Understanding complex interactions between a cell and its extracellular matrix (ECM) lies at the core of mechanobiology. For instance, stiffness of the ECM was previously shown to be highly heterogenous and deregulated in wound healing or during cancer progression. However, tools available for measuring and dynamically altering peri-cellular stiffness have been lacking and bulk measurements do not probe stiffness sensed by the cells. Thus, we have developed an optical tweezers active microrheology (AMR) system capable of multi-axes stiffness measurements in the peri-cellular region. In this thesis, multi-axes AMR system was used to investigate highly heterogenous and anisotropic stiffness landscapes established by dermal fibroblasts, human breast cancer and fibrosarcoma cells. Peri-cellular stiffness and anisotropy are shown to vary between the tested cell lines and with different treatments modifying cell behavior. Further, stiffness landscape and cell response to ECM vary with hydrogel source, concentration and fiber architecture of the local ECM. These studies underscore the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. Additionally, the method of patterned crosslinking is used to alter ECM topography by inducing fiber alignment and peri-cellular stiffness landscape. Following localized crosslinking, human breast cancer cells undergo contact guidance and durotaxis, as indicated by cell migration in a direction of fiber alignment and along an off-axis stiffness gradient, respectively. While these phenomena are widely known and studied on 2D substrates or in 3D synthetic hydrogels, patterned crosslinking allows for better understanding of processes governing directed migration of individual cells embedded inside naturally-derived fibrous hydrogels. Collectively, this thesis work investigates how cells respond to and regulate their local ECM stiffness based on a variety of different factors. Local ECM stiffness landscape established by the cells and probed using multi-axes AMR differs with the addition of different treatments regulating cell behavior and it is also strongly dependent on biochemical, mechanical and topographical properties of the ECM.