Cancer cell stiffness: integrated roles of three-dimensional matrix stiffness and transforming potential

Abstract

While significant advances have been made toward revealing the molecular mechanisms that influence breast cancer progression, much less is known about the associated cellular mechanical properties. To this end, we use particle-tracking microrheology to investigate the interplay among intracellular mechanics, three-dimensional matrix stiffness, and transforming potential in a mammary epithelial cell (MEC) cancer progression series. We use a well-characterized model system where human-derived MCF10A MECs overexpress either ErbB2, 14-3-3ζ, or both ErbB2 and 14-3-3ζ, with empty vector as a control. Our results show that MECs possessing ErbB2 transforming potential stiffen in response to elevated matrix stiffness, whereas non-transformed MECs or those overexpressing only 14-3-3ζ do no exhibit this response. We further observe that overexpression of ErbB2 alone is associated with the highest degree of intracellular sensitivity to matrix stiffness, and that the effect of transforming potential on intracellular stiffness is matrix-stiffness-dependent. Moreover, our intracellular stiffness measurements parallel cell migration behavior that has been previously reported for these MEC sublines. Given the current knowledge base of breast cancer mechanobiology, these findings suggest that there may be a positive relationship among intracellular stiffness sensitivity, cell motility, and perturbed mechanotransduction in breast cancer.

Figure 1

Matrix stiffness influences Brownian dynamics of tracer beads embedded within MECs that possess ErbB2 transforming potential. MSD of 1 μm tracer beads embedded within 10A.ErbB2.ζ (A) and 10A.vec (B) cells that reside within three-dimensional Type I collagen matrices of stiffness 104 (○), 271 (---), and 391 (—) Pa. Error bars omitted for figure clarity.

Figure 2

MECs that possess ErbB2 transforming potential stiffen in response to elevated matrix stiffness. Intracellular apparent stiffness G′_p increases as matrix stiffness G′_c increases for cell lines that overexpress ErbB2 (B and D) but does not significantly increase for 10A.vec (A) or 10A.14-3-3ζ cells (C). Error bars represent mean ± SE. P-values (^∗, p ≤ 0.05; †, p ≤ 0.10) determined from t-tests for unpaired samples; p-values are with respect to the lowest intracellular stiffness of the given cell line.

Figure 3

ErbB2 sensitizes the intracellular mechanical state to matrix stiffness. Wide bars (red) represent the % increase in intracellular apparent stiffness G′_p as matrix stiffness G′_c is increased from 104 to 391 Pa. P-values (^∗, p ≤ 0.05; †, p ≤ 0.10) determined from t-tests for unpaired samples; p-values reflect the significance of the highest intracellular stiffness relative to the lowest intracellular stiffness of the given cell line as reported in Fig. 2. Thin bars (14) represent the number of migrated cells counted per field of view, after a 6 h trans-well cell migration assay. Initial cell seeding count was maintained at 1 × 10⁵ cells for all cell lines (migration data retrieved from (14)). Error bars represent mean ± SE.

Figure 4

Matrix stiffness affects the relationship between transforming potential and intracellular stiffness. Intracellular apparent stiffness G′_p of MCF10A sublines for matrices of stiffness 104 (A), 271 (B), and 391 (C) Pa. Error bars represent mean ± SE. (D) Difference between G′_p of 10A.vec cells and G′_p of 10A.ErbB2.ζ cells as a function of matrix stiffness G′_c.. P-values (^∗, p ≤ 0.05; †, p ≤ 0.10) determined from t-tests for unpaired samples; p-values are with respect to intracellular stiffness of 10A.vec cells.

Figure 5

14-3-3ζ alters MCF10A single-cell morphology in three-dimensional matrices. Images of live, individual 10A.vec (A), 10A.ErbB2 (B), 10A.14-3-3ζ (C), and 10A.ErbB2.ζ (D) cells embedded wholly within three-dimensional Type I collagen matrices of stiffness 391 Pa. Scale bar = 20 μm. (E) Sphericity Ψ of MCF10A sublines embedded wholly within three-dimensional Type I collagen matrices of stiffness 391 Pa. Error bars represent mean ± SE. P-values (^∗∗∗, p ≤ 0.001; ^∗∗, p ≤ 0.01) determined from t-tests for unpaired samples; p-values are with respect to the sphericity of 10A.vec cells.