No abstract

Many 3D in vitro models induce breast cancer spheroid formation; however, this alone does not recapitulate the complex in vivo phenotype. To effectively screen therapeutics, it is urgently needed to validate in vitro cancer spheroid models against the gold standard of xenografts. A new oxime‐crosslinked hyaluronan (HA) hydrogel is designed, manipulating gelation rate and mechanical properties to grow breast cancer spheroids in 3D. This HA‐oxime breast cancer model maintains the gene expression profile most similar to that of tumor xenografts based on a pan‐cancer gene expression profile (comprising 730 genes) of three different human breast cancer subtypes compared to Matrigel or conventional 2D culture. Differences in gene expression between breast cancer cultures in HA‐oxime versus Matrigel or 2D are confirmed for 12 canonical pathways by gene set variation analysis. Importantly, drug response is dependent on the culture method. Breast cancer cells respond better to the Rac inhibitor (EHT‐1864) and the PI3K inhibitor (AZD6482) when cultured in HA‐oxime versus Matrigel. This study demonstrates the superiority of an HA‐based hydrogel as a platform for in vitro breast cancer culture of both primary, patient‐derived cells and cell lines, and provides a hydrogel culture model that closely matches that in vivo.

Toxins efficiently deliver cargo to cells by binding to cell surface ligands, initiating endocytosis, and escaping the endolysosomal pathway into the cytoplasm. We took advantage of this delivery pathway by conjugating an attenuated diphtheria toxin to siRNA, thereby achieving gene downregulation in patient-derived glioblastoma cells. We delivered siRNA against integrin-β1 (ITGB1)—a gene that promotes invasion and metastasis—and siRNA against eukaryotic translation initiation factor 3 subunit b (eIF-3b)—a survival gene. We demonstrated mRNA downregulation of both genes and the corresponding functional outcomes: knockdown of ITGB1 led to a significant inhibition of invasion, shown with an innovative 3D hydrogel model; and knockdown of eIF-3b resulted in significant cell death. This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and phenotypic effects in cancer cells.

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.

Bioengineered hydrogels enable systematic variation of mechanical and biochemical properties, resulting in the identification of optimal in vitro three-dimensional culture conditions for individual cell types. As the scientific community attempts to mimic and study more complex biologic processes, hydrogel design has become multi-faceted. To mimic organ and tissue heterogeneity in terms of spatial arrangement and temporal changes, hydrogels with spatiotemporal control over mechanical and biochemical properties are needed. In this prospective article, we present studies that focus on the development of hydrogels with dynamic mechanical and biochemical properties, highlighting the discoveries made using these scaffolds.