For the purpose of comparison and proof of Folinic acid calcium salt pentahydrate principle, we also conjugated an integrin binding RGD peptide and a fibronectin-derived heparin-binding peptide to the gel to investigate their effects on breast CSC maintenance in vitro. We chose those two peptides because fibronectin is one of the major components of ECM that mediates cell adhesion, and integrins are the major receptors on the cell surface that sense the environmental cues. Our results show that conjugation of FHBP to the gel matrix enhanced tumorsphere formation by the encapsulated 4T1 breast cancer cells while CD44BP and IBP abolished sphere formation in vitro. Furthermore, results demonstrate that the inert PEGDA hydrogel can be used as a model 3D matrix to study the role of individual Butenafine hydrochloride factors in the tumor microenvironment on tumorigenesis and maintenance of CSCs. The concept of CSC niche is based on the evidence that both normal stem cells and CSCs utilize similar signaling pathways within a unique microenvironment to maintain stemness. Signaling pathways that have been identified within the stem cell niche include Notch, Hedgehog, PI3K, Wnt, STAT and TGF-b. Processes such as inflammation, EMT, hypoxia and angiogenesis within the microenvironment regulate those pathways to sustain the rare population of CSCs. The cell microenvironment is composed of many cellular and non-cellular components such as cell binding proteins, growth factors, and nutrients. In addition, cells also respond to mechanical properties of the microenvironment such as stiffness and porosity of the ECM. Therefore, cell fate is determined by the specific combination of signals presented in its microenvironment. Due to the complex biochemical composition of the niche, it is difficult to study the role of individual factors in cell behavior with in vitro models. We have developed an inert hydrogel as a 3D matrix that supports the proliferation and maintenance of breast CSCs in a certain range of elastic moduli without the interference of proteins, peptides, and other biomolecules in the ECM. In this study, we investigated the effect of conjugating cell-binding peptides to the inert gel with controlled stiffness on the maintenance of stemness of breast CSCs without the interference of other factors. CD44 is the most widely used marker to identify breast CSCs. CD44 is a cell surface proteoglycan that functions in cell-cell and cell-matrix adhesion. It binds to many ECM ligands including hyaluronic acid, osteopontin, fibronectin and collagen. It also binds to matrix metalloproteinases and growth factors to promote tumor invasion and growth. Therefore, CD44 utilizes many signaling pathways to regulate cell behavior, and its activity depends on conformational changes and posttranslational modifications after ligand binding. CD44BP is a peptide derived from the D-domain of laminin a5 chain. It binds to CD44 and inhibits lung colonization of tumor cells in vivo but does not inhibit tumor cell proliferation when added to the culture medium. In this study, we found that CD44BP inhibits tumorsphere formation only when conjugated to the gel. Dissolving the peptide in the gel or in the medium did not have an effect on tumorsphere formation. This result suggests that CD44BP does not act as a soluble chemokine to block or activate CD44 signaling.