Development of a Breast Cancer Cell Microenvironment in Three-Dimensional Hyaluronic Acid Based Hydrogels

Breast cancer cell invasion is influenced by the tumor microenvironment including the extracellular matrix, mechanical properties, stromal cells, and bioactive factors. To elucidate the role of the microenvironment on cell invasion, defined 3-dimensional hydrogel models are required because xenograft models are too complex and 2-dimensional models lack a matrix for cell invasion and often do not recapitulate the appropriate mechanical properties. Breast cancer cell invasion is also influenced by gradients of epidermal growth factor (EGF); however, studying the influence of EGF gradients is challenging due to their transient nature and convoluted in vivo models. In this work, a hyaluronic acid (HA) based hydrogel crosslinked with matrix metalloproteinase (MMP) cleavable peptides (HA/MMPx) was developed to study the role of the microenvironment on breast cancer cell invasion. Specifically, crosslink density, adhesive ligand density, and MMP cleavable peptides were evaluated. Defined EGF gradients were photochemically immobilized into HA/MMPx hydrogels to evaluate the invasion and drug response of breast cancer cells with different EGF receptor (EGFR) expression levels. The crosslink and ligand densities of HA/MMPx hydrogels were decoupled to elucidate their effect on the invasion of MDA-MB-231 breast cancer cells. Increasing crosslink density decreased MDA-MB-231 invasion because more crosslinks had to be degraded for the cells to move through the hydrogel. Increasing adhesive ligand (GRGDS) density did not effect MDA-MB-231 invasion, but did increase cell number. The MMP cleavable crosslinker increased MDA-MB-231 cell invasion relative to a non-cleavable control, confirming that invasion is facilitated by cell-mediated matrix degradation. Immobilized EGF gradients in the HA/MMPx hydrogels differentially affected breast cancer cells with varying levels of EGFR expression: EGF gradients increased the invasion of moderate EGFR expressing MDA-MB-231 cells, decreased the invasion of high EGFR expressing MDA-MB-468 cells, and had no effect on low EGFR expressing MCF-7 cells. The EGFR inhibitor cetuixmab reversed the effects of EGF, decreasing MDA-MB-231 and increasing MDA-MB-468 cell invasion on EGF gradients. With these studies, we developed a versatile platform to study the influence of the microenvironment on breast cancer cell invasion and demonstrated the importance of including microenvironmental factors in culturing platforms when evaluating drugs that target these interactions.