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

Abstract Detection of tumor‐associated natural killer cells (TANKs) is crucial for evaluating cancer immunotherapy because they are found to be associated with improved overall survival rate. However, existing analytical methods relying on tissue biopsy are invasive and static, restricting their capacity to deliver dynamic information. Herein, we report an activatable near‐infrared fluorescence (NIRF)/photoacoustic (PA) macromolecular reporter (BhCyNK) for real‐time imaging of TANKs. To optimize the PA performance, the fluorophore scaffold of BhCyNK is screened. Among four hemicyanine derivatives, BhCy S with the longest absorption maximum (∼800 nm) as well as the highest photothermal conversion efficiency (71.13%) and PA brightness is constructed into BhCyNK, which specifically triggers its NIRF signal (by 10‐fold increase) and PA signal (by 8.3‐fold increase) in the presence of a TANK‐overexpressed protease. 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. The imaging results reveal that the increasing intratumoral signal of BhCyNK after combination immunotherapy correlates well with the increasing population of TANKs. Thus, this study not only reports a molecular strategy to develop efficient PA fluorophores but also provides an ideal tool for noninvasive monitoring of immune cells.