(610a) The Impact of Varying RGD Concentration in a Hyaluronic Acid Hydrogel on Dormancy Versus Proliferation in Brain Metastatic Breast Cancer Cells

A majority of deaths from breast cancer occur because of metastasis to distant organ sites. In particular, breast cancer metastasis to the brain is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death and detection. These dormant cancer cells can later become proliferative, leading to disease recurrence and a poorer prognosis for patients. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of these dormant cancer cells. However, studying the contributions of these cues using in vivo mouse models is challenging, as they provide limited control of the local microenvironment. To this end, we previously reported a biomimetic hyaluronic acid (HA) hydrogel-based in vitro platform to investigate dormancy mediated by biophysical cues in brain metastatic breast cancer cells. In this study, we investigated the impact of biochemical cues, particularly varying RGD concentration, on dormancy versus proliferation in brain metastatic breast cancer cells.

We utilized HA hydrogels with a stiffness of ~0.4 kPa to mimic the brain extracellular matrix. We applied varying concentrations of RGD peptide (0, 1, 2, and 4 mg/mL) to the surfaces of HA hydrogels. We confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA-MB-231Br cells (brain metastatic variant of triple negative breast cancer cell line MDA-MB-231) were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded morphology to a spindle-like morphology. Single-cell area quantification revealed that cell spreading area increased with increasing RGD concentration. In addition, an increase in RGD concentration resulted in an increase in cell proliferation, as quantified via EdU staining.

To evaluate cellular dormancy, we quantified the ratio of extracellular signal-regulated kinase 1/2 (p-ERK) to phosphorylated p38 (p-p38) positivity, as a low ratio of p-ERK to p-p38 has been reported to be a characteristic feature of the dormant state. Our results indicated that the ratio of p-ERK to p-p38 positivity was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to the hydrogels functionalized with higher RGD concentration. Finally, we demonstrated that the HA hydrogel-induced cellular dormancy was reversible. Specifically, when cells cultured on HA hydrogels without RGD incorporation for 5 days were transferred to RGD-enriched HA hydrogels, they exhibited a proliferative phenotype by the end of day 10. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.