CD44 receptor through a mechanism like receptor clustering. In previous studies, another CD44 binding peptide, A6, was found to enhance cell adhesion to HA and induce FAK and 
MEK phosphorylation in a CD44 dependent manner in breast and ovarian cancer cells. A6 also inhibited cancer cell migration and metastasis in vivo. It is unclear whether CD44BP binding also causes phosphorylation of these kinases in a CD44 dependent manner. ECM signaling mainly occurs through the BAY 73-4506 VEGFR/PDGFR inhibitor integrin family of proteins. The main integrin receptor is aVb3 and it has been implicated in the pathogenesis of several types of cancer. Downregulation of aVb3 integrin sensitized cancer cells to radiotherapy. Inhibition of aVb3 integrin blocked the CSC driven tumor formation in a prostate xenograft model. Further studies suggest that blocking aVb3 integrin leads to redistribution of bcatenin from the nucleus to the cytoplasm. Nuclear localization of b-catenin is known to be one of the mechanisms for maintaining stemness. Therefore, aVb3 integrin-mediated signaling is required for the maintenance of CSCs. RGD peptide, a well-known ligand of aVb3 integrin receptor, blocks the integrin mediated cell adhesion. In this study, RGD peptide without conjugation to the hydrogel did not affect tumorsphere formation. Since the PEGDA hydrogel did not have cell-binding motifs, blocking cell adhesion sites by RGD might not have a significant effect. However, the matrix rigidity is sensed by the actin cytoskeleton through integrin receptors. A recent study showed that membrane-bound RGD can induce the clustering of integrin receptors when cells are seeded on RGD peptide conjugated lipid membranes. Furthermore, integrin receptor clustering stimulates local actin polymerization and leads to cytoskeleton remodeling. We speculate that RGD peptide conjugated to the PEGDA hydrogel causes clustering of the integrin receptors, which in turn, alters cells’ ability to sense matrix rigidity. Since tumorsphere formation in PEGDA hydrogel is rigidity dependent, it would be interesting to determine whether breast cancer cells can form spheres in the RGD conjugated PEGDA hydrogel under a different elastic modulus. The RGD integrin binding peptide is present in many ECM components, but there are other binding motifs in the ECM. For example, fibronectin has a RGD-independent heparin-binding domain in the C-terminus that binds to heparin sulfate proteoglycans on the surface of tumor cells. In this study, we found that unlike CD44 and RGD binding peptides, FHBP conjugated to the gel enhanced tumorsphere formation. It has been shown that FHBP promotes focal adhesion formation in culture cells and it likely activates the focal adhesion kinase. Several lines of evidence have indicated the role of FAK in promoting breast cancer invasion and metastasis and FAK is required for the survival of breast cancer cells in the absence of cell attachment. The Paclitaxel expression of FAK dominant negative mutant in breast cancer cells leads to deactivation and degradation of endogenous FAK and cell apoptosis without matrix attachment. Therefore, FHBP, unlike CD44BP and IBP that block the receptor signaling, may activate FAK and promote CSC survival. As a result, 4T1 cells encapsulated in the gel conjugated with FHBP formed larger and greater number of spheres. Fibronectin is a mesenchymal marker and its expression is increased during the process of EMT.