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The ability to diffusely infiltrate as single cells through the highly confined extracellular spaces of the brain parenchyma and along blood vessels is a pathological hallmark of Glioblastoma Multiforme (GBM), which makes these tumors surgically incurable. The fact that GBM cells are capable of moving through diverse brain microenvironments raises the possibility that GBM cells may possess multiple mechanisms for invasion. At present, little is known about how GBM cells gain traction and generate sufficient contractile forces to overcome the mechanical challenge of migrating through the confined interstitial spaces of the brain. The purpose of this study is to gain cellular, biochemical and biophysical insights into GBM migration. To accomplish this we generated 1-D micro-patterned arrays that approximate the topography of brain microvasculature and microfluidic devices that approximate brain interstitial spaces. High-resolution 3D time lapse imaging is then used to track actin, myosin-II and alpha-tubulin dynamics during cell migration. We found that the geometry of micro-patterned laminin lanes in the absence of chemotactic, hapatotactic or durotactic gradients, is sufficient to trigger sustained (integrin) adhesion-dependent migration that is driven by actin polymerization at the leading edge and supported by acto-myosin-II cable-like structures that extend along the ventral and lateral edges of spindle shaped cells. A similar pattern of adhesion-dependent (mesenchymal) migration was seen in 3D channels with aspect ratio (15x15um) larger than the cell. In confined (5x5um) channels, GBM cells adopted interchangeable modes of migration, waxing between blebbing and mesenchyal migration (approximately 50:50). Blebbing migration was characterized by acto-myosin-II cable-like structures that extended the length of the cell along both ventral and dorsal surfaces. In contrast, mesenchymal phase migration was characterized by a cytoskeleton organization pattern that resembled 1-D migration. Exposure of cells in confined channels to actin inhibitors (latrunculin B and CK-666) caused the cells to adopt a blebbing predominant mode of migration, while myosin-II inhibitor (blebbistatin) induced a mesenchymal predominant pattern. While neither class of inhibitors blocked migration as a single agent, the combination produced cell arrest. GBM cells were also able to migrate through confined channels in the absence of any ECM coating by adopting a blebbing exclusive pattern of migration. This observation raises the possibility that in the absence of integrin signaling, GBM cells can use hydrostatic pressure to gain traction in confined spaces. Taken together these results suggest that GBM can interchangeably adopt adhesion dependent and independent modes of migration as they encounter different sized ECM topography and variable 3-D confinement. Citation Format: James O. Nyagilo, Sara Picirrillo, Yuxiao Sun, Loan T. Bui, Nanda Regmi, Jamie L. Wright, Donald Thevalingam, Armin Soltan Zadi, Young-tae Kim, Charles Choung, Digant P. Dave, Robert Bachoo. Extracellular matrix geometry and 3D spatial confinement trigger diverse mechanisms of primary human glioblastoma cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A54.