(432g) Hyaluronic Acid Hydrogels Modified Via Laminin and Laminin-Derived Peptides to Study Regulation of Dormancy Versus Proliferation in Metastatic Breast Cancer Cells

Breast cancer is the most common malignancy among women and the second leading cause of cancer-related deaths worldwide, with metastasis to distant organs responsible for approximately 90% of these fatalities. Among all metastatic sites, the brain presents an exceptionally difficult challenge and metastasis to the brain is considered one of the most aggressive forms, associated with a poor prognosis and a survival rate of less than one year. Breast cancer cells that metastasize to the brain can enter a state of dormancy for extended time periods. The tumor microenvironment plays an important role in regulating tumor cell behavior during metastasis. As a key component of the tumor microenvironment, the extracellular matrix (ECM) provides structural support along with biomechanical and biochemical cues that influence metastasis. Although our understanding of how ECM-derived cues affect breast cancer metastasis has improved, the specific role of biochemical signals in regulating dormancy at metastatic sites remains limited. Studying these contributions using in vivo mouse models is challenging because of the limited control over the local microenvironment. This lack of understanding continues to hinder the development of effective treatments. To address this, we previously developed a hyaluronic acid (HA) hydrogel based in vitro platform to investigate dormancy regulated by fibronectin-derived RGD binding peptide in brain metastatic breast cancer cells. In the current study, we examined the impact of ECM-derived biochemical cues, particularly varying concentrations of laminin and laminin derived peptides IKVAV and YIGSR, on dormancy and proliferation in brain metastatic breast cancer cells.

We utilized HA hydrogels with a stiffness of ~0.4 kPa to mimic the native brain ECM. To investigate the influence of ECM-derived biochemical cues, we applied a range of concentrations of laminin (0, 0.5, and 1 mg/mL), and laminin-derived peptides, IKVAV (0, 5, and 10 mg/mL), and YIGSR (0, 10, and 20 mg/mL) to functionalize the surfaces of these hydrogels. The degree of surface modification was verified using the Pierce BCA protein assay to quantify laminin binding, and fluorescence labeling was used to confirm peptide conjugation for both IKVAV and YIGSR. Following surface functionalization, ~ 10,000 MDA-MB-231Br cells, a brain-seeking subline of the triple negative breast cancer cell line MDA-MB-231, were seeded onto the hydrogel surfaces and maintained in culture for 5 days. We observed that as the concentration of laminin and IKVAV increased, cell morphology shifted from a rounded shape to a more elongated, spindle-like shape. In contrast, cells cultured on YIGSR-modified hydrogels maintained a rounded morphology regardless of the peptide concentration. Quantification of single cell areas indicated enhanced cell spreading in response to higher levels of laminin and IKVAV. Furthermore, cell proliferation was also elevated with increasing laminin and IKVAV concentration, as measured by EdU incorporation. However, the presence of YIGSR, even at higher concentrations, did not significantly increase cell proliferation when compared to YIGSR-deficient controls. To explore the mechanisms by which laminin influences cell behavior through cell-matrix interactions, we investigated the involvement of integrin αVß3, known for its role in mediating adhesion to matrix proteins such as laminin, through blocking studies. Our findings demonstrated that integrin aVß3, at least, in part, contributes to the enhancement of cell spreading, adhesion, and proliferation on HA hydrogels functionalized with laminin.

To assess cancer cell dormancy, we measured the percentage of cells positive for phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) and phosphorylated p38 (p-p38) under all experimental conditions. Our findings showed that p-ERK positivity significantly increased in the cells cultured on HA hydrogels functionalized with laminin or IKVAV. In contrast, p-p38 positivity was markedly reduced in those same conditions, particularly at higher laminin levels and the highest IKVAV concentration, when compared to protein/peptide-deficient controls. On the other hand, the presence of YIGSR did not result in any notable changes in the expression of either p-ERK or p-p38. We also demonstrated that the dormancy induced by protein/peptide-deficient HA hydrogel was reversible. In particular, when cells initially cultured on hydrogels without protein or peptide incorporation for 5 days were transferred to IKVAV-modified hydrogels, they exhibited a transition from dormant to a proliferative state by day 10. In sum, our findings highlight the role of ECM-derived biochemical cues in determining dormancy versus proliferation in brain metastatic breast cancer cells. In the future, such hydrogel-based platforms could provide a tool to elucidate the impact of various biochemical cues in the tumor microenvironment in regulating dormancy versus proliferation.