Understanding the complex tumor microenvironment of glioblastoma multiforme (GBM) is essential for developing more effective therapies. As the most aggressive primary brain tumor in adults, GBM presents unique challenges primarily in its high cellular heterogeneity and GBM stem cell (GSC) resistance to treatment. Traditional 2D culture models fall short of capturing this complexity, while animal models are limited in translation. In our work, we address this gap by developing glioblastoma organoids (GBOs) that not only grow to a clinically relevant size, over 4 mm diameter, but also exhibit hallmark features of the GBM microenvironment, including transdifferentiation into endothelial cells (ECs). Since our GBOs are fully self-assembled from GSCs without the need of exogenous matrices like Matrigel™ or serum supplements, we are also able to understand the spatial organization of these ECs with respect to self-secreted extracellular matrix (ECM). Once the GBOs surpass the 1 mm, we observe a shift from uniform cell spheres to highly organized structures that include vascular-like networks of ECs supported by fibronectin. This platform opens new doors not only for studying GBM progression and therapeutic response but also for exploring the role of the native extracellular matrix in modulating early tumor growth and vascular development. Ultimately, our approach offers a robust, scalable model that brings us one step closer to clinically relevant in vitro systems for translational medicine.
