Synergistic Probe Combining Lysosome Anchoring and Tumor Defense System Targeting for Precise Tumor Visualization

ABSTRACT Precise tumor imaging is essential for accurate intraoperative decision‐making, thereby directly influencing patient prognosis. Optical molecular probes enabling non‐invasive, dynamic assessment of cancerous lesions are clinically crucial. However, current optical molecular probes face challenges in dodging false‐positive and false‐negative signals at once during imaging, limiting their clinical diagnostic use. Here, we introduce a class of lysosome‐targeted, activatable optical probes (Dx‐NH 2 ), leveraging the increased lysosomal abundance during tumor metabolic reprogramming. Lysosomal protonation retains probes for better single‐cell resolution and fewer false‐negative signals. To enable more high‐precision tumor imaging, we synthesized probe CN‐D‐GGT from CN‐D‐NH 2 by introducing a γ‐glutamyltransferase substrate, following our group's prior offensive and defensive integration strategy. Due to its smart design, CN‐D‐GGT, upon activation, shows marked changes in fluorescence and photoacoustic signals, enabling its application for multi‐modal imaging. It also has high specificity, distinguishing cancer cells in co‐culture (∼6‐fold) and clearly differentiating tumors from normal and inflamed tissues (T/N or T/I signal ratios > 3.5). Importantly, it can also differentiate cancerous from adjacent tissues in clinical samples. This work has developed a probe that can accurately light tumors in complex pathological environments, with the potential to assist in intraoperative resection decision‐making, avoid excessive or missed resection.