103 A Medulloblastoma brain slice culture radiotherapy platform to dually assess therapeutic efficacy and toxic
Abstract
Medulloblastoma is the most common malignant childhood brain tumour. Radiotherapy (RT) forms a key treatment pillar but is associated with significant late effects. Ex vivo brain slice cultures present a translational model that overcomes limitations of existing in vitro and in vivo alternatives. Slice cultures provide a native “scaffold”, with representative cellular architecture and extracellular matrix (ECM), upon which cancer cell growth and invasion can be tracked in real-time. We describe an organotypic brain slice culture (OSC) model for Group3 medulloblastoma (MBGrp3), amenable to long-term culture and irradiation. MBGrp3 spheroids (g-MYC-tdTomato) were cultured to an optimised diameter of 250-350µm in ultra-low-attachment plates. Murine cerebellar OSCs were prepared using a vibratome and cultured on transwell inserts. Co-cultures were established at 72-hours, and RT delivered 48 hours later using a Small-Animal-Radiation-Research-Platform (SARRP). 1, 3 or 5 daily fractions (1.8Gy) or a single cumulative dose (9Gy) was delivered. Tissue toxicity was assessed through live/dead staining and ToxiLight® assay. Growth and invasion was quantified in real-time (live multi-photon-microscopy) and spheroid volume/invasion analysed (Huygen’s). OSCs were fixed at endpoint for confocal microscopy to assess DNA damage (γH2AX) and cellular architecture. Control OSCs maintained viability long-term (21-days) with preserved cellular architecture and ECM. Live/dead staining revealed dose-dependent tissue toxicity following RT, with 5 fractions or a single cumulative dose significantly reducing viability (p < 0.05). DNA damage accumulated over time in culture and was dose-dependent. Our multi-photon pipeline provided repeat live volumetric quantification of spheroid growth. Fractionated RT of OSCs is feasible, demonstrating dose-dependent DNA damage. This model serves as a basis for future studies to test radio-sensitising agents wherein cancer cell growth and invasion can be modelled in tandem with tissue toxicity.