Gencturk Elif, Kasim Muge, Morova Berna, Kiraz Alper, Ulgen Kutlu O
Department of Chemical Engineering, Boǧaziçi University, Biosystems Engineering Laboratory, Istanbul 34342, Turkey.
Department of Physics, Koç University, Sariyer, 34450 Istanbul, Turkey.
ACS Omega. 2022 May 2;7(19):16323-16332. doi: 10.1021/acsomega.1c06569. eCollection 2022 May 17.
Droplet-based microfluidic devices are used to investigate monocytic THP-1 cells in response to drug administration. Consistent and reproducible droplets are created, each of which acts as a bioreactor to carry out single cell experiments with minimized contamination and live cell tracking under an inverted fluorescence microscope for more than 2 days. Here, the effects of three different drugs (temsirolimus, rifabutin, and BAY 11-7082) on THP-1 are examined and the results are analyzed in the context of the inflammasome and apoptosis relationship. The ASC adaptor gene tagged with GFP is monitored as the inflammasome reporter. Thus, a systematic way is presented for deciphering cell-to-cell heterogeneity, which is an important issue in cancer treatment. The drug temsirolimus, which has effects of disrupting the mTOR pathway and triggering apoptosis in tumor cells, causes THP-1 cells to express ASC and to be involved in apoptosis. Treatment with rifabutin, which inhibits proliferation and initiates apoptosis in cells, affects ASC expression by first increasing and then decreasing it. CASP-3, which has a role in apoptosis and is directly related to ASC, has an increasing level in inflammasome conditioning. Thus, the cell under the effect of rifabutin might be faced with programmed cell death faster. The drug BAY 11-7082, which is responsible for NFκB inhibition, shows similar results to temsirolimus with more than 60% of cells having high fluorescence intensity (ASC expression). The microfluidic platform presented here offers strong potential for studying newly developed small-molecule inhibitors for personalized/precision medicine.
基于微滴的微流控装置用于研究单核细胞THP-1细胞对药物给药的反应。可产生一致且可重复的微滴，每个微滴都充当生物反应器，以进行单细胞实验，将污染降至最低，并在倒置荧光显微镜下对活细胞进行超过2天的跟踪。在此，研究了三种不同药物（替西罗莫司、利福布汀和BAY 11-7082）对THP-1的影响，并在炎性小体与细胞凋亡关系的背景下分析了结果。以绿色荧光蛋白（GFP）标记的凋亡相关斑点样蛋白（ASC）衔接子基因作为炎性小体报告基因进行监测。因此，提出了一种系统的方法来解读细胞间的异质性，这是癌症治疗中的一个重要问题。具有破坏mTOR通路和触发肿瘤细胞凋亡作用的药物替西罗莫司可使THP-1细胞表达ASC并参与细胞凋亡。利福布汀可抑制细胞增殖并引发细胞凋亡，其处理首先使ASC表达增加，然后使其降低。在细胞凋亡中起作用且与ASC直接相关的半胱天冬酶-3（CASP-3）在炎性小体调节中水平升高。因此，处于利福布汀作用下的细胞可能更快地面临程序性细胞死亡。负责抑制核因子κB（NFκB）的药物BAY 11-7082显示出与替西罗莫司相似的结果，超过60%的细胞具有高荧光强度（ASC表达）。本文介绍的微流控平台为研究用于个性化/精准医学的新开发小分子抑制剂提供了强大的潜力。
