Investigating the mechanistic influence of the tumor microenvironment on cancer cell migration and membrane blebbing is crucial in the understanding and eventual arrest of cancer metastasis. In this study, we investigate the effect of suspended and aligned nanofibers on the glioma cytoskeleton, cell shape, migration and plasma membrane blebbing dynamics using a non-electrospinning fiber-manufacturing platform. Cells attached in repeatable shapes of spindle on single fibers, rectangular on two parallel fibers and polygonal on intersecting fibers. Structural stiffness (N m(-1)) of aligned and suspended nanofibers (average diameter: 400 nm, length: 4, 6, and 10 mm) was found to significantly alter the migration speed with higher migration on lower stiffness fibers. For cells attached to fibers and exhibiting blebbing, an increase in cellular spread area resulted in both reduced bleb count and bleb size with an overall increase in cell migration speed. Blebs no longer appeared past a critical cellular spread area of approximately 1400 mu m(2). Our results highlighting the influence of the mechanistic environment on the invasion dynamics of glioma cells add to the understanding of how biophysical components influence glioma cell migration and blebbing dynamics.