Untangling the response of bone tumor cells and bone forming cells to matrix stiffness and adhesion ligand density by means of hydrogels

Abstract

How cancer cells and their anchorage-dependent normal counterparts respond to the adhesion ligand density and stiffness of the same extracellular matrix (ECM) is still not very clear. Here we investigated the effects of ECM adhesion ligand density and stiffness on bone tumor cells (osteosarcoma cells) and bone forming cells (osteoblasts) by using poly (ethylene glycol) diacrylate (PEGDA) and methacrylated gelatin (GelMA) hydrogels. By independently changing the PEGDA and GelMA content in the hydrogels, we achieved crosslinked hydrogel matrix with independently tunable stiffness (1.6, 6 and 25 kPa for 5%, 10%, 15% PEDGA, respectively) and adhesion ligand density (low, medium and high for 0.05%, 0.2%, 0.5% GelMA respectively). By using a series of biochemical and cell biological characterizations as well as in vivo studies, we confirmed that osteosarcoma and osteoblastic cells responded differently to the stiffness and adhesion ligand density within 3D ECM. When cultured within the 3D PEGDA/GelMA hydrogel matrix, osteosarcoma cells are highly dependent on the matrix stiffness via regulating the integrin-mediated focal adhesion (FA) pathway, whereas osteoblasts are highly sensitive to the matrix adhesion ligand density through regulating the integrin-mediated adherens junction (AJ) pathway. However, when seeded on the 2D surface of the hydrogels, osteosarcoma cells behaved differently and became sensitive to the matrix adhesion ligand density because they were “forced” to attach to the substrate, similar to anchorage-dependent osteoblasts. This study might provide new insights into rational design of scaffolds for generating in vitro tumor models to test anticancer therapeutics and for regenerating tissue to repair defects.

Effect of matrix stiffness and adhesion ligand density on chondrogenic differentiation of mesenchymal stem cells

“…Six types of PEGDA/GelMA hydrogels were fabricated (Table ), and desired stiffness (1.6, 6.0, and 25.0 kPa) and adhesion capacity (low or high) hydrogels were made by using the mixture of PEGDA and GelMA as previously reported (Jiang et al, ). Briefly, 0.5% (w/v) GelMA, PEGDA monomers, and 1% (w/v) Irgacure 2959 (BASF, Germany) were mixed in a centrifuge tube with fixed component contents to fabricate composited hydrogels with different range of adhesion ligand densities and ECM stiffness.…”

“…Polyethylene glycol diacrylate (PEGDA) gels, which have hydrophilic and bio-inert properties, were widely used as a model system to change hydrogel crosslinking density and subsequent material stiffness, without altering ligand /receptor density and the bioactivity of the hydrogels (Huang et al, 2019;Jiang et al, 2019). Owing to arginine-glycine-aspartic acid (RGD) motif in its sequence and its ability to bind cells, methacrylated gelatin (GelMA) exhibited unique advantages over nonadherent materials by modulating cell functions in anchorage-dependent cells, in part, simply because of increased cell attachment (Li, Dou, Feng, & Schonherr, 2018).…”

Understanding and Modeling Metastasis Biology to Improve Therapeutic Strategies for Combating Osteosarcoma Progression

“…Increasing sophistication of hydrogel-based models can be achieved through a combination of chemical engineering and biologic layering, including the construction of soluble mediator (growth factors, chemokines, peptidyl signaling molecules) gradients or combinatorial co-culturing of cancer cells with stromal cells including endothelial cells, fibroblasts, and immune cells. While hydrogels have been explored as a controlled drug release scaffold strategies for OS therapy (156)(157)(158), the study 3D scaffold tumor models for unraveling OS biology and metastasis remains limited, with some investigations describing differences in behavioral phenotype of malignant OS cells compared to non-transformed osteoblasts based upon matrix rigidity and elasticity (159,160). In addition to hydrogel scaffolds, chitosan, silk, and synthetic polymers have served as adhesive constructs for 3D OS modeling and have illuminated mechanisms behind viral permissiveness (161), hypoxia-induced angiogenic mediator secretions (162), drug resistance (163), and maintenance of stem cell phenotype (164).…”

Targeting Mechanotransduction in Osteosarcoma: A Comparative Oncology Perspective

“…Increasing the stiffness of the hydrogel while keeping the ligand density constant led to an increase in proteins related to focal adhesion signalling, namely, integrin beta 1, talin-1, FAK, paxillin and vinculin and the increase in mRNA levels of downstream pro-tumourigenic factors: hypoxia inducible factor 1 alpha (HIF1 alpha), vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 and 9 (MMP2 and 9). When these hydrogel scaffolds were injected in vivo, the tumour volume was greatest in mice inoculated with hydrogel scaffolds with the greatest stiffness, providing evidence that stiffness impacts tumour growth in vivo [ 94 ].…”