Three-dimensional quantitative phase imaging for the evaluation of glioblastoma targeting CAR T-cell efficacy

Glioblastoma (GBM) is a highly aggressive brain tumor with limited treatment options and poor patient outcomes. While patient-derived spheroids offer a platform for studying GBM treatment, existing imaging modalities are often destructive or limited to endpoint analyses. Here, we present quantitative Oblique Back- illumination Microscopy (qOBM) as a novel, label-free imaging technique for real-time, non-invasive monitoring of GBM spheroids treated with tumor targeting chimeric antigen receptor T-cells (CAR T-cells). qOBM overcomes existing imaging limitations by providing high-resolution, 3D quantitative phase imaging through epi-illumination, enabling longitudinal visualization of cellular and subcellular processes within thick spheroids. Here, we apply qOBM to monitor the responses of GBM spheroids derived from three unique patient cell lines to targeted CAR T-cell immunotherapy and non-targeted controls. With this, we capture changes in the spheroids over 72 hours of treatment, allowing for visualization of processes such as CAR T-cell infiltration, mitosis, and apoptosis. Further, using images from GBM/CAR T cell cocultures, we develop a machine learning model for predicting spheroid cytotoxicity rates. Our findings establish qOBM as a powerful, non-destructive tool for longitudinal GBM spheroid toxicity monitoring and demonstrate its potential to advance personalized cancer therapy by providing real-time insight into treatment efficacy.