs are abundant and prevalently found in most body fluids carrying important genetic information stored in nucleic acids which are protected from degradation due to its surrounded lipid bilayer. Despite its clinical importance, current methods of EV isolation and analysis are challenging because of complicated procedures with long processing time. In this thesis, methods for isolation and analysis of EV in lab-on-a-disc platforms were developed for liquid biopsy application. The most commonly used method is ultracentrifugation (UC) to isolate EVs which requires time-consuming steps involving centrifugation and acquire of large sample volumes. In chapter 2, Exodisc, a lab-on-a-disc for size-selective EV isolation and analysis, were demonstrated. It allows efficient EV isolation from both CCS and bladder cancer patient urine samples as well as their subpopulations. This enabled isolation and purification of EVs from raw samples within 30 min with high recovery rates of enriched EVs (>95% recovery of EVs and >100-fold higher mRNA concentrations of GAPDH, CD9, PSA and PSMA as compared UC results). With this system, the subsequent on-disc ELISA after EV isolation was demonstrated with the urine samples from bladder cancer patients. Although we demonstrated a different protein expression level of EVs in bladder cancer patients and normal, only classical EV markers are analyzed which is not a cancer protein marker. In chapter 3, as a contribution in liquid biopsy using EVs, we studied a biomarker, androgen receptor splice 7 (AR-V7) which is the most studied case among all AR splice variants to investigate progression of prostate cancer and patient’s prognosis, from the EVs isolated from urine of prostate cancer patients. Urine-derived EVs were isolated by a lab-on-a-disc integrated with six independent nanofiltration units (Exo-Hexa) allowing simultaneous processing of six individual samples. Exo-Hexa was further compared with other density-based EV isolation method including ultracentrifugation and precipitating reagents-based kits, showing higher yield in spiked urine sample. Using Exo-Hexa, we demonstrated higher AR-V7 and lower AR-FL expressions were detected in urine-derived EVs from 14 patients with castration-resistant prostate cancer than in those from 22 patients with hormone-sensitive prostate cancer. Furthermore, we compared the EVs isolated from urine and plasma of same patient, showing urinary EV gives higher detection rate of AR-V7. In conclusion, lab-on-a-disc platforms to isolate and analyze EVs have been developed and applied for liquid biopsy. To be specific, size-selective isolation of EV and their subpopulation have been demonstrated with the contribution in the liquid biopsy.