P02.05.B MAPPING MICROGLIAL DIVERSITY AND TUMOR HETEROGENEITY IN PRIMARY GLIOBLASTOMA AND PATIENT-DERIVED ORGANOIDS

Abstract BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, marked by extensive inter- and intratumoral heterogeneity and pronounced therapy resistance. Microglia is a major contributor to this complexity and plays a pivotal role in shaping tumor behavior and modulating responses to therapy. Yet its precise phenotypic and functional states across distinct tumor regions and model systems remain poorly defined. In this study, we employ patient-derived organoids (PDOs) alongside matched primary tissue (PT) to characterize microglial diversity and investigate how microglia contributes to the cellular inter- and intratumoral heterogeneity of GBM. MATERIAL AND METHODS Fresh tumor tissue was obtained from 13 GBM patients and sampled from six spatially distinct regions per tumor. PDOs were generated from these regions and compared to matched PT to evaluate microglial presence, distribution, and morphology. To assess intertumoral heterogeneity, we compared microglial characteristics across patients, while intratumoral heterogeneity was examined by analyzing differences between regions within individual tumors. Subsequently, PDOs were treated with temozolomide (TMZ), and microglia was visualized and quantified using Iba1 immunofluorescence to evaluate treatment-induced morphological changes and shifts in microglial abundance. RESULTS Microglia in PT exhibited a microglia morphology more similar to PDOs than to healthy brain tissue. Distinct microglial distribution patterns emerged across PDOs, with microglia typically arranged in one of four configurations: (1) perinuclear, (2) ring-like, (3) at the PDO margin, or (4) diffusely dispersed throughout the PDO. In 5/13 PDOs we found significantly higher microglial abundance compared to PT (PPDO 4 = 0.0013, PPDO 7 = 0.0424, PPDO 8 = 0.0153, PPDO 10 = 0.0162, PPDO 12 = 0.0083). Analysis across patients revealed significant intertumoral heterogeneity in microglial characteristics (p = 0.0405). In terms of intratumoral heterogeneity, regional differences in microglial abundance and morphology were observed within individual PDOs, with statistically significant variation only found in PDO 10 (p = 0.0211). Following TMZ treatment, 4/13 PDOs showed a significant recline in microglial abundance (PDO 2: p < 0.0001; PDO 4: p = 0.0041; PDO 7: p = 0.0015; PDO 10: p = 0.0297). CONCLUSION Our findings reveal significant inter- and intratumoral microglial heterogeneity in PDOs. Distinct microglial distribution patterns and variable responses to TMZ highlight the complex and patient-specific nature of microglial involvement in the GBM microenvironment. These results underscore the relevance of PDOs as a model system for studying tumor-immune interactions and therapeutic responses in a personalized context.