Abstract 251: Engineered matrix to study the effect of microenvironment on cancer stem cell maintenance.

Introduction: Tumors are highly heterogeneous. The heterogeneity of the tumor tissue is rooted in the existence of cancer stem cells (CSCs). Therefore, understanding the mechanism of CSC maintenance and its regulation by the microenvironment is critical to cancer prevention and treatment. Since cancer takes many years to grow, it is important to develop in vitro models to study the molecular basis of tumorigenesis and progression. 3D cell culture systems with biologic materials that support adhesion and growth of many cell types have emerged as another approach to cancer stem cell research. However, it is not possible to isolate individual factors in the microenvironment and the effect on cell response with naturally derived materials. In an effort to control the cell microenvironment, we have developed novel inert permissive gels with controlled physical, mechanical, and biological properties that support the maintenance of CSCs and tumorsphere formation. The objective of this work was to investigate the effect of matrix stiffness and a CD44 binding peptide, conjugated to the inert hydrogel matrix, on tumorsphere formation and CSC maintenance. Methods: Mouse 4T1 and human MCF7 breast cancer cells were encapsulated in the inert PEGDA gel with conjugation of CD44BP. Control groups included dissolved CD44BP and the gel conjugated with mutant CD44BP. Tumorsphere size and density, and expression of CSC markers were determined with incubation time in tumor CSC culture medium. Effect of CD44BP conjugation on breast CSC maintenance was compared with those gels conjugated with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP). For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined with time. Results: The gel stiffness had a strong effect on tumorsphere formation and the effect was bimodal. Tumorsphere formation and expression of CSC markers by the encapsulated cells peaked after 8 days of incubation. 4T1 and MCF7 cells encapsulated in the gel with 5-kPa stiffness formed the largest and highest density of tumorspheres, and had highest expression of breast CSC markers CD44 and ABCG2. Conjugation of CD44 binding peptide to the inert gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP. Conclusion: The PEGDA hydrogel cell culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors. This model system can be used to understand the effect of individual factors in the microenvironment on epithelial to mesenchymal transition (EMT) and tumorigenesis. Citation Format: Xiaoming Yang, Samaneh Kamali, Esmaiel Jabbari. Engineered matrix to study the effect of microenvironment on cancer stem cell maintenance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 251. doi:10.1158/1538-7445.AM2013-251