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Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments. [ABSTRACT FROM AUTHOR]

Adipose-derived stem cells (ASCs) are abundantly present in the mammary microenvironment and can promote breast cancer malignancy by differentiating into myofibroblasts. However, it remains largely unclear which role tumor-derived extracellular vesicles (TEVs) play in this process. Here, we used microfabricated, type I collagen-based 3-D tissue culture platforms to investigate the effect of breast cancer cell-derived TEVs on ASCs myofibroblast differentiation and consequential changes in extracellular matrix remodeling and vascular sprouting. TEVs collected from MDA MB-231 human metastatic breast cancer cells (MDAs) promoted ASC myofibroblast differentiation in both 2-D and 3-D cultures as indicated by increased alpha smooth muscle actin (α-SMA) and fibronectin (Fn) levels. Correspondingly, TEV-treated ASCs were more contractile, secreted more vascular endothelial growth factor (VEGF), and promoted angiogenic sprouting of human umbilical vein endothelial cells (HUVECs). These changes were dependent on transforming growth factor beta (TGF-β)-related signaling and tumor cell glutaminase activity as their inhibition decreased TEV-related myofibroblastic differentiation of ASCs and related functional consequences. In summary, our data suggest that TEVs are important signaling factors that contribute to ASC desmoplastic reprogramming in the tumor microenvironment, and suggest that tumor cell glutamine metabolism may be used as a therapeutic target to interfere with this process. [ABSTRACT FROM AUTHOR]