The extracellular matrix (ECM) is known to provide various physicochemical cues in directing cell behavior including composition, topography, and dimensionality. Physical remodeling of the ECM has been documented in a variety of cancers. In breast cancer, the increased deposition of matrix proteins, their crosslinking, and alignment create a stiffer microenvironment that activates cell contractility and promotes cancer invasion. In this paper, we sought to study the collective influence of ECM composition and density on the contractile mechanics of human MDA-MB-231 cells making use of the recently established trypsin deadhesion assay. Using collagen and fibronectin-coated surfaces of varying density, we show that cell contractility is tuned in a density-dependent manner, with faster deadhesion on fibronectin-coated surfaces compared to collagen-coated surfaces under identical coating densities. The deadhesion responses are significantly delayed when cells are treated with the myosin inhibitor blebbistatin. By combining collagen and fibronectin at two different densities, we show that mixed ligand surfaces synergistically modulate cell contractility. Finally, we show that on fibroblast-derived 3D matrices that closely mimic in vivo matrices, cells are strongly polarized and exhibit faster deadhesion compared to the mixed ligand surfaces. Together, our results demonstrate that ECM composition, density, and 3D organization collectively regulate cell contractility. 1. Introduction Of the several hallmarks of tumor formation, the extracellular matrix (ECM) plays a central role in regulating evasion of apoptosis, uncontrolled proliferation, angiogenesis, and metastasis [1, 2]. The acquisition of these hallmarks is made possible through a series of continuous alterations in ECM composition and organization during tumor progression that is manifested in altered ECM mechanical properties. For example, tumors are significantly stiffer than normal tissue, and malignant transformation may be promoted by ECM stiffening. Such alterations in ECM properties lead to altered tensional homeostasis, that is, the force balance between individual cells and the ECM [3]. The ECM is composed of a heterogeneous network of collagen, fibronectin, laminin, glycoproteins, and proteoglycans, with its composition varying in a tissue-specific manner. ECM composition and organization are frequently altered in the context of cancer. For example, increased deposition of collagen I is associated with mammographic density and an increase in the development of breast cancer [4]. Further,
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