A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays

In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic ‘switching’. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.为高效表征靶向细胞周期的小分子的抗增殖效能与作用机制，我们开发了一种高通量成像检测分析法，用于测定细胞数量及细胞周期时相分布。借助该方法，我们针对一组细胞系，分析了具有不同作用机制的实验性与获批抗癌药物的作用效果，并将直接细胞计数与两种基于代谢的细胞活力/增殖检测方法进行了对比：ATP依赖型生物发光检测、MTS（3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑鎓）还原检测，以及全孔DNA结合染料荧光检测。研究表明，根据化合物的作用机制不同，基于代谢的替代检测方法往往存在两大缺陷：1）显著低估化合物的活性与效能；2）因浓度依赖性表型"切换"而出现非单调剂量反应曲线。具体而言，吉西他滨、依托泊苷等靶向DNA合成的药物，其抗增殖效能与活性会被ATP检测及MTS还原检测大幅低估。在同一成像检测体系中，我们还发现药物诱导的ATP含量升高，与细胞体积增大、线粒体含量及呼吸通量成比例增加存在关联，同时伴随细胞周期阻滞。因此，在筛选抗增殖活性化合物或分析细胞系组的药物敏感性时，若使用ATP或四唑鎓还原检测作为细胞数量的替代指标，化合物的作用机制差异以及细胞系特异性应答可能会产生极具误导性的结果。