Department of Technology R&D, Nexcelom Bioscience LLC, Lawrence, Massachusetts, 01843.
Cytometry A. 2017 Sep;91(9):883-892. doi: 10.1002/cyto.a.23143. Epub 2017 Jun 15.
The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better identify more qualified cancer drug candidates for drug discovery research. © 2017 International Society for Advancement of Cytometry.
近年来，用于癌症药物发现研究的三维（3D）多细胞肿瘤球体模型的发展有所增加。与二维（2D）测定相比，使用 3D 肿瘤球体模型可能更能代表复杂的体内肿瘤微环境。目前，3D 多细胞肿瘤球体的活力通常使用代谢试剂（如 CellTiter-Glo®）在标准平板读数器上进行终点分析来测量。或者，高内涵图像细胞仪已用于测量药物对球体大小和活力的影响。以前，我们已经使用 Celigo 图像细胞仪证明了一种用于 3D 多细胞肿瘤球体的新型终点药物筛选方法。为了更好地描述癌症药物的作用，还需要测量对 3D 多细胞肿瘤球体的动力学细胞毒性和细胞凋亡作用。在这项工作中，我们展示了使用 PI 和 caspase 3/7 染色实时测量 3D 多细胞肿瘤球体活力和凋亡的方法。该方法首先通过用 PI 和 caspase 3/7 染色不同类型的肿瘤球体并监测 16 和 21 天的荧光强度进行验证。接下来，将 PI 染色和非染色的对照肿瘤球体消化成单细胞悬浮液，以直接在 2D 测定中测量活力，从而确定 PI 的潜在毒性。最后，对相关时间依赖性 PI 和 caspase 3/7 荧光强度与球体大小和坏死核心形成进行了广泛的数据分析，以确定用于癌症药物测试的最佳起始时间点。实时测量活力和凋亡的能力对于开发用于筛选肿瘤球体的适当 3D 模型非常重要，这可以使研究人员确定通常不能通过终点测定捕获的时间依赖性药物作用。这将改进当前的肿瘤球体分析方法，从而有可能更好地识别更有资格的癌症药物候选物，用于药物发现研究。©2017 国际细胞分析协会。
