Dermal fibroblasts and triple-negative mammary epithelial cancer cells differentially stiffen their local matrix

Abstract

The bulk measurement of extracellular matrix (ECM) stiffness is commonly used in mechanobiology. However, past studies by our group show that peri-cellular stiffness is quite heterogeneous and divergent from the bulk. We use optical tweezers active microrheology (AMR) to quantify how two phenotypically distinct migratory cell lines establish dissimilar patterns of peri-cellular stiffness. Dermal fibroblasts (DFs) and triple-negative human breast cancer cells MDA-MB-231 (MDAs) were embedded within type 1 collagen (T1C) hydrogels polymerized at two concentrations: 1.0 mg/ml and 1.5 mg/ml. We found DFs increase the local stiffness of 1.0 mg/ml T1C hydrogels but, surprisingly, do not alter the stiffness of 1.5 mg/ml T1C hydrogels. In contrast, MDAs predominantly do not stiffen T1C hydrogels as compared to cell-free controls. The results suggest that MDAs adapt to the bulk ECM stiffness, while DFs regulate local stiffness to levels they intrinsically prefer. In other experiments, cells were treated with transforming growth factor-β1 (TGF-β1), glucose, or ROCK inhibitor Y27632, which have known effects on DFs and MDAs related to migration, proliferation, and contractility. The results show that TGF-β1 alters stiffness anisotropy, while glucose increases stiffness magnitude around DFs but not MDAs and Y27632 treatment inhibits cell-mediated stiffening. Both cell lines exhibit an elongated morphology and local stiffness anisotropy, where the stiffer axis depends on the cell line, T1C concentration, and treatment. In summary, our findings demonstrate that AMR reveals otherwise masked mechanical properties such as spatial gradients and anisotropy, which are known to affect cell behavior at the macro-scale. The same properties manifest with similar magnitude around single cells.

Cell contact guidance via sensing anisotropy of network mechanical resistance

“…Recent work by our group uses this multi-axis AMR system to measure collagen network stiffness heterogeneity and anisotropy surrounding dermal fibroblast and MDA-MB-231 malignant mammary epithelial cells cultured within collagen gel (21). This study used two-axis AMR for the measurement of material property anisotropy in the fibrillar network of a fibrin gel.…”

“…AMR. Measurements of fibrin resistance to deformation were conducted in acellular fibrin gels using an optical tweezers AMR system previously described (51) but modified to measure material properties along multiple axes (52), in which bead position is detected from backscattered laser beam illumination. For AMR measurements, fibrin gels were prepared at University of Minnesota using the same reagents as used for the cell related experiments.…”

The role of cell–matrix interactions in connective tissue mechanics

“…Several methods have recently been developed that can help shed light on this issue. For instance, optical micro-rheological techniques using tracer particles seeded in the tissue have been successfully employed to study local variations of the mechanical environment in the pericellular space [115]. Furthermore, metabolic labelling techniques based on the principle of click chemistry offer the opportunity to evaluate the dynamics of secreted extracellular components, while cytoskeletal dyes compatible with live microscopy offer the chance to observe cytoskeletal rearrangements during prolonged tissue culture [83].…”

Stiffness-Matched Extracellular Matrices Comprising Collagen or Fibrin Differentially Affect Cell-Mediated Remodeling and Peri-Cellular Stiffness

“…Past research by our group has shown that 24 hours after hydrogel polymerization, cells alter local and peri-cellular stiffness by a few orders of magnitude, even on a single cell level 10 . Further, cells can promote distinct stiffness anisotropies which vary with cell line, T1C concentration and biochemical treatments 11 . Nonetheless, more comprehensive understanding of bi-directional relationship between cell behavior and ECM stiffness is still lacking.…”

“…In the rst set of experiments, cells were cultured inside rat tail T1C hydrogels prepared at 4 different concentrations (1.0, 1.5, 2.0 and 3.0 mg/ml). AMR measurements were conducted 48 hours after sample preparation to investigate if local stiffness levels established by the cells are in uenced by the initial concentration of hydrogels or whether cells have a stiffness setpoint and remodel their local environment to promote stiffness levels they intrinsically prefer, as previously suggested by our group 11 .…”