Engineering patterned tumor microtissues in 3D microwells via stress relaxation-regulated cell–matrix interactions

Abstract

The geometric architecture of solid tumors is correlated with tumor progression. In vivo studies reveal that non-spherical tumors with high interfacial curvature facilitate cell detachment and invasion while precisely recapitulating these geometric features and constructing 3D patterned tumor microtissues in vitro remains challenging. While tumor spheroids and scaffold-based cell assemblies enable 3D microtissue modeling, limitations in spherical homogeneity and scaffold confinement hinder the investigation of the relationship between the geometrical complexity and physiological development. In this study, we developed a standardized method to engineer 3D patterned tumor microtissues using alginate gel-based microwells with precisely controlled geometries and mechanical properties. Up to 85% of microtissues formed in slow-relaxing microwells (τ_1/2 = 1710 ± 120 s) achieved well-defined stable architectures with uniform cell distribution and high cellular proliferation. Conversely, fast-relaxing microwells (τ_1/2 = 392 ± 35 s) induced structural collapse in 81% of microtissues and decreased cellular proliferation by 27%, exhibiting edge-accumulated cells and central cavitation. This difference was determined by stress relaxation-mediated changes between cadherin-mediated cell–cell cohesion and integrin-mediated cell–matrix adhesion, where fast relaxation amplified integrin-dependent actomyosin overexpression by 2.9-fold. Notably, actomyosin inhibition reduced integrin expression and rescued the microtissue formation across stress relaxation regimes. Our findings highlighted the crucial role of stress relaxation in regulating adhesion-driven multicellular organization and established a standardized 3D tumor platform for investigating the influence of tumor geometries on tumor progression.