Characterization of Native Extracellular Matrix of Patient-Derived Glioblastoma Multiforme Organoids

Model systems play a crucial role in biological and biomedical research, especially in the search for new treatments for challenging diseases such as glioblastoma multiforme (GBM). Organoids are 3D in vitro multicellular "middle-ground" model systems that recapitulate highly organized and heterogeneous in vivo organ-like systems, often through stem cell differentiation. Incorporating Matrigel™ or other exogenous extracellular matrices (ECMs) that do not naturally occur in the human body is common practice for organoid generation, ignoring the role of dynamic reciprocity between the cells and the ECM in tissue development. In this study, we describe a method to develop GBM organoids (GBOs) from cells without the need for exogenous ECM encapsulation and without cell culture media changes to produce stable tissue-like organoids that reach a 4 mm diameter in as little as 6 weeks. We observed a transition from homogenous cell populations to tissue-like structures when GBOs were larger than 1 mm in diameter. Transcriptomic analysis revealed that the greatest gene expression changes occurred when GBOs were 2 mm in diameter, with collagen VI as the most upregulated ECM-related gene. Quantitative and histochemical assessments further supported native ECM synthesis with significantly higher levels of glycosaminoglycans and collagen in GBOs compared with spheroids. To our knowledge, this study presents the first reproducibly large GBOs with natively produced ECMs. Organoids with natively synthesized ECMs promise to eliminate artifacts and variability from aged, homogeneic, or xenogeneic scaffolds and to provide insights for ECM-targeted drug development. Impact Statement Glioblastoma multiforme (GBM) is the most common and deadly brain tumor due to its complex tissue heterogeneity. Drug development for GBM is difficult because GBM models are not very translatable and are limited, leading to the need of GBM organoids (GBOs). Current GBO development is highly laborious and of questionable relevance because of the reliance on non-native, animal-derived extracellular matrix (ECM). This study describes a scalable and reproducible method of developing GBOs with natively generated ECMs. These GBOs allow for both the study of the early stages of GBM that are currently inaccessible and a quicker and more translatable tool for GBM drug screening and development.