Cancer progresses when individual cancer cells invade through physical tissue barriers in the extracellular matrix surrounding the tumor microenvironment and subsequently spread to different niches in the body.  This process requires a complex synergy of traction force generation against the ECM, mechanosensitive feedback, and subsequent cytoskeletal rearrangement.  Two distinct modes of cancer cell invasion, known as mesenchymal and amoeboid invasion, have been identified, and the structure and composition of the ECM can dictate which route cancer cells utilize.  This plasticity of invasion mode is a clinical challenge as it can limit the effectiveness of targeted chemotherapy.  To investigate this transition, microchannels with widths between 3 and 10 μm and lengths over 150 μm were fabricated, necessitating three-dimensional cell-ECM interactions, self-imposed cellular confinement in two axes, and complete movement of the cell into the channel for permeation.  Cancer cell lines from three different tissue origins were observed interacting with the channels.  Altogether, five different cell lines were found to successfully permeate through the narrow 3 μm channels.  In four of these lines, migration was faster in the narrow 3 μm channels than in the wide 10 μm channels.  Furthermore, cells navigating narrow channels exhibited blebs when exiting and had smooth leading edge profiles, suggesting an ECM-induced transition from mesenchymal invasion to amoeboid invasion.  Consistent with this, a reduction in focal adhesion protein localization was observed in narrow channels, suggesting that confined migration is adhesion independent.  ECM protein coating-dependent changes in cell permeation speed were also observed.  Cells were also found to permeate narrow channels even in the absence of cell-binding ECM proteins. Chemical inhibition of the Rho/ROCK (amoeboid) and Rac (mesenchymal) pathways revealed that amoeboid invasion through confined environments may rely on both Rho and Rac in a time- and ECM-dependent manner. SiR-Actin live cell labeling was used to reveal distinct patterns of cytoskeletal organization inside wide and narrow channels, and a mechanosensing period was identified in which the cell determines the channel to be too narrow for mesenchymal-based migration, reorganizes its cytoskeleton, and proceeds using an amoeboid phenotype.  These results suggest that cancer cell invasion through the ECM is dependent on the physical mechanics of the matrix, and provides a platform for future testing of clinical strategies against cancer invasion.