Abstract
This study focuses on advancing the understanding of breast cancer through 3D in vitro models, which provide biomimetic environments superior to many 2D cultures and animal models. Ex vivo analyses show that malignant breast tissues exhibit increased stiffness with higher tumour grade. Tumour stiffening is associated with altered cell phenotype, promoting progression, invasion, and metastasis. This research aims to design 3D models that mimic the evolving tumour microenvironment to study how matrix stiffness affects breast cancer cell behaviour. Using gelatin-methacryloyl (GelMA) hydrogels, we investigated the phenotypic responses of MCF7 and MDA-MB-231 cells in 3D models of clinically relevant stiffness. A visible-light photoinitiation system enabled precise control of hydrogel mechanics while supporting biocompatibility and long-term cell viability. Over a 21-day culture period, MCF7 cells exhibited partial epithelial-mesenchymal transition in stiff hydrogels, showing altered morphology, downregulating E-cadherin and upregulating N-cadherin and Vimentin. Comparatively, MDA-MB-231 cells showed no such changes. Phenotype remained stable in soft hydrogels for both cell lines. This study demonstrates the impact of microenvironmental stiffness on breast cancer cell phenotype and highlights 3D GelMA hydrogels as a platform to investigate tumour microenvironment dynamics. The findings provide insights into how matrix stiffness influences EMT and breast cancer behaviour in biomimetic settings.