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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\u2010cell 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\u2010cadherin\/\u03b2\u2010catenin interactions, triggering nuclear \u03b2\u2010catenin and stemness programs. Using this insight, we print Janus microsphere sandwiches using a coaxial co\u2010flow capillary microfluidic device, confining cells between mechanically distinct hydrogel layers to encode cellular\u2010scale stiffness gradients, a feature unattainable in conventional single\u2010layer microspheres or bulk\u2010embedded 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.<\/jats:p>","DOI":"10.1002\/smll.75385","type":"journal-article","created":{"date-parts":[[2026,8,22]],"date-time":"2026-08-22T09:36:58Z","timestamp":1787391418000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering Biomimetic Tumor Organoids Inspired by Janus Stiffness\u2013Driven Cell Stemness"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7680-5292","authenticated-orcid":false,"given":"Ziyu","family":"Zhu","sequence":"first","affiliation":[{"name":"The Affiliated Hospital of Stomatology School of Stomatology Zhejiang University School of Medicine and Key Laboratory of Oral Biomedical Research of Zhejiang Province  Hangzhou Zhejiang 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