Glioblastoma is the deadliest and most common form of primary brain tumour. Its aggressive and infiltrative spread is facilitated by microstructures in the brain such as neuronal axons and small blood vessels, which act as tracts to guide and enhance widespread migration/invasion. Unfortunately, such structures are rarely incorporated into pre-clinical in vitro models investigating mechanisms of cellular movement and drug responses. This thesis aims to evaluate the suitability of existing in vitro models whilst also proposing innovative approaches to create new models using principles of tissue engineering, including the use of hydrogel scaffolds and 3D printing/bioprinting. Results demonstrated that the choice of research model significantly influenced cell drug responses and gene silencing outcomes. For example, spheroid-based migration assays using aligned nanofibers revealed an increase in cell migration when exposed to a cocktail of anti-migratory drugs (Rhosin Hydrochloride and CCG-1423), unlike the scratch and spheroid-collagen hydrogel assay where significant reductions in migration/invasion were observed. Furthermore, significant increases in migration/invasion were observed using aligned nanofibers following knockdown of the Rho GTPase activating protein ARHGAP29, whereas no significant differences were observed when using other assays. A focus on cell morphology in the different assays under different drug/genetic conditions also reiterated the importance of the cellular environment on governing and regulating actin dynamics and cell shape via multiple Rho-related signalling pathways, which play a major role in regulating migration/invasion events. Two novel approaches were then developed to mimic brain-like microstructures in vitro, with a focus on incorporating important microstructures, maintain high viability, and mimicking key mechanical properties of natural brain tissue. Both methods used collagen (type I) for the tracts, chosen primarily for printability, cell-friendly crosslinking mechanisms and cell adhesion. Other materials were also explored to act as the non-cell adhesive portion of the model, such as agarose, sodium alginate and low acyl gellan gum, which were each closely evaluated for their respective suitability in the proposed models. The first method included 3D printing custom moulds for hydrogel casting, including using segments of plastic filaments to create subtractive channels for subsequent spheroid and collagen inclusion. The second approach instead employed 3D bioprinting for greater precision and flexibility when creating microstructural collagen-based tracts. Among the materials explored for 3D bioprinting, gellan gum outperformed alginate due to its reduced dependence on extracellular cations, minimising cytotoxicity, as well as its ability to mimic viscoelastic properties of natural brain tissue by carefully adjusting crosslinking mechanisms. Overall, these findings contribute to the development of more physiologically relevant in vitro models of GBM invasion, offering improved platforms for studying disease mechanisms and testing novel and personalised anti-cancer therapies.
| Date of Award | 6 Jun 2025 |
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| Original language | English |
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| Sponsors | Engineering and Physical Sciences Research Council |
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| Supervisor | Alan Smith (Main Supervisor) & Barbara Conway (Co-Supervisor) |
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