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In the present study, we investigate the mechanobiological responses of human lung cancer that may occur through their interactions with two different types of gold nanoparticles: nanostars and nanospheres. Hyperspectral images of nanoparticle-treated cells revealed different spatial distributions of nanoparticles in cells depending on their morphology, with nanospheres being more uniformly distributed in cells than nanostars. Gold nanospheres were also found to be more effective in mechanobiological modulations. They significantly suppressed the migratory ability of cells under different incubation times while lowering the bulk stiffness and adhesion of cells. This in vitro study suggests the potential applications of gold nanoparticles to manage cell migration. Nano-bio-interactions appeared to impact the cytoskeletal organization of cells and consequently alter the mechanical properties of cells, which could influence the cellular functions of cells. According to the results and migratory index model, it is thought that nanoparticle-treated cells experience mechanical changes in their body, which largely reduces their migratory potentials. These findings provide a better understanding of nano-bio-interaction in terms of cell mechanics and highlight the importance of mechanobiological responses in designing gold nanoparticles for cancer therapy.