A Scalable 3D High-Content Imaging Protocol for Measuring a Drug Induced DNA Damage Response Using Immunofluorescent Sub-nuclear γH2AX Spots in Patient Derived Ovarian Cancer Organoids

• Published in: bioRxiv
• Main Authors: Keles, Hakan, Schofield, Christopher A, Rannikmae, Helena, Edwards, Erin Elizabeth, Mohamet, Lisa
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 19.09.2022
• Subjects: Deoxyribonucleic acid; DNA; DNA damage; DNA repair; Etoposide; Image processing; Immunofluorescence; Morbidity; Organoids; Ovarian cancer; Patients; Tumor cell lines
• DOI: 10.1101/2022.09.15.508096

The high morbidity rate of ovarian cancer has remained unchanged during the past four decades, partly due to 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 clinic is often lacking. The use of patient derived organoids is closing that translational gap yet the 3D nature of organoid cultures present challenges for assay measurements beyond viability measurements. In particular, high-content imaging has the potential for screening at scale providing a better understanding of mechanism of action of drugs or genetic perturbagens. In this study we report a semi-automated and scalable immunofluorescence imaging assay utilising the development of a 384-well plate based subnuclear staining and clearing protocol and optimisation 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 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. Competing Interest Statement The authors have declared no competing interest. Footnotes * Minor corrections in text.