A DNA Damage Analysis of Breast Cancer Cells Under Cyclic Mechanical Actuation

Metastasis is the leading cause of mortality in breast cancer, yet tumor cell behavior and their response to mechanical cues in the pre-metastatic niche remains poorly understood. This study investigates how breast cancer cells respond to strain mimicking lung tissue dynamics, with a focus on DNA damage. Using a custom-designed micro-actuating lung model, we cultured mApple at p53BP1 and H2B-GFP reporter cells on fibrillar fibronectin-coated substrates subjected to cyclic mechanical forces that simulate respiratory motion. A group of cells were treated with escalating concentrations of Niraparib (0.1–10 µM), a PARP inhibitor, as a positive control. To visualize and quantify DNA damage in real-time, we tracked the red nuclear foci upon DNA double-strand break formation through confocal microscopy. Following treatme nt, cells were lysed directly from the fibronectin band and collected for protein quantification of γ-H2AX. This approach allows us to analyze how mechanically primed tumor cells in a lung-like niche modulate their DNA repair capacity in response to chemotherapeutic stress. These findings contribute to understanding how mechanical environments influence therapeutic resistance and could inform strategies to target metastatic progression at early, mechanosensitive stages.