Cytoskeletal stiffness, friction, and fluidity of cancer cell lines with different metastatic potential

Abstract

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g') and internal friction (g″) were measured over a wide frequency range. The dependencies of g' and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x = 1) to fluid-like (x = 2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.

Fig. 1

Histogram of the logarithm of bead displacement amplitude d₀ for 253J cells at 0.1 Hz. The histogram is truncated at low displacements because beads whose amplitude is below the level of system noise were excluded from analysis. Using the mean and standard deviation computed from the truncated distribution produces normal curves that are shifted to higher displacement values (dashed line). Fitting the normal curve to the truncated distribution produced lower error between data and normal curve (solid line) measured by the sum of the squared differences at each bin midpoint

Fig. 2

Glass-adherent a PC3M and b PC3MLN4 cells with attached RGD-coated magnetic beads (black circles) for OMTC measurements. Beads came into sharp focus above the apical cell surface indicating that the beads were sitting on top of the cells

Fig. 3

Bead displacement amplitude d₀ at the low frequency f = 0.1 Hz for all cell lines tested. Mean bead displacement was near 100 nm (dashed line)

Fig. 4

Mean and standard deviation of cytoskeletal stiffness g′ and friction g″ of a 253J (black symbols) and 253JB5 (white symbols) cells, b MCF10-V (black), MCF10A-ErbB (white), MCF10A-ζ (light grey), and MCF10A-ErbB2-ζ (dark grey), c 253J (black) and 253JB5 (white) cells, d PC3M (black) and PC3MLN4 (white) cells, e A375P (black) and A375SM (white) cells, f SN12C (black) and SN12PM6 (white) cells. Stiffness increased as a power law of the oscillatory frequency f as seen by the nearly linear dependence of g′ on f on log–log scales. Friction g″ at low frequencies increased, remained constant, or decreased with increasing frequency. All cells except the MCF10 group (b) exhibited strong frequency dependence at high frequencies consistent with viscous dissipation

Fig. 5

Cytoskeletal stiffness g′ at the lowest measured frequency f = 0.1 Hz for 253J cells was log-normally distributed, thus a the logarithm of g′ is distributed nearly normally (dashed line). Logarithm of g′ of all cell lines appeared normally distributed, so mean values of the logarithm of stiffness were compared by z test between cells within each group. b Comparison of logarithm of stiffness g′ at the lowest measured frequency f = 0.1 Hz of all cell line groups (mean ± standard deviation). Significant difference in mean values* (P = 5 %)

Fig. 6

Cytoskeletal friction g″ at the lowest measured frequency f = 0.1 Hz for 253J cells was log-normally distributed. a The logarithm of g″ is distributed nearly normally (dashed line). Logarithm of g″ of all cell lines appeared normally distributed, so mean values of the logarithm of friction were compared by z test between cells within each group. b Comparison of logarithm of friction g″ at the lowest measured frequency f = 0.1 Hz for all cell line groups (mean ± standard deviation). Significant difference in mean values* (P = 5 %)

Fig. 7

a Distribution of cytoskeletal fluidity x for 253J cells with superimposed normal curve (dashed line). The distribution of x for other cell lines was similar. b Normal probability plot of cytoskeletal fluidity x for 253J cells. Deviations from linearity at high x indicate a heavier right tail than would be expected for a normal distribution. c Comparison of cytoskeletal fluidity x between cell lines within each group by z test (mean ± standard deviation). Significant difference in mean values* (P = 5 %)