P01.08.B CAPTURING FUNCTIONAL INTRATUMORAL HETEROGENEITY IN GLIOBLASTOMA: MAPPING REGIONAL METHYLOME AND TEMOZOLOMIDE RESPONSE USING PATIENT-DERIVED ORGANOIDS

Abstract BACKGROUND Glioblastoma (GBM) is the most common malignant primary brain tumor in adults and characterized by marked inter- and intratumoral heterogeneity, contributing to poor therapeutic response and high recurrence rates. In this study, we specifically investigate regional differences in methylation profile and temozolomide (TMZ) sensitivity using spatially derived primary tumor tissue (PT) and corresponding patient-derived organoids (PDOs). By comparing epigenetic patterns and drug response across multiple regions, we aim to better understand the functional implications of intratumoral variation in GBM. MATERIAL AND METHODS Tumor specimens from 15 GBM-patients were collected across six spatially distinct regions during surgical resection to capture intratumoral heterogeneity. PDOs were generated and matched PTs and PDOs used for their characterization by methylation profiling. Subsequently, PDOs were treated with 3.3 mM TMZ for 7 days, and cell viability was assessed on day 8 using the alamarBlue assay. RESULTS We observed intertumoral differences in the methylome between patients, as well as intratumoral heterogeneity across distinct regions within individual PTs and PDOs. When assessing the response to TMZ in PDOs, we identified significant intertumoral variability in drug sensitivity (p < 0.0001). In addition, marked intratumoral differences in TMZ response were observed between spatially separated tumor regions within individual patients (p < 0.0001). CONCLUSION Our study reveals a striking degree of intratumoral heterogeneity in both the methylome and TMZ response in GBM. Spatially derived PDOs faithfully captured these regional differences and preserved functional diversity. These findings underscore the importance of multi-regional sampling, as single-biopsy approaches risk overlooking clinically relevant subpopulations. This platform offers a powerful tool for dissecting tumor complexity and adopting individualized therapeutic strategies.