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Pancreatitis-Associated Extracellular Vesicle Identification through an Allosteric Probe-Initiated Cascade Amplification System.

ABSTRACT: As an emerging class of noninvasive biomarkers, accurate quantification of circulating extracellular vesicles (EVs) is essential to both the basic biological research and early diagnosis of pancreatitis. We report here an allosteric probe-initiated cascade amplification system for a highly sensitive detection of serum-circulating EVs. In this method, the special recognition of EVs by the allosteric probes triggers allosterism of the probe and thus induces the release of a signal amplification initiator. Through the following multiple rounds of cascade amplifications, a large number of fluorescence moiety are released, generating an enhanced fluorescence signal. This method exhibits a large dynamic range of 5 orders of magnitude. In addition, this strategy could also be performed under isothermal conditions in a wash-free way, indicating its potential applications in early diagnosis and prognosis of pancreatitis.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC8296005 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Simple Summary MYCN gene amplification, the strongest prognostic marker of aggressive neuroblastoma, is detected on invasive biopsy tissues. This study aimed to establish a less invasive method to detect MYCN status based on MYCN mRNA contents in large extracellular vesicles or microvesicles. MYCN mRNA-containing microvesicles were detectable in three distinct MYCN-amplified neuroblastoma cell lines but absent in three neuroblastoma cells with MYCN-non-amplification. The feasibility of this EV-based workflow was successfully demonstrated by using the simulated samples (prepared by pulsing neuroblastoma MVs into the normal human serum) and bone marrow plasma specimens obtained from nine patients at various disease stages. Taken together, this study established the novel EV-based method for detecting MYCN status in pediatric neuroblastoma. Abstract MYCN amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect MYCN 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 MYCN 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 MYCN mRNA. As a result, MYCN mRNA was detectable in the MVs, but not exosomes, of MYCN-amplified NB cells. MYCN mRNA-containing MVs (MYCN-MV) were successfully detected in three distinct MYCN-amplified NB cell lines but absent in three MYCN non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. MYCN–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 MYCN-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 MYCN status of pediatric NB based on MYCN mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance.

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Pancreatitis-Associated Extracellular Vesicle Identification through an Allosteric Probe-Initiated Cascade Amplification System.

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