Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here we apply a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacological inhibition of myosin II increases invasiveness, while increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By demonstrating that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.

Loss of expression of the TGF-␤ superfamily coreceptor, the type III TGF-␤ receptor (T␤RIII or betaglycan), occurs in a broad spectrum of human cancers including breast, lung, ovarian, pancreatic, prostate, and renal cell cancer. T␤RIII suppresses cancer progression in vivo, at least in part, by reducing cancer cell motility. However, the mechanism by which T␤RIII regulates migration is unknown. Here, we demonstrate an unexpected TGF-␤ signaling independent role for T␤RIII in activating Cdc42, altering the actin cytoskeleton and reducing directional persistence to inhibit random migration of both cancer and normal epithelial cells. Functionally, T␤RIII through its interaction with the scaffolding protein ␤-arrestin2, activates Cdc42 and inhibits migration. These studies identify a TGF-␤ independent homeostatic function for T␤RIII in regulating cell migration.C ell migration is a complex process required for physiological functions including embryonic development and wound healing. Alterations in cell migration are also critical to disease pathogenesis, including inflammatory and vascular diseases, tumor cell invasion, and metastases (1, 2). The ability of cells to migrate is regulated both by the speed and the directionality of migration that can be triggered by external cues (i.e., chemotaxis) or because of the intrinsic property of cells to migrate (i.e., intrinsic persistence) that are in turn regulated by the Rho family of GTPases, integrins, the actin cytoskelton, and microtubules (3, 4). Several migratory processes in development and tissue remodeling occur without any evidence of extrinsic chemotactic signaling, relying instead on intrinsic cell migratory properties (5).The type III TGF-␤ receptor (T␤RIII/betaglycan), an 849 aa heparan sulfate proteoglycan, is the most abundant and ubiquitously expressed TGF-␤ superfamily coreceptor. T␤RIII has the potential to increase or decrease TGF-␤ signaling through mechanisms yet to be fully defined (6-8). T␤RIII is classically thought to function as a coreceptor, presenting TGF-␤ superfamily ligands to their respective signaling receptors (8). Recent studies have suggested essential, nonredundant roles for T␤RIII in regulating signaling through T␤RII and T␤RI as well as independently of T␤RII and T␤RI (9). T␤RIII null embryos die on embryonic day 13.5, exhibiting hepatic and cardiovascular defects (10). An essential role for T␤RIII has also been demonstrated in mesenchymal transformation during chick embryonic heart development (11, 12) and in mediating TGF-␤ resistance in intestinal goblet cells (13). We have defined essential roles for the cytoplasmic domain of T␤RIII in mediating TGF-␤ signaling independent of the ligand presentation role (14), along with regulating cell-surface levels of T␤RIII and T␤RII through interactions with G␣-interacting protein-interacting protein, C terminus (GIPC) (15) and ␤-arrestin2 (16).Loss of T␤RIII expression has been reported in multiple cancers, with loss of expression correlating with disease progression, advanced stage, or ...