NIR-IIb fluorescence ratiometric imaging of tumor-associated neutrophils for immunotherapy efficacy monitoring and strategy optimization.

Imaging the immune activation status of tumor-associated neutrophils (TANs) provides insights into cancer immunotherapeutic strategy improvement. Here we show a ratiometric nanoprobe responding to tumor microenvironment and enabling NIR-IIb quantitative fluorescence imaging of TANs with overexpression of neutrophil elastase (NE). The ratiometric nanoprobe integrates erbium-based nanoparticles (ErNPs) with NE-cleavable peptide-caged near-infrared-II (NIR-II) fluorescence dye. Upon systemic delivery, the nanoprobe actively targets tumor microenvironment, where NE cleaves the caging peptide to trigger energy transfer within ErNPs under 808 nm excitation, yielding enhanced NIR-IIb fluorescence emission at 1532 nm. In contrast, the NE-independent fluorescence of ErNPs at 1532 nm remains stable when excited at 980 nm, providing a self-calibrated signal. Across Hepa1-6 and H22 tumor models during immunotherapy, the nanoprobe translates TANs' phenotype shift into precise ratiometric signals, enabling quantitative dynamic imaging of TANs' immune status changes. Subsequently, the responders to immunotherapy among mice could be effectively identified by fluorescence ratiometric imaging of TANs. This strategy offers a non-invasive way to track immune status in real time and could guide timely adjustments to immunotherapy strategies, potentially improving outcomes for patients.