Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines

Abstract

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 applied 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 pharmacologic inhibition of myosin II increases invasiveness, whereas 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 showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.

Figure 1. Invasive cancer cells have the highest compliance

A) Invasion assays were performed on the indicated cancer cell lines (Methods) and percent invasion (I) relative to each other presented. Data represent the mean ± SEM of three independent experiments. B) Invasion of primary cancer cells as described in (A). C) Maximum compliance for all the cell lines is presented. The three boxes encompassed by dashed lines indicate the different scored regions based on relative invasion, with cell lines within a box not being statistically significant from each other mechanically (p≥0.05). (Dash dot box (--)- high invasion, I≥0.4, Solid box (-)- medium invasion, 0.2

Figure 2. Stiffness correlates with invasion

Stiffness calculated by fitting a modified Kelvin Voigt model (Figure S1) to compliance curves for ovarian cancer cell lines (A) and primary ovarian cancer cells (B) and mapped with the relative invasion from Figure 1A and 1B. The boxes represent the same scored regions as in Figure 1C and 1D. C) Power law showing the correlation between the stiffness of ovarian cancer cell lines and their invasion. IGROV when treated with blebbistatin (open circle, solid arrow) and Ovca429Neo with Ovca429TβRIII (triangle, dash dot arrow) move on the line, consistent with the correlation. (INSET) Power law on log log plot. D) Power law correlation for the primary ovarian cancer cells. (INSET) power law on a log log plot.

Figure 3. Highly invasive and stiff cancer cells express cortical actin and myosin

Immunoflourescence images of cells stained either for (A) actin using rhodamine conjugated to phalloidin or with an antibody to (B) phosphorylated myosin light chain (pMLC). Quantification of fluorescence intensity using Image J software across the lines shown in the corresponding panels on the left are indicative of stress fiber density in the case of actin or cortical pMLC localization. C) Stiffness of the respective ovarian cancer cell lines.

Figure 4. Myosin II function and TβRIII alter stiffness and invasion

A) Invasion assays of Ovca429-Neo and Ovca429-TβRIII was performed as described in Methods and Figure 1. Data are a composite of two independent experiments performed in duplicate. Each column represents the mean ± SEM. B) Stiffness for the corresponding cell type in (A) obtained as described in Methods and Figure 2. C) Effect of blebbistatin treatment on the invasion of IGROV and Ovca429-TβRIII cell types. D) Stiffness for the corresponding cell type and treatments in (C)(** -p<0.01,*=p<0.05).