Extracellular Vesicle-Based Method for Detecting Amplification Status of Pediatric Neuroblastoma

amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect status requires invasive procedures. Extracellular vesicles (EVs) contain molecular signatures of originated cells, present in biofluids, and serve as an invaluable source for cancer liquid biopsies. This study aimed to establish an EV-based method to detect the status of NB. Two EV subtypes, i.e., microvesicles (MVs) and exosomes, were sequentially isolated from the culture supernatant by step-wise centrifugation, ultrafiltration, and size-exclusion chromatography. Quantitative RT-PCR was performed to detect mRNA. As a result, mRNA was detectable in the MVs, but not exosomes, of -amplified NB cells. mRNA-containing MVs (-MV) were successfully detected in three distinct -amplified NB cell lines but absent in three non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. -MV detection in the simulated samples showed a less interfering effect from the human blood matrix. Validation using clinical specimens (2 mL bone marrow plasma) obtained from patients at various disease stages showed a promising result. Five out of six specimens of -amplified patients showed positive results, while there were no false positives in four plasma samples of the MYCN non-amplification group. This study communicated a novel EV-based method for detecting the status of pediatric NB based on mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance.