Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface. We extracted an average tension of T ∼ 20.25 nN/μm, which agrees with macroscopic experimental methods. We then measured cortical mechanical properties in nonadherent human foreskin fibroblasts and THP-1 human monocytes before and after pharmacological perturbations of actomyosin activity. Our results show that myosin II activity and actin polymerization increase cortex tension and intracellular pressure, whereas branched actin networks decreased them. Interestingly, myosin II activity stiffens the cortex and branched actin networks soften it, but actin polymerization has no effect on cortex stiffness. Our method is capable of detecting changes in cell mechanical properties in response to perturbations of the cytoskeleton, allowing characterization with physically relevant parameters. Altogether, this simple method should be of broad application for deciphering the molecular regulation of cell cortical mechanical properties.

Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. Cells confined in a non-adhesive environment migrate in the direction of a very large ‘leader bleb.’ Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation. Eps8 capping and bundling activities act antagonistically to organize actin within leader blebs, and Erk mediates this effect. An Erk biosensor reveals concentrated kinase activity within leader blebs. Bleb contents are trapped by the narrow neck that separates the leader bleb from the cell body. Thus, Erk activity promotes actin bundling by Eps8 to enhance cortex tension and drive the bleb-based migration of cancer cells under non-adhesive confinement.