Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

Abstract

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

Circulating Extracellular Vesicles Contain Liver-Derived RNA Species as Indicators of Severe Cholestasis-Induced Early Liver Fibrosis in Mice

“…Thus, the combined use of miRNAs and mRNAs may assist in finding the origin of circulating EVs, in detecting in a noninvasive way any pathological changes occurring in the tissues of origin, and in developing assays such as those based on flow cytometry methodology to allow multiple biomarkers detection in a single setting ( Fig. 10 ) ( 51 ). Overall, these findings suggest that, while miR122-5p can be regarded as a generic hepatocellular toxicity marker, circulating EV-contained miR192-5p, miR194-5p, miR22-3p, and miR29a-3p can be specifically predictive of severe cholestasis-induced liver fibrosis.…”

Blood Nanoparticles – Influence on Extracellular Vesicle Isolation and Characterization

“…Standardized reporting can improve quantitative comparisons of results from different laboratories, and support the development of new instruments and assays ( Welsh et al, 2020a ). For deeper insight into EV flow cytometry, please also consult the following articles ( Arraud et al, 2016 ; Welsh et al, 2017 ; Welsh et al, 2020a ; van der Pol et al, 2018 ; Görgens et al, 2019 ; Tian et al, 2020 ; Yang and Rhee, 2021 ).…”

Multiplexed Profiling of Extracellular Vesicles for Biomarker Development

“…To profile both EV proteins and miRNA within one configuration, Rhee and co-workers have explored the combination of fluorescent antibodies and molecular beacons for targeting membrane proteins and internal miRNAs, respectively [76]. As a proof of concept, Alexa Fluor® 488-conjugated anti-CD63 antibody and molecular beacon-21 targeting miR-21 were investigated for multiplexed biomarker detection in healthy vs cancer-derived EVs using flow cytometry (Fig.…”

“…b Simultaneous in situ detection of EV membrane protein and internal miRNA using dye conjugated molecular beacons and dye conjugated antibodies, respectively. Reproduced with permission from Ref [76]…”