Abstract 7530: Comparative analysis of therapy responses and stem cell marker expression in glioblastoma neurospheres verus monolayer cultures using imaging, cytotoxicity assays, and flow cytometry

Abstract Background: Glioblastoma (GBM) is an aggressive and lethal primary brain tumor that is resistant to conventional therapies. Despite advances in treatment strategies, the standard of care and median patient survival have remained largely unchanged for nearly two decades. One major obstacle to therapeutic progress is the complexity of the GBM tumor microenvironment, which is poorly recapitulated by most GBM cell lines when used in animal models and in vitro screening assays. Three-dimensional (3D) neurosphere cultures of GBM cell lines are thought to improve model performance over traditional two-dimensional (2D) monolayer systems by enriching for glioblastoma stem-like cells (GSC), which, when implanted orthotopically, produce tumors that more closely resemble human disease. Consequently, in vitro drug screens using neurospheres more accurately reflect the spatial architecture, cell-cell interactions, and diffusion gradients improving predictive values of these types of studies. Here, we sought to further characterize the phenotype of U-87 MG-Luc2 cells grown in 2D or as neurospheres and to assess their drug sensitivity in vitro and as xenografts. Methods: U-87 MG-Luc2 cells grown in 2D or as neurospheres were treated with temozolomide, lomustine, and bortezomib, individually and in combination, to assess drug sensitivity in vitro. In addition, these cells were stained for the GSC markers CD133, CD15, CD49f, and CD44 and analyzed by flow cytometry before and after intracranial implant. Orthotopic cell growth was tracked by IVIS imaging. Results: Cytotoxicity assays revealed that neurospheres were significantly less sensitive to treatment compared to monolayers, consistent with enhanced resistance mechanisms supported by 3D architecture and GSC. Flow cytometry indicated differences in cell size, granularity, and GSC markers between the two models. Importantly, both culture types successfully engrafted and proliferated as murine orthotopic xenografts. Conclusion: These findings suggest that 3D neurosphere models more accurately reflect the in vivo tumor environment and drug response, while 2D monolayers remain a valuable tool for initial therapeutic screening. Neurosphere culture conditions impacted U-87 MG-Luc2 GSC surface marker expression. Tracking changes in frequency and expression of GSC markers after drug treatment may be useful for selecting promising drug candidates. Head-to-head comparisons underscore the importance of model selection in preclinical GBM research. Citation Format: Daniel S. Costa, Joseph P. Kolb, Olivia Mankos, Karsten E. Fynboe, Stephanie M. Fogerson, William D. Culp, Kathryn R. Meshaw, . Comparative analysis of therapy responses and stem cell marker expression in glioblastoma neurospheres verus monolayer cultures using imaging, cytotoxicity assays, and flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7530.