Establishment of a Drug Screening Platform Using Patient-Derived Organoids from Paediatric Brain Tumours

Abstract

Paediatric brain tumours (PBTs) are the leading cause of non-accidental death in children. Their biological and molecular heterogeneity, combined with the lack of experimental models capable of rapidly provide evidences of drug sensitivity, are major challenges for the development of new therapeutic approaches for these children. Patient-derived organoids (PDOs) have been recognized as powerful preclinical models, as they preserve the morphological, genetic, and phenotypic characteristics of the primary tumour while faithfully recapitulating intratumoral heterogeneity. Therefore, our main aim was to establish a drug screening platform using PDOs derived from fresh PBT surgical samples. To this end, we cultured spheroids derived from immortalised human astrocytes (NHA) and patient-derived glioblastoma (BIT14) cells, to optimise culture conditions, identify the most suitable readouts, and define the key parameters for drug screening. These optimised conditions were then applied to PDOs using a compound library (Prestwick Chemical Library) and additional clinically approved compounds. As a result, we developed a reproducible protocol based on enzymatic dissociation of PDOs with TrypLETM or AccutaseTM, followed by homogeneous seeding of 2000 cells per well in U-bottom plates, enabling consistent PDO re-formation prior to testing. Among the different viability assays evaluated, the CellTiter-Glo® 3D assay proved to be the most reliable method for assessing drug responses. Benzethonium chloride was defined as the positive control, and Z' score analysis confirmed assay robustness. Importantly, this optimised workflow was validated in PDOs derived from two ependymomas and one WNT-activated medulloblastoma, using clinically relevant drugs administered to the corresponding patients. In summary, we successfully established and validated a robust and scalable drug screening protocol using PDOs from PBT samples. This platform enables the assessment of patient-specific drug sensitivities within clinically relevant timeframes, representing an important step towards integrating PDO assays into preclinical research and advancing personalised treatment strategies for children with PBTs.