Longitudinal, label-free, high-resolution imaging of glioblastoma spheroid response to therapy: a translational tool for preclinical evaluation of chemotherapy, radiation, and immunotherapy

Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and poor patient survival, underscoring the need for novel, to our knowledge, therapeutic strategies and improved preclinical models. Patient-derived tumor spheroids (PDTSs) offer a physiologically relevant <i>in vitro</i> platform for evaluating treatments such as chimeric antigen receptor (CAR) T cell therapy, chemotherapy, and radiation. However, significant challenges remain in monitoring the complex three-dimensional (3D) microenvironment of the GBM PDTSs. Current imaging techniques used for this purpose are primarily endpoint analyses which lack critical real-time, non-invasive capabilities that ultimately preclude longitudinal and continuous monitoring. In this study, we introduce quantitative oblique back-illumination microscopy (qOBM) as a label-free and non-invasive imaging approach for longitudinal and continuous, high-resolution monitoring of GBM PDTSs during treatment. qOBM enables real-time visualization of cellular processes, including apoptosis, cell migration, and T cell-mediated cytotoxicity by leveraging tomographic refractive index-based quantitative imaging. We construct a compact qOBM system that fits within common incubators and apply it to study the effects of radiation, chemotherapy, and immunotherapy on three patient-derived GBM cell lines, extracting both static and dynamic image features over a 72 h treatment period. Additionally, we develop machine learning models to predict spheroid viability and cytotoxicity, demonstrating the potential of qOBM to enhance treatment evaluation. Our findings establish qOBM as a powerful tool for longitudinal and continuous spheroid monitoring, offering a non-destructive, high-resolution alternative to conventional endpoint assays and improving the evaluation of preclinical treatments for GBM.