Breast cancer is the most common type of cancer, which accounts for 23% of all cancers in women worldwide. Breast tumors are highly heterogeneous, in which cells with self-renewal and highly invasive capacity coexist with cells that are more differentiated and non-invasive. Increasing Tulathromycin B evidence suggests that the heterogeneity of the tumor tissue is rooted in the existence of cancer stem cells. Consistent with this notion, the triple negative breast cancer, which is one of the most aggressive types of breast cancer, contains a high fraction of CSCs. Therefore, understanding the mechanism of CSC maintenance is critical for breast cancer prevention and treatment. The maintenance of CSCs, like that of normal stem cells, is regulated by the microenvironment. Interactions between the stem cells and support cells, interactions between stem cells and extracellular matrix, the composition of ECM and the physicochemical properties of the environment are key contributing factors in stem cell maintenance. Two major factors hinder the study of microenvironment on tumor development in vivo. First, the process of cancer development takes many years, which makes it difficult to follow. Second, it is difficult to study the effect of a specific factor in the microenvironment while keeping all other factors unchanged, as cancer cells are affected by many factors simultaneously. Many in vitro studies have provided insight on the regulation of CSC fate by the microenvironment. However, most in vitro studies use 2-dimensional tissue 4-(Benzyloxy)phenol culture plates coated with ECM components to investigate cell signaling and behavior, which may not reflect those conditions under 3-dimensional physiological environment. Therefore, the 3D in vitro cell culture system has emerged as another approach to investigate the interaction between the microenvironment and cancer cells. Most commonly used matrices for 3D cell culture are type I collagen and Matrigel. However, these matrices contain many cell regulatory factors, which make it difficult to determine the role of individual environmental factors on cell behavior. We have developed an inert polyethylene glycol diacrylate based in vitro 3D cell culture system which does not have cell interaction ligands, thus providing a unique tool to study tumor microenvironment in vitro. More importantly, the CSCs of breast cancer can maintain their stemness and proliferate while the growth of nonCSCs is inhibited when encapsulated in the PEGDA gel within a certain range of elastic moduli. In this study, the inert PEGDA hydrogel, in a certain range of moduli, was used as a 3D cell culture model to investigate the role of cell binding peptides in the maintenance of breast CSCs. More specifically, we investigated the effect of CD44 binding peptide conjugated to the gel or dissolved in the gel on the maintenance of CSCs, because CD44 expression is the most widely used marker for characterization and identification of breast CSCs. CD44 is a cell membrane glycoprotein involved in cell migration and adhesion. CD44 has been used for CSC detection and targeting but the mechanism of its involvement in the maintenance of CSCs is not clear. Antibodies against CD44 inhibit breast tumor growth and prevent cancer recurrence. Anti-CD44 antibodies are found to induce the differentiation of acute myeloid leukemia stem cells.