(582a) Investigating Glioblastoma Stem Cell Behaviors in Three-Dimensional Hyaluronic Acid Hydrogels

Glioblastoma multiforme (GBM), the most common type of brain tumor in adults, is extremely malignant and lethal. GBM tumors are highly heterogeneous, being composed of tumor cells as well as glioblastoma stem cells (GSCs; also known as brain tumor initiating cells) that contribute to drug resistance and tumor recurrence following surgery. To improve treatment efficacy, targeting GSCs and their surrounding microenvironment is a promising approach. The brain tumor microenvironment provides physical, chemical, and cellular cues that influence GSC behavior and thus promote tumorigenesis, drug resistance, and tumor recurrence. A mechanistic understanding of the impact of these microenvironmental cues on GSCs is crucial to develop an improved therapeutic approach. Here, we employed three dimensional (3D) hyaluronic acid (HA) hydrogels that allow incorporation of brain microenvironment cues to investigate GSC behaviors.

In this study, U87 cell line and patient derived D456 cells were cultured adherently (in the presence of serum) and/or as suspension culture (serum-free). To prepare HA hydrogels, HA was functionalized with methacrylate groups to form HA methacrylate. A 5% w/w solution of HA methacrylate was made in Neurobasal-A medium (for suspension cells) and Eagle’s Minimum Essential Medium (EMEM) with serum (for adherent cells), to which cells and cross-linker dithiothreitol (DTT) were added to encapsulate the cells in the resulting HA hydrogels. We observed that all the seeded single cells (serum grown U87, serum-free U87 and serum-free D456) expanded and formed spheres, and the size of the spheres increased with time (from day 7 to day 14) indicating the proliferation of GBM cells in the HA hydrogels. The addition of integrin binding peptide (RGD) in the HA hydrogels did not significantly affect the sphere sizes of GBM cells as compared to only HA hydrogels. Further, increasing the initial cell seeding density of serum-free U87 and D456 cells influenced the sphere distribution in HA hydrogels. Interestingly, clonal expansion of serum-free U87 and D456 cells was observed in HA hydrogels as compared to when grown as suspension culture on tissue culture polystyrene (TCPS). Evaluation of GBM stemness markers when cultured in HA hydrogels as compared to adherent and/or suspension culture indicated higher expression of Nestin and Sox2. In addition, CD133, Nanog and OCT-4 expression were also altered. Finally, we demonstrated that HA hydrogels can support long term GSC culture (up to 60 days) with retention of stemness markers. Overall, such biomimetic culture systems could further our understanding of microenvironmental regulation of GSC phenotypes.