Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells.

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.

Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine.

Extracellular vesicles (EVs)-comprising a heterogeneous population of cell-derived lipid vesicles including exosomes, microvesicles, and others-have recently emerged as both mediators of intercellular information transfer in numerous biological systems and vehicles for drug delivery. In both roles, EVs have immense potential to impact tissue engineering and regenerative medicine applications. For example, the therapeutic effects of several progenitor and stem cell-based therapies have been attributed primarily to EVs secreted by these cells, and EVs have been recently reported to play direct roles in injury-induced tissue regeneration processes in multiple physiological systems. In addition, EVs have been utilized for targeted drug delivery in regenerative applications and possess unique potential to be harnessed as patient-derived drug delivery vehicles for personalized medicine. This review discusses EVs in the context of tissue repair and regeneration, including their utilization as drug carriers and their crucial role in cell-based therapies. Furthermore, the article highlights the growing need for bioengineers to understand, consider, and ultimately design and specifically control the activity of EVs to maximize the efficacy of tissue engineering and regenerative therapies.

In situ generation of cell-laden porous MMP-sensitive PEGDA hydrogels by gelatin leaching.

Proteolytically degradable poly(ethylene) glycol (PEG) hydrogels have been investigated as tissue engineering scaffolds; however, cell invasion and tissue regeneration are limited by the rate of cell-mediated degradation due to the small mesh size of the resultant crosslinked network. Gelatin leaching is combined with photopolymerization to form porous matrix-metalloproteinase (MMP)-sensitive PEG scaffolds under cytocompatible conditions in the presence of cells. Gelatin leaching allows control over pore size and porosity through selectivity of gelatin bead particle size and porogen loading, respectively. Increases in porogen loading lead to increased porosity, decreased compressive modulus and degradation time, and enhanced proliferation of encapsulated vascular smooth muscle cells.

Label-free nondestructive imaging of vascular network structure in 3D culture.

Three-dimensional (3D) cell culture assays are important tools in the study of vessel assembly. Current techniques for quantitative analysis of vascular network structure have provided important insight into 3D vessel assembly. However, these methods typically require immunohistochemical staining, which requires sample destruction, or fluorescent cell labeling, which may alter cell behavior. The methods also may require sophisticated and expensive microscopy. More robust, easily quantifiable techniques are needed for imaging vascular networks non-invasively. We present an imaging method based on widefield optical sectioning and digital deconvolution (WOSD) that enables imaging of vascular networks in 3D culture without the use of cell labeling, staining, or sample destruction. WOSD can be performed using a standard optical microscope and allows non-invasive 3D monitoring of vascular network formation. This method is illustrated by imaging vascular networks in a 3D hydrogel system. WOSD enabled production of quantifiable 3D images of the network structure. Accuracy of the technique was evaluated by comparing data from WOSD with confocal images of fixed and fluorescently stained samples. Data for vessel length, diameter, and density are consistent between the two methods. The WOSD approach can be applied using standard laboratory equipment and shows great promise for use in analysis of 3D vascular network formation.

Effective tuning of ligand incorporation and mechanical properties in visible light photopolymerized poly(ethylene glycol) diacrylate hydrogels dictates cell adhesion and proliferation.

Cell behavior is guided by the complex interplay of matrix mechanical properties as well as soluble and immobilized biochemical signals. The development of synthetic scaffolds that incorporate key functionalities of the native extracellular matrix (ECM) for support of cell proliferation and tissue regeneration requires that stiffness and immobilized concentrations of ECM signals within these biomaterials be tuned and optimized prior to in vitro and in vivo studies. A detailed experimental sensitivity analysis was conducted to identify the key polymerization conditions that result in significant changes in both elastic modulus and immobilized YRGDS within visible light photopolymerized poly(ethylene glycol) diacrylate hydrogels. Among the polymerization conditions investigated, single as well as simultaneous variations in N-vinylpyrrolidinone and precursor concentrations of acryl-PEG3400-YRGDS resulted in a broad range of the hydrogel elastic modulus (81-1178 kPa) and YRGDS surface concentration (0.04-1.72 pmol cm(-2)). Increasing the YRGDS surface concentration enhanced fibroblast cell adhesion and proliferation for a given stiffness, while increases in the hydrogel elastic modulus caused decreases in cell adhesion and increases in proliferation. The identification of key polymerization conditions is critical for the tuning and optimization of biomaterial properties and the controlled study of cell-substrate interactions.

