Gelatin‐Hyaluronan Click‐Crosslinked Cryogels Elucidate Human Macrophage Invasion Behavior

Abstract Hydrogel models of metastasis traditionally focus on the invasion of cancer cells; however, other cells in the tumor microenvironment that are associated with metastasis also have the ability to migrate. Macrophage phenotype plays a key role in the tumor microenvironment, yet understanding their migration within tunable 3D in vitro models has been limited. To gain a greater understanding of macrophage invasive behavior, stable and transparent oxime‐crosslinked cryogels comprised of click‐crosslinked gelatin‐oxyamine and hyaluronan‐aldehyde (GELox‐HAa) are synthesized. Fibronectin‐derived, oxyamine‐modified PHSRN‐RGDSP peptides are incorporated to further mimic the tumor extracellular matrix without impacting cryogel mechanics. It is found that primary human macrophages migrate to a greater depth in cryogels with greater porosity and pore size. To better understand the mechanism of migration, cells are treated with either inhibitors of matrix metalloproteinases (MMPs) or rho‐associated protein kinase (ROCK) and a predominantly MMP‐mediated mechanism of invasion is found. Macrophage polarization studies reveal that anti‐inflammatory, interleukin‐4/13 (IL4/IL13)‐treated macrophages migrate through cryogels to a greater extent than pro‐inflammatory, interferon‐gamma/lipopolysaccharide (IFNγ/LPS)‐treated cells. Interestingly, polarized macrophages move through cryogels using a combination of amoeboid and mesenchymal migration. These findings of macrophage invasion in this cryogel platform set the stage for their further study in a biomimetic tumor microenvironment.