Cancer cells' ability to mechanically adjust to extracellular matrix stiffness correlates with their invasive potential

Abstract

Increased tissue stiffness is a classic characteristic of solid tumors. One of the major contributing factors is increased density of collagen fibers in the extracellular matrix (ECM). Here, we investigate how cancer cells biomechanically interact with and respond to the stiffness of the ECM. Probing the adaptability of cancer cells to altered ECM stiffness using optical tweezers-based microrheology and deformability cytometry, we find that only malignant cancer cells have the ability to adjust to collagen matrices of different densities. Employing microrheology on the biologically relevant spheroid invasion assay, we can furthermore demonstrate that, even within a cluster of cells of similar origin, there are differences in the intracellular biomechanical properties dependent on the cells' invasive behavior. We reveal a consistent increase of viscosity in cancer cells leading the invasion into the collagen matrices in comparison with cancer cells following in the stalk or remaining in the center of the spheroid. We hypothesize that this differential viscoelasticity might facilitate spheroid tip invasion through a dense matrix. These findings highlight the importance of the biomechanical interplay between cells and their microenvironment for tumor progression.

FIGURE 1:

The ability to adjust cytoplasmic viscoelasticity to mechanical properties of the environment correlates with cancer cell invasiveness. (A) Illustration of the optical trapping setup. (B) Schematic illustration of possible origins of different α values: α→1 describes tracer movement in a purely viscous environment; α→0 describes confined tracer motion, e.g., in a densely packed, more elastic cytoplasm; α > 1 indicates superdiffusion, possibly mediated by active motor transportation of a granule along microtubuli. (C) Overview of the cancer cell pairs used in the microrheological measurements. (D) Young’s moduli of 1 and 4 mg/ml collagen I matrices determined by shear rheology. (E) Representative confocal images of mEmerald-lifeAct-7–labeled 4T1 and 67NR breast cancer and KP^R172HC and KP^flC pancreatic cancer cells cultured in matrices of 1 or 4 mg/ml rat-tail collagen I. Scale bars: 10 µm. (F) Scaling exponents, α, characterizing the intracellular lipid granule diffusion in human breast cancer cell lines MDA-MB-231 and MCF7 at different matrix stiffnesses. The noninvasive cell line (MCF7) is depicted in gray, the highly invasive (MDA-MB-231) in red. (G) Same as F, but for pancreatic cancer cell lines KP^R172HC (red, invasive) and KP^flC (gray, noninvasive). (H) Same as F, but for mouse breast cancer cell lines 4T1 (red, invasive) and 67NR (gray, noninvasive). (I) Same as F, but for colorectal cancer cell lines SW620 (red, invasive) and SW480 (gray, noninvasive). Box plot of 5th to 95th percentile. ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant in a Mann-Whitney test (two-tailed).

FIGURE 2:

The ability to adjust cellular elasticity to mechanical properties of the environment correlates with cancer cell invasiveness. (A) Schematic of RT-DC of cancer cells cultured on matrices of different collagen I concentration. After detachment, suspended cells experience high shear forces when entering a 20-μm channel. The resulting deformation is imaged by a high-speed camera. (B–E) Overview of the median apparent Young’s moduli of pairs of human breast cancer (B), pancreatic cancer (C), mouse breast cancer (D), and colorectal cancer (E) cell lines after 24-h culture on different collagen I matrices. Error bars denote 1 SD, n = 4. p Values are derived from a paired Student’s t test.

FIGURE 3:

Cancer cells show a differential increase in viscosity during the process of 3D invasion. (A) Image of a 4T1 spheroid 72 h after being embedded within a matrix of 4 mg/ml collagen I. The different regions (“Center,” “Stalk,” and “Tip”) are indicated in red. (B, C) Assessment of the scaling exponent, α, characterizing intracellular lipid granule diffusion in 4T1 (B) and KP^R172HC (C) in the center, in the stalk, or at the tip of an invading branch of a spheroid embedded in matrices of 1 or 4 mg/ml collagen I. *, p < 0.05 in an ordinary one-way analysis of variance followed by a Holm-Sidak’s multiple-comparisons test.