P01.24.B CORE2EDGE: A HUMAN GLIOBLASTOMA ORGANOID AND BRAIN SLICE CO-CULTURE MODEL CAPTURING TUMOR INFILTRATION AND TRANSCRIPTIONAL HETEROGENEITY FROM CORE TO SINGLE-CELL DISPERSION

Abstract BACKGROUND Glioblastomas function as intricate cellular networks that extend into the surrounding brain tissue enabling long distance communication. This malignant connectivity spans from the tumor core to remote infiltration zones, supporting the concept of glioblastoma as a whole-brain disease. With growing ethical considerations in biomedical research and the limitations inherent in cross-species comparisons between animal studies and human glioblastoma models, human tumor platforms remain constrained in their ability to replicate the full infiltration spectrum from the tumor core to single-cell dispersion. Here, we present a three-dimensional, fully human ex-vivo glioblastoma model (,,Core2Edge”) that replicates this extensive infiltration range while preserving the intratumoral heterogeneity of the original tumor. This model involves implanting fluorescently labeled human glioblastoma organoids (GBOs) into organotypic human brain slices, maintaining the genetic integrity and cytoarchitecture of both brain and tumor. MATERIAL AND METHODS The Core2Edge model involves a comprehensive workflow completed over 7-12 days. Key steps include brain slice preparation (~4-6 hours, depending on quantity), one day for initial culture before GBO staining and transplantation, a variable culture period (up to ten days), and fixation with different techniques (~8 hours). Following model preparation, downstream analyses such as imaging, sequencing, and proteomics were performed. RESULTS Multiplex immunofluorescence confirmed cell composition consistent with histological staining. Spatial transcriptomics enabled detailed glioblastoma cell subtyping based on Neftel states. Advanced imaging, combining tissue expansion and light-sheet fluorescence microscopy, facilitated super-resolution, three-dimensional visualization of the GBO-brain slice system. This approach allows for the study of initial infiltration steps, in-depth analysis of the invasive front, exploration of cell-cell interactions between tumor cells and the tumor microenvironment, and offers a platform for drug screening and testing, reducing the need for animal models. CONCLUSION The,,Core2Edge“model represents an innovative ex-vivo platform replicating glioblastoma infiltration and heterogeneity. By preserving genetic integrity and cytoarchitecture, this fully human model bridges critical gaps in glioblastoma research, offering unparalleled insights into tumor behavior and potential therapeutic interventions.