Figure 6:

Increased fibronectin in the local ECM is critical to breast cancer cell invasion in 12 kPa scaffolds. A.) Two-photon microscopy showed increased fibronectin deposition around actively invading cells in spheroids embedded in high stiffness scaffolds. Scale bars=50 and 25 μm (higher magnification of outlined region). B.) The addition of cellular fibronectin to high stiffness gels increased invasion (green is phalloidin, blue is DAPI, scale bar=150 μm), while C.) siRNA knockdown of FN1 decreased fibronectin intensity and spread in both 2 and 12 kPa gels. Scale bar=150 μm. D.) In high stiffness scaffolds, the addition of cellular fibronectin doubled invasion at 72 hours post embedding while siRNA knockdown of FN1 drastically decreased invasion over the same period. Neither intervention affected invasion in low stiffness hydrogels. * denotes p<0.05 relative to untreated control of same stiffness, n=9–11 and 16–18 spheroids per conditions for the cFN and siFN experiments, respectively.