Programming Temporal Stiffness Cues Within Extracellular Matrix Hydrogels for Modelling Cancer Niches

Extracellular matrix (ECM) stiffening is a common occurrence during the progression of many diseases, such as breast cancer. To accurately mimic the pathophysiological context of disease within 3D models, there is high demand for smart biomaterials which replicate the dynamic and temporal mechanical cues of diseased states. This study describes a preclinical disease model, using breast cancer as an example, which replicates the dynamic plasticity of the tumour microenvironment by incorporating temporal (3-week progression) biomechanical cues within a tissue-specific hydrogel microenvironment. The composite hydrogel formulation, integrating adipose-derived decellularised ECM (AdECM) and silk fibroin, was initially crosslinked using a visible light-mediated system, and then progressively stiffened through spontaneous secondary structure interactions inherent between the polymer chains (∼10-15 kPa increase, with a final stiffness of 25 kPa). When encapsulated and cultured , MCF-7 breast cancer cells initially formed numerous, large spheroids (>1000 μm in area), however, with progressive temporal stiffening, cells demonstrated growth arrest and underwent phenotypic changes resulting in intratumoral heterogeneity. Unlike widely-investigated static mechanical models, this stiffening hydrogel allowed for progressive phenotypic changes to be observed, and fostered the development of mature organoid-like spheroids, which mimicked both the organisation and acinar-structures of mature breast epithelium. The spheroids contained a central population of cells which expressed aggressive cellular programs, evidenced by increased fibronectin expression and reduction of E-cadherin. The phenotypic heterogeneity observed using this model is more reflective of physiological tumours, demonstrating the importance of establishing temporal cues within preclinical models in future work. Overall, the developed model demonstrated a novel strategy to uncouple ECM biomechanical properties from the cellular complexities of the disease microenvironment and offers the potential for wide applicability in other 3D disease models through addition of tissue-specific dECM materials.