Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release

Abstract

Extracellular vesicles (EVs) are identified as mediators of intercellular communication and cellular regulation. In the immune system, EVs play a role in antigen presentation as a part of cellular communication. To enable drug discovery and characterization of compounds that affect EV biogenesis, function, and release in immune cells, we developed and characterized a reporter cell line that allows the quantitation of EVs shed into culture media in phenotypic high-throughput screen (HTS) format. Tetraspanins CD63 and CD9 were previously reported to be enriched in EVs; hence, a construct with dual reporters consisting of CD63-Turbo-luciferase (Tluc) and CD9-Emerald green fluorescent protein (EmGFP) was engineered. This construct was transduced into the human monocytic leukemia cell line, THP-1. Cells expressing the highest EmGFP were sorted by flow cytometry as single cell, and clonal pools were expanded under antibiotic selection pressure. After four passages, the green fluorescence dimmed, and EV biogenesis was then tracked by luciferase activity in culture supernatants. The Tluc activities of EVs shed from CD63Tluc-CD9EmGFP reporter cells in the culture supernatant positively correlated with the concentrations of released EVs measured by nanoparticle tracking analysis. To examine the potential for use in HTS, we first miniaturized the assay into a robotic 384-well plate format. A 2210 commercial compound library (Maybridge) was then screened twice on separate days, for the induction of extracellular luciferase activity. The screening data showed high reproducibility on days 1 and 2 (78.6%), a wide signal window, and an excellent Z′ factor (average of 2-day screen, 0.54). One hundred eighty-seven compounds showed a response ratio that was 3SD above the negative controls in both day 1 and 2 screens and were considered as hit candidates (approximately 10%). Twenty-two out of 40 re-tested compounds were validated. These results indicate that the performance of CD63Tluc-CD9EmGFP reporter cells is reliable, reproducible, robust, and feasible for HTS of compounds that regulate EV release by the immune cells.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

“…We next analyzed the same VLPs by single vesicle flow cytometry (vFC, Cellarcus Biosciences), which uses a fluorogenic membrane probe to detect and size vesicles, and fluorescent MAbs to measure vesicle surface cargo by immunofluorescence [ 114 , 115 ]. This revealed a modest, but consistent proportion of Env+ particles bound PGT121 (13.25–21.58%) in samples transfected with WITO Env ( Fig 10C , columns 2–4 and D, right plot).…”

“…A new, state-of-the-art method [ 128 ] revealed that only ~25% of particles in VLP preparations are Env+, the rest being bald [ 114 , 115 ]. Some particles may derive from FBS that carries ubiquitous vesicles.…”

“…Particle concentration, size, Env+ fraction and spike density were determined by single vesicle flow cytometry [ 114 , 115 ], using a commercial kit (vFC Assay kit, Cellarcus Biosciences, La Jolla, CA) and flow cytometer (CytoFlexS, Beckman Coulter, Indianapolis, IN). Briefly, samples were stained with the fluorogenic membrane stain vFRed TM and anti-Env MAb PGT121, labeled with AlexaFluor647 (Thermo Fisher) for 1h at RT and analyzed using membrane fluorescence to trigger detection.…”

Homosalate boosts the release of tumor-derived Extracellular Vesicles with anti-anoikis properties

“…2C-D), highlighting a differential effect of this MVB-modulating drug on CD63 or CD9 positive EV secretion. In another study, the use of a dual reporter system of CD63-Turbo-Luciferase-CD9-Emerald-Green allowed to identify several compounds regulating the secretion of CD63-CD9 double positive EVs by immune cells [51]. Turbo-Luciferase could be particularly suitable for high-throughput screenings because it is less sensitive to chemical compound interference [52].…”

A Triple High Throughput Screening for Extracellular Vesicle Inducing Agents With Immunostimulatory Activity

“…Recently, we developed and characterized a human monocytic leukemia THP-1 reporter cell line engineered with a fusion construct for the expression of EV-associated tetraspanins (CD63 and CD9) linked to Turbo-luciferase (Tluc) and Emerald Green Fluorescent Protein (EmGFP) (CD63 Tluc-CD9-EmGFP THP-1 cells) to quantitatively measure release of EVs in culture supernatants ( Shpigelman et al, 2021 ). Using this reporter cell line, we described that Tluc activity levels correlated with concentrations of released EVs in the culture supernatant as measured by nanoparticle tracking ( Shpigelman et al, 2021 ). Here, we utilized this cell line for a high throughput screening (HTS) of a library of 27,895 compounds.…”