Abstract With breast cancer incidence rising worldwide—including in younger Korean women in their 40s—there is an urgent need for advanced disease models that capture mechanobiological features unique to this population. Post-surgical hormonal alterations and the high prevalence of bone metastases further complicate outcomes, underscoring the need for strategies addressing both primary tumor biomechanics and skeletal microenvironments. To meet this need, we developed a three-dimensional (3D) breast cancer spheroid model using biomimetic extracellular matrix materials to replicate native tissue mechanics and architecture, supported by collagen-producing stem cells. High-resolution confocal microscopy, immunofluorescent labeling of F-actin, β-Tubulin, and Vimentin, and computational analysis enabled detailed visualization of cytoskeletal organization and intracellular changes. Mechanobiology-driven evaluation identified membrane-type 1 matrix metalloproteinase (MT1-MMP) as a key regulator of invasive behavior in TNBC spheroids, mediating actin-based protrusions such as filopodia and lamellipodia. Functional assays showed that targeted MT1-MMP inhibition altered cellular mechanics and significantly enhanced chemotherapy efficacy in the 3D model.To validate this platform, dose-dependent accumulation and spatial distribution of doxorubicin were analyzed using confocal Z-stack imaging. Quantitative radial intensity profiling showed approximately 20% decrease in fluorescence signal from the spheroid edge to core at higher concentrations, indicating measurable penetration barriers. Channel-to-channel distance analyses confirmed heterogeneous but quantifiable drug distribution patterns. We also assessed drug distribution and viability using Calcein AM and fluorescence imaging. MT1-MMP inhibitor alone maintained high viability (∼80% live-cell area) with spheroid diameters of ∼180-200 µm and minimal drug uptake. Chemotherapy monotherapy reduced viability to ∼45% with high drug uptake and decreased spheroid integrity (∼160-180 µm). Simultaneous combination improved uptake but reduced viability to ∼40%, with similar spheroid size. Notably, sequential MT1-MMP inhibitor pre-treatment preserved higher viability (∼15%) while maintaining comparable drug uptake and spheroid size (∼180 µm), indicating improved penetration with moderated cytotoxicity.To further characterize the model, XF Seahorse extracellular flux analysis was used to evaluate metabolic responses under different treatment conditions. By quantifying glycolytic activity and oxidative phosphorylation (OXPHOS), we identified shifts in ATP production pathways across monotherapy, combination, and sequential regimens. Sequential MT1-MMP inhibitor pre-treatment induced a more balanced metabolic profile, confirming that MT1-MMP inhibition can modulate and enhance control over OXPHOS activity. Protein marker analysis further supported the mechanism, showing that MT1-MMP-centered mechano-regulation can modulate chemosensitivity in this 3D spheroid system.Therefore, this advanced 3D spheroid system represents an essential platform for Mechanomedicine research and evaluation, closely replicating clinical tumor architecture and mechanical microenvironments. It enables precise study of mechanobiological drivers of metastasis and drug response, providing a foundation for identifying and validating Mechanomedicine targets like MT1-MMP and developing Mechanomedicine candidate therapeutics to improve precision oncology outcomes in breast cancer. Citation Format: J. Yang, M. Ku. Mechanomedicine targeting of MT1-MMP in TNBC spheroids abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-04-15.
Yang et al. (Tue,) studied this question.