Shpigelman Jonathan, Lao Fitzgerald S, Yao Shiyin, Li Chenyang, Saito Tetsuya, Sato-Kaneko Fumi, Nolan John P, Shukla Nikunj M, Pu Minya, Messer Karen, Cottam Howard B, Carson Dennis A, Corr Maripat, Hayashi Tomoko
Moores Cancer Center, University of California, San Diego, La Jolla, CA, United States.
School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, China.
Front Pharmacol. 2021 Apr 16;12:668609. doi: 10.3389/fphar.2021.668609. eCollection 2021.
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.
细胞外囊泡（EVs）被确定为细胞间通讯和细胞调节的介质。在免疫系统中，EVs作为细胞通讯的一部分，在抗原呈递中发挥作用。为了实现药物发现以及鉴定影响免疫细胞中EV生物发生、功能和释放的化合物，我们开发并表征了一种报告细胞系，该细胞系能够以表型高通量筛选（HTS）形式对分泌到培养基中的EVs进行定量。四跨膜蛋白CD63和CD9先前被报道在EVs中富集；因此，构建了一种由CD63- Turbo荧光素酶（Tluc）和CD9-翡翠绿色荧光蛋白（EmGFP）组成的双报告基因构建体。该构建体被转导到人单核细胞白血病细胞系THP-1中。通过流式细胞术将表达最高EmGFP的细胞分选成单细胞，并在抗生素选择压力下扩增克隆池。传代四次后，绿色荧光变弱，然后通过培养上清液中的荧光素酶活性追踪EV生物发生。培养上清液中从CD63Tluc-CD9EmGFP报告细胞分泌的EVs的Tluc活性与通过纳米颗粒跟踪分析测量的释放的EVs浓度呈正相关。为了检验在HTS中的应用潜力，我们首先将检测小型化到机器人384孔板形式。然后在不同的日子对一个2210种商业化合物库（Maybridge）进行了两次筛选，以检测细胞外荧光素酶活性的诱导情况。筛选数据显示第1天和第2天具有高重现性（78.6%）、宽信号窗口和出色的Z'因子（两天筛选的平均值为0.54）。187种化合物在第1天和第2天的筛选中显示出比阴性对照高3倍标准差的响应率，被视为命中候选物（约10%）。40种重新测试的化合物中有22种得到了验证。这些结果表明，CD63Tluc-CD9EmGFP报告细胞的性能可靠、可重现、稳健，并且对于筛选调节免疫细胞释放EVs的化合物的HTS是可行的。
