Photonic Crystal Cellular Force Microscopy Reveals the Dynamic Mechanical Responses of Colorectal Cancer Cells to Low-Intensity Pulsed Ultrasound

Low-intensity pulsed ultrasound (LIPUS) has antitumor potential, but its effects on tumor cell mechanics remain poorly understood. In this study, photonic crystal cellular force microscopy (PCCFM) enabled time-resolved mechanical measurements of colorectal cancer cells after LIPUS treatment. Serial PCCFM structural-color and corresponding bright-field images were acquired to reconstruct the vertical stress and substrate deformation in cell-associated regions; integration of the reconstructed stress over defined regions yielded quantitative collective and single-cell vertical force measurements LIPUS altered the time course of collective cellular force after seeding exhibiting distinct collective response patterns. Single-cell analysis further showed marked intercellular heterogeneity in force magnitude and temporal profiles. These mechanical changes were accompanied by altered F-actin organization, fewer peripheral protrusions, and reduced migration and invasion indices, indicating concordant changes in cellular force and motility-related structural and functional phenotypes. In vivo, xenografts derived from LIPUS-pretreated cells had reduced final xenograft volume and tumor weight. Piezo1 immunofluorescence and Ca2+ imaging suggested an association between mechanosensitive-channel signaling and the observed mechanical changes. Overall, PCCFM resolved temporal vertical force responses to LIPUS and revealed associations among LIPUS treatment, cellular mechanics, and motility- and growth-related phenotypes. Therefore, PCCFM provides a label-free, visual, and quantitative cellular-force sensing approach for assessing the dynamic responses of tumor cells to external mechanical stimulation.