Abstract LB-492: CD44 binding peptide attached to an engineered matrix prevents the formation of CSC tumorspheres

Abstract

Abstract Objective: Cancer recurrence is related to the existence of cancer stem cells (CSCs) in the tumor tissue that are highly resistant to conventional therapies. Self-renewal and differentiation of CSCs are regulated by tumor microenvironment. Naturally derived matrices have been used for immobilization of cancer stem cells but, due to their many ligand-receptor interactions, those matrices cannot isolate the effect of individual factors in the microenvironment on CSCs. We hypothesized that an inert biodegradable hydrogel, conjugated with bioactive peptides, that allow precise control of mechanical, physiochemical, and biological properties are ideal for investigating the effect of microenvironmental on maintenance of CSCs. The objective of this work was to investigate the effect of CD44 binding peptide RLVSYNGIIFFLK conjugated to an engineered inert but permissive gel (SPELA gel) on viability and maintenance of 4T1 CSCs. Methodology: 4T1 cancer cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml PEN and 100 μg/ml STREP. After 70% confluence, 4T1 tumor cells were suspended in DMEM-F12 medium supplemented with 0.4% BSA, 5 μg/ml insulin, 40 ng/ml bFGF, 20 ng/ml EGF, 5% horse serum (CSC medium). Next, 4T1 cells were seeded in ultra-low attachment plates at a density of 100K cells/mL. After 7-10 days, the cultures were analyzed for tumorsphere formation. The acrylamide-terminated CD44-binding peptide RLVSYNGIIFFLK was synthesized in the solid phase by Fmoc chemistry. 4T1 cell-laden SPELA gels were produced by photopolymerization and cultured in CSCS medium. At each time point, samples were analyzed by fluorescent microscopy and mRNA analysis for the formation of CSCs. Data and Results: The 4T1 CSCs were characterized by inoculation in the back of Balb/c mice. 500 CSCs inoculated in mice formed larger tumors in mice than 50,000 tumor cells after 14 days. When tumor cells were encapsulated in the inert SPELA gel matrix, tumorspheres began to form and continued to grow in size with incubation, but tumorsphere formation strongly depended on the gel modulus. For example, tumorsphere size in gels with moduli in the 5-10 kPa range increased from 25 to 100, and 225 after 2, 6, and 10 days of incubation. Interestingly, the covalent attachment of CD44 ligand to the inert gel prevented the formation of tumorspheres and maintenance of CSCs. Inert engineered matrices are very powerful tools for understanding the effect of tumor microenvironment on maintenance of CSCs and tumorsphere formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-492. doi:1538-7445.AM2012-LB-492