<p> Durotaxis is a mechanism of directed cell migration in which cells respond to gradients of extracellular stiffness. While durotaxis has been predominantly characterized in a subset of mesenchymal cells, the potential for cancer cells to durotax has not been well defined. Numerous studies have demonstrated the role of diffusible factors in cancer cell migration and metastasis. However, given the gradual stiffening of many tumor microenvironments, we hypothesized that a durotactic mechanism might also contribute to the migration of cancer cells. We evaluated the durotactic potential of multiple cancer cell lines by employing a stiffness gradient that mirrors the physiological stiffness encountered by cells throughout a variety of tissues. Customized MATLAB software permitted rapid acquisition of positional data for migrating cells. The automation of cell tracking allowed for large sample sizes and therefore a more robust statistical analysis than previously used to evaluate durotaxis. Durotaxis assays identified two glioblastoma lines, a metastatic breast cancer line, and fibrosarcoma line that are responsive to changes in extracellular stiffness. Of interest was our finding that cancer cells showed strong durotactic behavior when occupying the softest region of the stiffness gradient with decreasing responsiveness as cells occupied increasingly stiff regions of the gradient. These observations suggest that durotaxis is influenced by the stiffness of a cell&rsquo;s local environment, with soft substrates increasing durotaxis efficiency in the cancer cell lines evaluated. Furthermore, we determined that PI3K inhibition was sufficient to inhibit glioblastoma chemotaxis but not durotaxis, suggesting that alternative signaling is used to respond to durotactic directional cues. Lastly, we evaluated the protrusion and retraction dynamics of cells on a stiffness gradient and have identified a unique mode of discontinuous migration exhibited by these cells. Based on our observations, we developed a durotaxis model to suggest how discontinuously migrating cells can respond to a gradient by moving towards regions of increasing stiffness.</p><p>