Protease degradable, hyaluronic acid based hydrogels provide a platform for breast cancer cell invasion

摘要

Introduction: The role of the microenvironment in cancer invasion is not well understood. The microenvironment is a complex composition of extracellular matrix (ECM), signaling molecules, and stromal cells. Current in vitro models of the tumor microenvironment consist of cells suspended in ill- defined or synthetic matrices, which limit experimental reproducibility and fail to recapitulate the native microenvironment. To improve these in vitro matrices, hydrogels must be well defined and easily modified to incorporate features of the microenvironment. In the current study, a defined hyaluronic acid (HA) hydrogel crossliked with ECM mimetic peptides was developed to elucidate the role of the tumor microenvironment on breast cancer progression Materials: MA was purchased from Lifecore Biomedical. Fmoc protected amino acids were purchased from AnaSpec. Collagenase IV was purchased from Sigma-Aldrich. GM6001 MMP inhibitor was purchased from EMD Millipore. Methods: HA hydrogels were formed via Diels Alder click chemistry by reacting furan functionalized HA with bis-maleimide peptide crosslinkers containing either an matrix metalloproteinase (MMP) cleavable (MMPx) or non-cleavable (GAGx) sequence. Peptides were synthesized using Fmoc chemistry. Crosslink density was tuned by altering HA furan substitution. Invasive MDA-MB-231 breast cancer cells were seeded on HA/MMPx with 32,40, and 55% furan substation (low, medium, and high crosslink density) and invasion was measured. To confirm MMP sensitivity, HA/MMPx and HA/GAGx were treated with collagenase and the change in mass was measured. MDA-MB-231 cells were seeded on HA/GAGx or HA/MMPx with or without GM6001 and invasion was measured. Results: Increased HA/MMPx crosslink density significantly decreased invasion (Figure 1). Figure 1: MDA-MB-231 invasion in low, medium and high crosslink density HA/MMPx. A) Normalized to medium crosslink density (n=3, day 4). B) Reconstruction of MDA-MB-231 invasion. HA/MMPx degraded in collagenase while HA/GAGx had little change in mass (not shown). MDA-MB-231 cells invaded further into HA/MMPx compared to HA/GAGx (Figure 2). GM6001 decreased MDA-MB-231 invasion into HA/MMPx (not shown). Figure 2: MDA-MB-231 invasion in HA/MMPx and HA/GAGx. A) Normalized to HA/MMPx (n=3, day 4). B) Reconstruction of MDA-MB-231 invasion. Discussion: To invade into ECM, cells must either squeeze through matrix pores or use proteases to locally degrade the ECM. Invasion into high crosslink density HA/MMPx was reduced relative to low crosslink density due to more crosslinks needing to be locally degraded. The degradation of HA/MMPx in collagenase confirmed that MMPx is sensitive to MMPs. MDA-MB-231 cells secrete MMPs and invaded twice as far into HA/MMPx compared to HA/GAGx. Invasion into HA/MMPx in the presence of GM6001 was reduced, but not eliminated due to the ability of MDA-MB-231 cells to switch between proteolytic-dependent and -independent invasion. Conclusion: An HA based hydrogel was developed for use as an in vitro breast cancer microenvironment. MDA-MB-231 invasion was enhanced by MMP cleavable crosslinks and low crosslink density. Future work will incorporate additional signaling molecules into HA/MMPx to elucidate the role of components of the microenvironment on breast cancer invasion.