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Abstract

Breast cancer is a leading cause of global cancer-related mortality, which is characterized by significant biological heterogeneity and aggressive subtypes such as triple-negative breast cancer (TNBC). While traditional oncology has focused heavily on genetic mutations and biochemical signaling, the physical microenvironment is increasingly recognized as an active driver of malignancy. For example, extracellular matrix (ECM) remodeling imposes intense mechanical confinement and solid stress on cancer cells, acting as a selective pressure that triggers malignant adaptive responses. Despite its importance, conventional 2D in vitro models fail to recapitulate these 3D mechanical constraints. Therefore, the development of biomimetic systems capable of simulating physical stress beside biochemical variables is necessary. To address this limitation, this dissertation utilizes a tumor-like 3D culture system composed of alginate-gelatin microcapsules (MCs). These MCs are designed to simulate the specific elastic properties and physical confinement characteristic of a solid breast tumor microenvironment and the experimental setup allows for the systematic investi gation of how solid stress impacts cancer cell activity. This work demonstrates that 3D mechanical confinement greatly influences the phenotypic and biomechanical behavior of breast cancer cells. Specifically, restrictive encapsulation within the 3D MCs acts as a potential physical trigger for the formation of polynucleated and giant cancer cells through cytokinesis failure and a transition to a slow-cycling, stress-adapted proliferative state. Upon release from confinement, these cells exhibit accelerated migration speeds and en hanced directional persistence on 1D guidelines. Such aggressive migratory phenotype is attributed to a redistribution of biomechanical forces, characterized by enhanced traction force and accelerated focal adhesion turnover, suggesting that 3D confinement induces invasiveness to favor rapid dissemination. At the structural level, mechanical confinement lead to remodeling of the nuclear lamina, revealed by lamin B depletion and lamin A/C accumulation which increases nuclear stiffness. However, this architectural reinforcement is insufficient to maintain integrity, as evidenced by elevated DNA damage and genomic instability. Collectively, this work illustrates that physical confinement is not merely a passive factor of tumor growth but a critical driver of malignancy that influences cancer cells for persistent invasiveness and cancer progression. Furthermore, this 3D MC system offers a simple alternative for therapeutic development as a scalable tool for future drug screening applications.