Cell surface anchored ratiometric probes for heparan sulfate enable live cell bioimaging and suppression of cancer cell migraion

Heparan sulfate (HS) proteoglycans are essential molecular components of the cell surface glycocalyx that play critical roles in tumor growth, cell migration, and cancer invasion. Despite their biological significance, the development of fluorescent probes capable of selectively and quantitatively visualizing HS in live cells has been highly challenging due to poor water solubility, limited cell-surface retention, and insufficient molecular selectivity. Here, we synthesized a rationally designed peptide-based fluorescent probe (probe 2) that selectively binds to HS on live cell membranes. This probe exhibits prolonged membrane localization and produces a distinct ratiometric fluorescence response. Under physiological conditions, probe 2 achieves stable anchoring on the cell surface and enables quantitative detection of HS through red-shifted emissions upon aggregation. Importantly, this probe allows real-time visualization of HS downregulation in live cells following siRNA-mediated gene silencing or chemical inhibition of HS biosynthesis. Moreover, probe 2 significantly inhibited cell migration and wound healing in vitro, suggesting that HS modulation by the probe may influence tumor-associated cellular behaviors. Collectively, this study introduces a new class of membrane-anchored fluorescent probes that enable dynamic and quantitative imaging of HS in live cells. These findings suggest that probe 2 is both a diagnostic imaging tool for heparan sulfate and a molecular modulator of HS-related cancer biology, with potential implications for cancer research and therapeutic development.