Cancer Research UK Edinburgh Centre, Institute of Genetics & Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, United Kingdom.
Methods Appl Fluoresc. 2018 Oct 24;7(1):015001. doi: 10.1088/2050-6120/aae6f8.
Evasion of apoptosis is a hallmark of human cancer, and a desired endpoint of many anticancer agents is the induction of cell death. With the heterogeneity of cancer becoming increasingly apparent, to understand drug mechanisms of action and identify combination therapies in cell populations, the development of tools to assess drug effects at the single cell level is a necessity for future preclinical drug development. Herein we describe the development of pCasFSwitch, a genetically encoded reporter construct designed to identify cells undergoing caspase-3 mediated apoptosis, by a translocation of a GFP signal from the cell membrane into the nucleus. Anticipated cellular distribution was demonstrated by use of confocal microscopy and cleavage by caspase-3 was shown to be required for the translocation of the GFP signal seen in apoptotic cells. Quantification of apoptosis using the construct revealed similar levels to that obtained with a commercially available apoptosis imaging agent (22.6% versus 20.3%). Moreover, we demonstrated its capacity for use in a high-throughput setting making it a powerful tool for drug development pipelines.
细胞凋亡的逃避是人类癌症的一个标志，许多抗癌药物的理想终点是诱导细胞死亡。随着癌症异质性变得越来越明显，为了了解药物的作用机制并在细胞群体中确定联合治疗方法，开发在单细胞水平评估药物作用的工具对于未来的临床前药物开发是必要的。在这里，我们描述了 pCasFSwitch 的开发，这是一种基因编码的报告构建体，旨在通过 GFP 信号从细胞膜易位到细胞核来识别经历 caspase-3 介导的细胞凋亡的细胞。通过共聚焦显微镜证实了预期的细胞分布，并且表明 caspase-3 的切割对于在凋亡细胞中观察到的 GFP 信号的易位是必需的。使用该构建体进行的细胞凋亡定量显示与商业可用的细胞凋亡成像剂获得的水平相似（22.6%与 20.3%）。此外，我们证明了它在高通量环境中的应用能力，使其成为药物开发管道的有力工具。
