(19b) A Three-Dimensional Hyaluronic Acid Hydrogel Platform to Study the Mechanobiology and Invasion of Brain Metastatic Breast Cancer Cells

Narkhede, A., The University of Alabama
Crenshaw, J., The University of Alabama
Manning, R., The University of Alabama
Rao, S., The University of Alabama
A majority of deaths from breast cancer occur because of metastasis to vital organs such as bone, lungs, liver and brain. Out of all the metastatic sites, brain metastasis is the most aggressive, with the median survival period of only 4-9 months. The brain microenvironment plays a vital role in the metastasis process by providing mechanical (by modulating matrix stiffness), biochemical, and cellular cues to the metastatic breast cancer cells. The ultimate fate of metastatic breast cancer cells is controlled by their dynamic interaction with the brain microenvironment. However, our mechanistic understanding of these interactions remains obscure due to relatively few three dimensional (3D) in vitro culture platforms for studying breast cancer brain metastasis (Narkhede et al., Int. J. Cancer, 2017). To address this need, we recently utilized a biomimetic hyaluronic acid hydrogel platform to elucidate the mechanobiology of brain metastatic breast cancer cells (Narkhede et al., J Biomed Mater Res A, 2018). We chose hyaluronic acid as it is a glycosaminoglycan predominantly found in the brain and is known to interact with the CD44 receptors on metastatic breast cancer cells. Hyaluronic acid hydrogels of varying stiffness (0.2 kPa - 4.5 kPa), including the range relevant to the brain environment (0.2 kPa - 1 kPa) were prepared via dithiothreitol (DTT) crosslinking and further modified with a cell adhesive peptide (RGD) to promote cell adhesion. MDA-MB-231Br (brain metastatic) cells were used as model metastatic cancer cells and were cultured on top of hyaluronic acid hydrogels. In this system, we observed that the MDA-MB-231Br cell adhesion, spreading, proliferation, and migration increased with the hydrogel stiffness. Further, we showed that the focal adhesion kinase (FAK) - phosphoinositide-3 kinase (PI3K) pathway, in part, mediates the stiffness-based response of MDA-MB-231Br cells.

We have further adapted this system to study MDA-MB-231Br invasion in 3D in vitro. Specifically, we engineered degradable hyaluronic acid hydrogels by incorporating a degradable matrix metalloproteinases (MMP) - cleavable peptide crosslinker along with a non-degradable crosslinker (DTT). By tuning the ratio between the degradable and non-degradable crosslinker, we were able to achieve hyaluronic acid hydrogels of varying degradability but having similar stiffnesses and mesh size thereby enabling decoupling of the stiffness and mesh size from degradability. When MDA-MB-231Br cell spheroids were encapsulated and cultured in the degradable hyaluronic acid hydrogels, the cells invaded out of the spheroids whereas they did not invade in non-degradable (DTT crosslinker only) hydrogels. We are currently quantifying MDA-MB-231Br invasion and studying the impact of hydrogel degradability on invasion. Overall, such a 3D biomimetic platform would allow us to further our understanding of breast cancer brain metastasis and discover new therapeutic targets. Such a platform could also be adapted to evaluate the efficacy of cancer therapeutics.