Abstract 3991: A neural organoid glioblastoma model to assess tumor microenvironment interactions and tumor associated microglia and macrophage responses

Abstract Glioblastoma (GBM) is a treatment resistant brain tumor with a median survival post-diagnosis of only 18-20 months. One significant limitation in the development of effective therapeutics for GBM is the lack of suitable pre-clinical models that accurately replicate human GBM and the surrounding tumor microenvironment (TME) cell interactions. Tumor Associated Microglia and Myeloid-derived Macrophages, collectively TAMs, are of particular interest due to their essential role in tumor immunosuppression. We have adapted Stem Pharm’s neural organoid technology by incorporating GBM cell lines and PDX models to develop a GBM organoid model which enables interrogation of these complex, multicellular interactions within a 3D tumor organoid. Stem Pharm’s cerebral organoids contain neurons, astrocytes, endothelial cells, and microglia and leverage our proprietary synthetic hydrogel matrix to form highly reproducible organoids in a 96 well plate format. We have previously applied these organoids as a general model of neuroinflammation and developmental neurotoxicity. Microglia integrated into Stem Pharm’s neural organoids exhibit appropriate gene signatures, ramified morphology, tile, and are uniformly distributed throughout the organoid. Here we demonstrate that GBM cells integrate into and proliferate within the neural organoids and activate tumor associated microglia to an immunosuppressive phenotype. Using single cell RNA Sequencing (10X Genomics) we have characterized microglia gene expression in our model in the presence or absence of integrated GBM cells. We find microglia form distinct clusters based on the presence or absence of GBM cells in the organoids. Differentially expressed genes (DEGs) of microglia between GBM and control organoids were used for Gene Set Enrichment Analysis (GSEA). Gene sets related to migration, response to growth factors, and oxygen levels were upregulated in microglia exposed to GBM cells while sets related to inflammatory responses, cytokine production, and antigen presentation were downregulated, consistent with an anti-inflammatory, tumor-supportive microglia phenotype found within glioblastoma. This model will be useful to explore cellular interactions and modulation of the immunosuppressive tumor microenvironment of GBM in pursuit of improved therapeutics. Citation Format: Connie S. Lebakken, William Richards, Sophia Clark, Kailyn Parham, Lei Zhao, Jack Shireman, Mahua Dey. A neural organoid glioblastoma model to assess tumor microenvironment interactions and tumor associated microglia and macrophage responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3991.