Engineering Patterned Tumor Microtissues in 3D Microwells via Stress Relaxation-Regulated Cell-Matrix Interactions

Abstract

The geometric architecture of solid tumors correlates with tumor progression. In vivo studies reveal that non-spherical tumors with high interfacial curvature facilitate cell detachment and invasion, yet 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 microtissues 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. We found that alginate stress relaxation significantly influenced microtissues formation. Slow-relaxing microwells supported the formation of architecturally stable microtissues with uniform cell distribution, high viability, and cellular proliferation. Conversely, fast-relaxing microwells resulted in structural collapse of microtissues, characterized by 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. Notably, actomyosin inhibition reduced integrin expression and rescued the microtissues 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.