Real-time monitoring of pediatric high-grade glioma invasion using organotypic brain cultures reveals a developmentally dependent sensitivity

High-grade gliomas (HGGs) constitute a considerable portion of pediatric brain tumors, typically arising in the supratentorial cortex (median age, 13 years) or infratentorial/midline regions (median age, 7 years). About half are diffuse midline gliomas (DMGs), whose aggressive invasiveness contributes significantly to their lethality. Monitoring tumor behavior in its native environment, especially invasion, remains challenging. Although mouse models are used in glioma research, pediatric HGGs present unique difficulties. Multiphoton microscopy allows cortical invasion studies, but DMG analysis is hindered by deep anatomical locations and rapid mouse aging, which limits relevance to developmental stages. To overcome these barriers, we established a novel platform combining direct implantation of pontine DMG and cortical HGG cells into organotypic ex vivo brain slice cultures with spinning-disc confocal time-lapse imaging. This method enables accelerated, real-time monitoring of tumor invasion across multiple slice cultures under conditions that recapitulate some of the key features of the native microenvironment. Using this system, we demonstrated that the developmental stage of brain tissue strongly influences pontine DMG invasion and validated the platform’s utility for evaluating anti-invasive therapies.