Activatable Fluorescence/Photoacoustic Macromolecular Probe for Imaging of Tumor‐Associated Natural Killer Cells

The detection of tumor‐associated natural killer cells (TANKs) is crucial for evaluating cancer immunotherapy because they are associated with improved overall survival in cancer patients. However, existing analytical methods relying on tissue biopsy are invasive and static, restricting their capacity to deliver dynamic information. Herein, we report an activatable fluorescence/photoacoustic macromolecular reporter (BhCyNK) for real‐time monitoring of TANKs for cancer immunotherapy. To optimize the photoacoustic performance, the fluorophore scaffold of BhCyNK is screened via a molecular engineering strategy involving acceptor replacement and heavy atom incorporation. Among four hemicyanine derivatives, BhCyS with the longest absorption peak as well as the highest photothermal conversion efficiency and PA brightness is constructed into an activatable probe (BhCyNK), which specifically turns on its near‐infrared fluorescence signal (by 10‐fold increase) and PA signal (by 8.3‐fold increase) in the presence of a TANK overexpressed protease. Thus, BhCyNK effectively distinguishes natural killer cells from other immune cells including T cells, neutrophils, and macrophages. The high specificity of BhCyNK enables real‐time monitoring of TANKs population in the tumor of living mice amid cancer immunotherapies through systemic administration. The imaging results reveal that the increasing intratumoral signal of BhCyNK after combination immunotherapy correlates well with the increasing population of TANKs.