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Summary: High-content imaging of tumor organoids (TOs) treated with therapeutic agents provides detailed cell viability readouts at the organoid level. In contrast, most used protocols provide one number per well. While requiring the use of inverted microscopy with an automated stage, this protocol can provide critical information about heterogeneous responses of TOs to various treatments. This protocol describes a technique for culturing and drug testing TOs using fluorescent indicators of cell viability with high reproducibility.For complete details on the use and execution of this protocol, please refer to Larsen et al. (2021). : Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.