Real-Time Tracking of Tumor Invasion Dynamics in Organotypic Brain Slices at Subcellular Resolution

Replicating the cytoarchitecture and cell heterogeneity of the brain in vitro remains challenging. Although ex vivo organotypic slices preserve native tissue complexity, current culturing methods limit long-term, high-resolution imaging and restrict temporally controlled perturbations. Here, we present PHIROS, a microfluidic platform for high-resolution imaging of organotypic slices that supports static culturing of excised tissue at the air-liquid interface and provides continuous optical access for extended imaging at subcellular resolution. Controlled perfusion with oxygenated medium preserves tissue viability over several days, enabling, e.g., the monitoring of spontaneous and pharmacologically modulated astrocytic calcium activity. Using PHIROS we characterized medulloblastoma (MB) cell behavior in a physiological tumor microenvironment and observed dynamic F-actin-driven interactions with tissue-resident astrocytes, as well as leading edge localization of the immune-checkpoint marker B7-H3 in invading tumor cells. Quantitative assessment of mitochondria transfer across heterotypic actin-rich connections evidences the potential of PHIROS as a versatile system for mechanistic studies in a tissue context, enabling controlled compound exposure and high-resolution imaging in physiologically relevant tissue settings.