Multiplexed live-cell imaging for drug responses in patient-derived organoid models of cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to simultaneously quantitate cell health and apoptosis. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer. SUMMARY Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes. ### Competing Interest Statement KWT is a co-owner of Immortagen Inc. CJD is an employee of Agilent. JSdB has served on advisory boards and received fees from Amgen, Astra Zeneca, Astellas, Bayer, Bioxcel Therapeutics, Boehringer Ingelheim, Cellcentric, Daiichi, Eisai, Genentech/Roche, Genmab, GSK, Harpoon, ImCheck Therapeutics, Janssen, Merck Serono, Merck Sharp & Dohme, Menarini/Silicon Biosystems, Orion, Pfizer, Qiagen, Sanofi Aventis, Sierra Oncology, Taiho, Terumo, and Vertex Pharmaceuticals; is an employee of the Institute of Cancer Research (ICR), which have received funding or other support for his research work from AZ, Astellas, Bayer, Cellcentric, Daiichi, Genentech, Genmab, GSK, Janssen, Merck Serono, MSD, Menarini/Silicon Biosystems, Orion, Sanofi Aventis, Sierra Oncology, Taiho, Pfizer, and Vertex, and which has a commercial interest in abiraterone, PARP inhibition in DNA repair defective cancers, and PI3K/AKT pathway inhibitors (no personal income); was named as an inventor, with no financial interest for patent 8 822 438, submitted by Janssen that covers the use of abiraterone acetate with corticosteroids; has been the CI/PI of many industry-sponsored clinical trials; and is a National Institute for Health Research (NIHR) Senior Investigator. No other authors have any potential conflicts of interest to disclose.