(72a) Investigating Matrix Stiffness Mediated Dormancy in Brain Metastatic Breast Cancer Cells Using a Hyaluronic Acid Hydrogel Platform

Authors: 
Rao, S., The University of Alabama
Narkhede, A., The University of Alabama
Crenshaw, J., The University of Alabama
Breast cancer is known to relapse at secondary organ (metastatic) sites several years after surgical resection of the primary tumor. Breast cancer cells (BCCs) may remain dormant at the metastatic site (e.g., brain), which when activated results in metastasis. This transition marks the most advanced stage of the disease with a median survival period of only 4-9 months. The BCCs dynamically interact with the brain microenvironment which, in turn, mediates their phenotype. The brain microenvironment provides mechanical (by modulating matrix stiffness), biochemical, and cellular cues to the BCCs. Here, we investigated whether the brain microenvironmental (matrix) stiffness mediates dormancy in BCCs. To this end, we utilized a hyaluronic acid (HA) hydrogel platform, representative of the brain extracellular matrix to investigate if matrix stiffness mediates dormancy in BCCs. MDA-MB-231Br (brain metastatic variant of triple negative breast cancer cell line MDA-MB-231) and BT474Br (brain metastatic variant of human epidermal growth factor receptor 2 (HER2) positive breast cancer cell line BT474) cells were used as model metastatic cancer cells and were cultured on top of HA hydrogels.

We observed that BCCs cultured on soft (~ 0.4 kPa) HA hydrogel were largely EdU and Ki67 negative suggesting that they were dormant (non-proliferative) whereas, cells cultured on stiff (~ 4.5 kPa) HA hydrogel were proliferative as indicated by EdU as well as Ki67 positivity. In addition, we observed nuclear localization of p21 and p27 (proteins associated with cell cycle arrest/dormancy) in cells cultured on soft HA hydrogels in contrast to their cytoplasmic localization in cells cultured on stiff HA hydrogels. Further, we observed enhanced resistance to chemotherapy drugs such as Paclitaxel (Taxol) in BCCs cultured on soft HA hydrogels as compared to stiff HA hydrogels. When non-proliferative cells from the soft HA hydrogel were transferred onto the stiff HA hydrogel, they became proliferative, and vice versa, indicating that the stiffness mediated dormancy is reversible. Overall, these results indicate that the HA hydrogel stiffness mediates dormancy in BCCs. We further investigated the role of focal adhesion kinases (FAK) in stiffness mediated dormancy and observed that blocking FAK in cells cultured on stiff HA hydrogels resulted in decreased Ki-67 positivity, suggesting that FAK signaling, in part, mediates dormancy in BCCs. We are currently studying the differential gene expression in BCCs cultured on soft versus stiff HA hydrogels through RNA sequencing to investigate genes associated with dormant phenotype in BCCs cultured on soft HA hydrogels. Such a biomimetic HA hydrogel platform could be utilized to model breast cancer dormancy in vitro and may be adapted as a drug screening platform.