Controlled proteolytic cleavage site presentation in biomimetic PEGDA hydrogels enhances neovascularization in vitro.

The volume of tissue that can be engineered is limited by the extent to which vascularization can be stimulated within the scaffold. The ability of a scaffold to induce vascularization is highly dependent on its rate of degradation. We present a novel approach for engineering poly (ethylene glycol) diacrylate (PEGDA) hydrogels with controlled protease-mediated degradation independent of alterations in hydrogel mechanical and physical properties. Matrix metalloproteinase (MMP)-sensitive peptides containing one (SSite) or three (TriSite) proteolytic cleavage sites were engineered and conjugated to PEGDA macromers followed by photopolymerization to form PEGDA hydrogels with tethered cell adhesion ligands of YRGDS and with either single or multiple MMP-sensitive peptide domains between cross links. These hydrogels were investigated as provisional matrices for inducing neovascularization, while maintaining the structural integrity of the hydrogel network. We show that hydrogels made from SSite and TriSite peptide-containing PEGDA macromers polymerized under the same conditions do not result in alterations in hydrogel swelling, mesh size, or compressive modulus, but result in statistically different hydrogel degradation times with TriSite gels degrading in 1-3 h compared to 2-4 days in SSite gels. In both polymer types, increases in the PEGDA concentration result in decreases in hydrogel swelling and mesh size, and increases in the compressive modulus and degradation time. Furthermore, TriSite gels support vessel invasion over a 0.3-3.6 kPa range of compressive modulus, while SSite gels do not support invasion in hydrogels above compressive modulus values of 0.4 kPa. In vitro data demonstrate that TriSite gels result in enhanced vessel invasion areas by sevenfold and depth of invasion by twofold compared to SSite gels by 3 weeks. This approach allows for controlled, localized, and cell-mediated matrix remodeling and can be tailored to tissues that may require more rapid regeneration and neovascularization.

FGF-1 and proteolytically mediated cleavage site presentation influence three-dimensional fibroblast invasion in biomimetic PEGDA hydrogels.

Controlled scaffold degradation is a critical design criterion for the clinical success of tissue-engineered constructs. Here, we exploited a biomimetic poly(ethylene glycol) diacrylate (PEGDA) hydrogel system immobilized with tethered YRGDS as the cell adhesion ligand and with either single (SSite) or multiple (MSite) collagenase-sensitive domains between crosslinks, to systematically study the effect of proteolytic cleavage site presentation on hydrogel degradation rate and three-dimensional (3-D) fibroblast invasion in vitro. Through the incorporation of multiple collagenase-sensitive domains between cross-links, hydrogel degradation rate was controlled and enhanced independent of alterations in compressive modulus. As compared to SSite hydrogels, MSite hydrogels resulted in increased 3-D fibroblast invasion in vitro, which occurred over a wider range of compressive moduli. Furthermore, encapsulated soluble acidic fibroblast growth factor (FGF-1), a potent mitogen during processes such as vascularization and wound healing, was incorporated into SSite and MSite PEGDA scaffolds to determine its in vitro potential on fibroblast cell invasion. Hydrogels containing soluble FGF-1 significantly enhanced 3-D fibroblast invasion in a dose-dependent manner within the different types of PEG matrices investigated over a period of 15 days. The methodology presented provides flexibility in designing PEG scaffolds with desired mechanical properties, but with increased susceptibility to proteolytically mediated degradation. These results indicate that effective tuning of initial matrix stiffness and hydrogel degradation kinetics plays a critical role in effectively designing PEG scaffolds that promote controlled 3-D cellular behavior and in situ tissue regeneration.