Bioengineering of Heterogenous Glioblastoma Multiforme Microenvironment
Glioblastoma (GBM) is the most aggressive brain tumor that originates from glioblastoma stem cells (GSCs). In the brain, GSCs are supported by a tumor microenvironment (TME) wherein the perivascular niche and hypoxic region are present. The glioblastoma microenvironment (GBME) exhibits high heterogeneity, vast cell-to-cell interactions, and stiff mechanical properties. To produce in vitro models mimicking the GBME features, GBM organoid (GBO) models have been developed. Conventional organoid studies rely on growing them in serum-free media, resulting in sphere formation. However, this conventional method is not scalable and often fails in recapitulating inter- and intratumor heterogeneity. Also, the conventional method is not ideal to produce adequate quantities of GBOs to screen drugs for personalized medicine. Therefore, development of a reproducible and scalable GBO culture method can provide a better platform to simulate novel treatments.First, the bioreactor design was optimized by using different diameters of impellers and bioreactor vessels. Even with similar shear stresses, cell proliferation was inhibited or promoted depending on the ratio of the impeller diameters to the vessel diameters. With the optimized vessel geometry, shear stress and media supplements were optimized for GBO production. The bioreactor GBOs (bGBOs) were produced in uniform size, not by clonal aggregations, but by cell proliferation. With the optimal agitation rate, bGBOs displayed upregulation of genes involved in stemness, hypoxia, angiogenesis, proliferation, and migration. The statistical analysis revealed the synergetic effects of the high agitation rate and the size of the bGBOs. Next, bGBO models were characterized by their morphologies and transcriptional and translational profiles. The bGBOs exhibited high and strong cell-to-cell contact. Multivariate gene analysis found a significant correlation between gene expression and the size of the bGBOs. GBME was established and spatially organized in bGBOs greater than 800 Âµm in diameter. Hypoxic TME was developed in bGBOs greater than 400 Âµm in diameter. Inside the bGBOs, spatially separated features of the hypoxic niche and the perivascular niche were demonstrated. Also, the large bGBOs displayed angiogenesis features. Self-established GBME was organized by transdifferentiated GBM into endothelial cells, pericytes, and astrocytes. GBME containing necrotic regions displayed more spatially distinctive and hierarchically organized GSC niches. The GSCs in the niche were regulated by transcription factors involved in dedifferentiation. Hydrogels have been employed to further understand underlying mechanisms of the transformation of GBM in the bGBO model. Mechanical properties of GBME was engineered using hyaluronic acid (HA)-based hydrogels. Cell behavior in response to the hydrogel stiffness was examined. Transcription factors dedifferentiating GBM into GSCs were translocated to the nucleus in response to stiffer substrates. Collectively, these studies provided a small-scale model for a high-throughput production of GBOs that recapitulate in vivo GBM features, including high heterogeneity and high cell-to-cell interactions. Hydrogel model and the bioreactor culture conditions described here suggest that GBOs can be biomanufactured by modulating mechanical stress.