Engineering Biomimetic Tumor Organoids Inspired by Janus Stiffness-Driven Cell Stemness.

Organoids become powerful platforms for modeling cancer biology but poorly preserve stemness due to inadequate biophysical cues. Cancer stem cells (CSCs), crucial for tumor growth, are rare and lose stemness under conventional in vitro conditions, limiting drug screening. While chemical and genetic stemness regulators are identified, the role of spatial matrix mechanics remains unclear and largely absent from current organoid architectures. Here, we demonstrate that stemness depends not on stiffness alone but on 3D spatial stiffness differentials across distinct microenvironmental regions. Single-cell clustering and spatial localization analyses reveal that CSCs localize at tumor invasive fronts. In vitro mechanobiological studies further identify that Janus stiffness sustains stemness by altering E-cadherin/β-catenin interactions, triggering nuclear β-catenin and stemness programs. Using this insight, we print Janus microsphere sandwiches using a coaxial co-flow capillary microfluidic device, confining cells between mechanically distinct hydrogel layers to encode cellular-scale stiffness gradients, a feature unattainable in conventional single-layer microspheres or bulk-embedded organoids. This architecture mimics CSC spatial distribution in oral squamous cell carcinoma, maintaining stemness and enabling robust drug screening. By transforming a previously underrepresented mechanobiological mechanism into an engineerable organoid, we create new biomimetic tumor organoid where spatially programmed mechanics, not just biochemistry, governs stemness and therapeutic relevance.