Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based screening assays do not capture complexity of a (cytostatic or cytotoxic) intratumor heterogeneity that has been shown to be retained in PDTOs due bulk readout. Live-cell imaging is powerful tool overcome this issue visualize responses more in-depth. image analysis software often adapted three-dimensionality PDTOs, requires fluorescent viability dyes, compatible with 384-well microplate format. This paper describes semi-automated methodology seed, treat, high-throughput, format using conventional, widefield, live-cell systems. In addition, we developed marker-free quantify growth rate-based metrics improve reproducibility correct rate variations between different PDTO lines. Using normalized metric, which scores based on positive negative control condition, cell death dye, cytotoxic cytostatic can easily distinguished, profoundly improving classification responders non-responders. drug-response by quantified single-organoid identify potential, resistant clones. Ultimately, method aims prediction clinical capturing multiparametric signature, includes kinetic arrest quantification.