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.

Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a fibrin hydrogel. Laser scanning illumination activates ruthenium-catalyzed crosslinking of fibrin tyrosine residues, resulting in tunable stiffness changes spanning distances as small as a few microns and a localized compaction of the material. As probed by active microrheology, stiffness increases by as much as 25X, similar to previously observed stiffness changes around single cells in 3D culture. In summary, our method allows for selective modification of fibrin stiffness at the micron scale with the potential to create complex patterns, which could be valuable for the investigation of mechanotransduction in a biologically meaningful way. STATEMENT OF SIGNIFICANCE: Fibrin hydrogels are used as a naturally derived model to study interactions between cells and their surrounding extracellular matrix (ECM). ECM stiffness influences cell state. Cells in 3D culture considerably modify the stiffness of their pericellular space, which can be quite heterogeneous at the micron-scale. Here we present and evaluate a method to pattern stiffness within fibrin hydrogels using a laser scanning confocal microscope and selective photo crosslinking. We believe that this technique can aid future studies of cell-ECM interactions by enabling researchers to modify the pericellular distribution of stiffness.