Figure 7:

Local enrichment of EDA-fibronectin drives cell invasion in stiff environments. A.) Total fibronectin and EDA-fibronectin staining in low (left) and high (right) stiffness gels at 72 hr revealed the fibronectin halo that developed in 12 kPa gels consisted of the EDA-fibronectin isoform. Scale bar=150 μm. B.) Treatment with irigenin, a steric blocker of cell-EDA interaction, did not alter morphology in soft conditions but drastically inhibited the invasive morphology in the stiff gels. Scale bar=50 μm. C.) Invasion in high, but not low, stiffness hydrogels was decreased by irigenin treatment. * denotes p<0.05 relative to vehicle control for same stiffness, n=15–20 spheroids per condition. D.) Representative image of benign (left), DCIS (middle), and TNBC (right). Green is EDA-FN, pink is pan-cytokeratin, and blue is DAPI. Scale bar = 50 μm. E.) Mean EDA-FN fluorescence levels in benign (n=8), DCIS (n=30), TNBC (n=32), ER/PR+ (n=150), HER2+ (n=5), and TPBC (n=7). Each dot represents an individual patient, bars represent mean and SD. * denotes p<0.05 relative to benign samples by Dunnett’s test. F.) Summary of stiffness-related invasion mechanism.