Matrix Stiffness Directs Stemness Signatures in Breast Cancer

ABSTRACT

Phenotypic plasticity contributes to tumor progression and metastasis, with the tumor microenvironment playing a central role through dynamic cues such as extracellular matrix stiffness. In this study, 2D and 3D in vitro breast cancer models were developed to investigate how ECM mechanics regulate cancer cell behavior. Hydrogel micropatterning enabled the mimicry of spatial confinement and stiffness in 2D microtumors, while drop‐on‐demand bioprinting facilitated the fabrication of mechanically tuneable 3D matrices. Phenotypic characterisation was conducted using immunofluorescence staining for molecular markers of plasticity and stemness, and drug resistance was assessed with doxorubicin and enzalutamide, the latter chosen for its emerging relevance in targeting stem‐like cancer cell populations. Soft matrices promoted stem‐like phenotypes, elevated ALDH1 expression, and enhanced drug resistance, whereas stiff matrices maintained a more differentiated profile. CD44 isoform expression was stiffness‐dependent, with the CD44 standard isoform enriched in soft matrices and the CD44 variant 9 isoform enriched in stiff matrices. The 3D matrices reproduced the mechanical regulation observed in 2D, providing a physiologically relevant platform for high‐throughput investigation of biomechanics‐driven cancer progression. These findings highlight the role of matrix stiffness in driving breast cancer phenotypic heterogeneity and support the application of microengineered synthetic matrices for studying metastasis and drug resistance.