Assembly of 3D Tumor Spheroids by Tuning Matrix Mechanics

Many studies report that stiffness of an extracellular matrix may be pivotal in stimulating cancer malignancy. However, this seminal role of matrix stiffness is not well-understood because of the lack of an in vitro three dimensional (3D) cell culture platform with tunable stiffness and cell malignancy. Existing 3D culture platforms are also confounded by the interdependency between matrix properties such as stiffness, permeability and cell adhesion ligands. This study presents a new method to create tumor spheroids with tuned malignancy by altering matrix stiffness. We used a hydrogel consisting of collagen and poly(ethylene glycol)-di(succinic acid N-hydroxysuccinimide ester (PEG-diNHS) for 3D hepatocarcinoma cell (HCC) culture. Matrix stiffness was tuned by adjusting PEG-diNHS concentration and resulting matrix properties including stiffness, permeability and spatial distribution of fibrils were extensively characterized. HCCs were encapsulated in these 3D matrices with varied stiffness and the spheroid morphology, phenotype, angiogenic activities, and drug resistance was further examined both in vitro and in vivo. With this platform, we were able to control the stiffness in a physiologically relevant range while minimally affecting other matrix properties. In the range of elastic moduli examined, increasing gel stiffness with PEG-diNHS generated small and benign HCC spheroids, high hepatocyte-specific activities, and low angiogenic activities, while soft collagen gels generated large and malignant HCC spheroids. Overall, this platform will greatly assist efforts to better understand and modulate emergent behavior of cancer cells.