3R-Compliant Murine Ex Vivo Platform for Scalable Brain Cancer Modeling and Drug Screening

Abstract Translating in vitro findings into in vivo applications remains a major challenge in brain cancer drug discovery, largely due to inadequate models for assessing neurotoxicity and tumor microenvironment (TME) dynamics. Here, we present a novel, highly modular murine ex vivo platform based on adult neural stem cells (aNSCs), designed for scalable, medium-throughput drug screening and mechanistic studies for brain cancer research. This system integrates progressive 2D and 3D models, including assembled models for tumor migration, cancer core models mimicking tumor localization within neural tissue and advanced cancer core models incorporating immune cells to recapitulate key TME interactions. With high reproducibility, defined cell composition and rapid formation within 72 hours, these assembloids provide a translationally relevant and ethical alternative to animal models. Aligned with 3R principles, this platform bridges the gap between in vitro and in vivo studies, offering a powerful tool for innovating brain cancer drug discovery. Competing Interest Statement The authors declare the following competing interests: A European patent application (Application No. 24 219 517.0) has been filed, covering aspects related to core models as described in the manuscript. The patent application was submitted on December 12, 2024, to the European Patent Office. The applicant for this patent is the Tubingen University Hospital and the inventor designation is pending. The patent application is currently under examination.