High-grade gliomas are aggressive, lethal tumours of the central nervous system with no available cure. Both adult and paediatric high-grade gliomas (HGGs) are characterized by rapid proliferation and diffuse invasion of tumour cells into the surrounding stroma. Additionally, these tumours display a high degree of heterogeneity among patients, as well as within individual patients’ tumours, posing a significant challenge to effective treatment. Recent work has advanced our understanding of molecular variation among adult and paediatric HGGs, providing definitions of tumour subtypes according to genetic alterations and cell state. However, it is unclear how these molecularly-defined profiles align with other factors guiding tumour progression, including invasive behaviour, intracellular mechanical properties, and interactions of glioma cells with the tumour microenvironment. Here, we present an integrative analysis of molecular, mechanical, and environmental properties of distinct subtypes of high-grade glioma. We reveal that patient-derived glioblastoma (GBM) cells display inter- and intra-tumoral mechanical heterogeneity, and that these mechanical properties are associated with altered regulation of cytoskeletal dynamics. We demonstrate robust migration of adult GBM and paediatric diffuse midline glioma (DMG) cells in vivo in zebrafish and mouse models, respectively, including along clinically-relevant perivascular routes. In novel models of DMG, we describe genetic alteration-specific patterns of proliferation and invasion, and demonstrate subtype-specific interactions of tumour cells with the brain endothelium directing proliferation, invasion, and presence of neural stem cell markers. Finally, in each of these settings, we use targeted pharmacological interference to determine biological mechanism and potential therapeutic targets. This dissertation provides an example of multidisciplinary, multi-scale profiling of distinct subtypes of adult and paediatric HGG, and supports new strategies for patient stratification and therapeutic development.