Keles Hakan, Schofield Christopher A, Rannikmae Helena, Edwards Erin Elizabeth, Mohamet Lisa
Genome Biology, Genomic Sciences, R&D, GSK, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom.
Complex In Vitro Models, In Vitro In Vivo Translation, R&D, GSK, Gunnels Wood Road, Stevenage, SG1 2NY, United Kingdom.
ACS Pharmacol Transl Sci. 2022 Dec 12;6(1):12-21. doi: 10.1021/acsptsci.2c00200. eCollection 2023 Jan 13.
The high morbidity rate of ovarian cancer has remained unchanged during the past four decades, partly due to a lack of understanding of disease mechanisms and difficulties in developing new targeted therapies. Defective DNA damage detection and repair is one of the hallmarks of cancer cells and is a defining characteristic of ovarian cancer. Most in vitro studies to date involve viability measurements at scale using relevant cancer cell lines; however, the translation to the clinic is often lacking. The use of patient derived organoids is closing that translational gap, yet the 3D nature of organoid cultures presents challenges for assay measurements beyond viability measurements. In particular, high-content imaging has the potential for screening at scale, providing a better understanding of the mechanism of action of drugs or genetic perturbagens. In this study we report a semiautomated and scalable immunofluorescence imaging assay utilizing the development of a 384-well plate based subnuclear staining and clearing protocol and optimization of 3D confocal image analysis for studying DNA damage dose response in human ovarian cancer organoids. The assay was validated in four organoid models and demonstrated a predictable response to etoposide drug treatment with the lowest efficacy observed in the clinically most resistant model. This imaging and analysis method can be applied to other 3D organoid and spheroid models for use in high content screening.
在过去的四十年里，卵巢癌的高发病率一直没有变化，部分原因是对疾病机制缺乏了解以及开发新的靶向治疗存在困难。DNA损伤检测和修复缺陷是癌细胞的特征之一，也是卵巢癌的一个决定性特征。迄今为止，大多数体外研究都涉及使用相关癌细胞系进行大规模的活力测量；然而，往往缺乏向临床的转化。使用患者来源的类器官正在缩小这一转化差距，但类器官培养的三维性质给除活力测量之外的检测测量带来了挑战。特别是，高内涵成像具有大规模筛选的潜力，能更好地理解药物或基因干扰剂的作用机制。在本研究中，我们报告了一种半自动化且可扩展的免疫荧光成像检测方法，该方法利用了基于384孔板的亚核染色和清除方案的开发以及三维共聚焦图像分析的优化，用于研究人卵巢癌类器官中的DNA损伤剂量反应。该检测方法在四种类器官模型中得到验证，并显示出对依托泊苷药物治疗有可预测的反应，在临床上最具抗性的模型中观察到最低的疗效。这种成像和分析方法可应用于其他三维类器官和球体模型，用于高内涵筛选。
