The development of the 3D micropatterned ECM (extracellular matrix) models of glioma-astrocyte co-culture: Investigating glioma migration and astrocyte reactivity

Glioblastoma multiforme (GBM) is the most fatal type of primary malignant brain tumors, characterized by aggressive infiltration into brain tissue. GBM migration is a complex process affected by several factors, including the bilateral communication between the GBM tumor and its microenvironment (TME). Astrocytes, the star-shaped stromal components of the GBM TME, account for half of the brain cells; however, their exact role within GBM invasiveness remains not fully understood. The utilization of scaffold-based 3D cell culture platforms has emerged as a promising strategy to mimic the complexity of the GBM TME. Among them, hydrogels with tunable biochemical and mechanical properties are ideal for replicating the low-stiffness and well-hydrated extracellular matrix (ECM) of the brain. The photocrosslinkable, semi-synthetic hydrogel, gelatin methacrylate (GelMA) offers advantages of cell-responsiveness and biodegradability, simultaneously providing a fine level of control over its mechanical properties, including matrix stiffness and porosity. In this thesis, it was aimed to establish an in vitro 3D co-culture model of the GBM TME. To accomplish this goal, a two-step photolithography technique with photomasks was employed to surround high-stiffness U87 GBM cells encapsulated in GelMA microspheres, resembling glioma tumors, by lower-stiffness GelMA matrices, mimicking the healthy brain parenchyma. This technique facilitated the establishment of two distinct astrocyte-glioma co-culture configurations, demonstrating the versatility of the proposed microfabrication technique in precisely localizing various cell types. The quantitative analysis of time-lapse confocal microscopy images revealed the differential impact of the immortalized normal human astrocyte (I-NHA) co-culture configuration on GBM migration. Encapsulating I-NHA cells in vicinity to U87 GBM cells resulted in extensive invasion of the surrounding GelMA matrix by GBM cells. In contrast, surrounding U87 GBM microspheres with an astrocyte-dispersed matrix led to the establishment of the astrocytic network, which inhibited the tumor cell migration. To further validate the physiological relevance of the microfabricated platform, we performed the immunofluorescent staining and qPCR analysis of proteins and genes related to GBM migration and malignancy as well as astrocyte reactivity. The immunocytochemical staining confirmed the upregulation of GFAP expression by interconnected astrocytic networks around U87 microspheres, suggesting the reactive transformation of astrocytes and the formation of the glial scar in the presence of GBM cells. Additionally, culturing U87 cells in the 3D environment of GelMA microspheres resulted in the upregulation of genes linked to GBM invasiveness, including MMP family proteases (MMP-2, MMP-9, MMP-14), hypoxia-related factors (VEGF, HIF-1α), and epithelial-to-mesenchymal transition (EMT) markers (CD44, FN1, TGF-β). In conclusion, this thesis presents a 3D micropatterned co-culture model of the GBM TME, offering a novel biomimetic in vitro tool for advancing our understanding of GBM migration and the intricate dynamics within the GBM TME.