Precision oncology has been shown to greatly improve outcomes of cancer patients, with tailored treatment approaches that consist of patient-directed therapies on the molecular characteristics of a patient. Despite this, chemo- and radiotherapy are still the basis of most standard treatment regimens, especially for gastrointestinal (GI) cancer patients. Importantly, there are significant differences in how GI cancer patients respond to standard-of-care (SOC) chemotherapy (CT) and chemoradiation (CRT), resulting in a majority of patients experiencing either over- or undertreatment and a delay in starting the optimal treatment. Tailored treatment approaches for SOC CT/CRT to enable precision oncology for these standard therapies is of high interest in order to improve quality-of-life and survival of GI cancer patients. With no existing predictive biomarkers for CT/CRT, and genomic profiling falling short on this front, there is therefore a clear unmet medical need for a novel model that can distinguish CT/CRT responders and non-responders in GI cancer patients. Patient-derived tumor organoids (PDOs), a functional precision oncology strategy, are 3D vivo models generated from individual patient tumor tissue and have recently emerged as a promising tool for predicting CT/CRT responses in cancer patients. PDO-guided treatment has not yet been implemented in the clinic, because some limitations need to be overcome first. With this study, we aim to overcome the most important limitations by developing a multiparametric, live-cell imaging-based drug response signature for ex vivo PDO screenings that enables monitoring of the true PDO drug response. We hypothesize that this will drastically improve the predictive value of PDOs and feasibility of using PDO drug screenings in routine clinical practice. To test this and as proof-of-concept we will also perform a multicentric prospective observational cohort study with our novel PDO screening platform for prediction of neoadjuvant CT/CRT response in rectal and esophageal cancer patients in regional hospitals. If successful, we aim to set up a prospective clinical phase-1 trial in the future, and on the long term implement our PDO drug response signature as a tool to help guide clinical decision-making of CT/CRT treatment choices for GI cancer patients.