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.
Highlights
Bulk stiffness of the extracellular matrix (ECM) has been previously shown to regulate cellular processes and correspond to invasiveness of cancer cells.[1,2,3] ECM stiffness is a measure of ECM resistance to deformation and is primarily regulated by ECM remodeling, strain stiffening, degradation, and deposition carried out by cells in response to a variety of biochemical cues.[1]
Treatment media included Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 25 mM glucose, 10 ng/ml transforming growth factor-b1 (TGF-b1), or 20 lM Y27632. j of cell-free hydrogels in control conditions increased with concentration (p ( 0.01; nbeads 1⁄4 136 for 1.0T1C and nbeads 1⁄4 127 for 1.5T1C)
Cell contractile forces were previously shown by our group to establish highly heterogeneous j distributions around individual Dermal fibroblasts (DFs) with significant ECM stiffening in the peri-cellular region as compared to cell-free hydrogels.[10]
Summary
Bulk stiffness of the extracellular matrix (ECM) has been previously shown to regulate cellular processes and correspond to invasiveness of cancer cells.[1,2,3] ECM stiffness is a measure of ECM resistance to deformation and is primarily regulated by ECM remodeling, strain stiffening, degradation, and deposition carried out by cells in response to a variety of biochemical cues.[1]. Past research in our laboratory has shown that the peri-cellular stiffness on a single cell level can span orders of magnitude.[10] These findings prompted us to investigate how cells remodel their local stiffness in correlation with bulk (e.g., cell-free) ECM stiffness and other mechanical and biochemical cues
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