Real-Time In Situ Imaging of Immunotherapy Efficacy: A DNA Nanowire-Based Biosensor for Granzyme A-Mediated APE1 Inactivation.

Real-time monitoring of natural killer (NK) cell-mediated cancer immunotherapy efficacy at the cellular and organismal levels remains a critical challenge, primarily due to the ultra-low abundance of Granzyme A (GzmA) delivered into target cells, its rapid spatiotemporal dynamics, and the scarcity of suitable in situ imaging probes. Here, we present a DNA nanowire-based biosensor that leverages a spatially confined catalytic hairpin assembly (SCCHA) system for the real-time in situ imaging of immunotherapy efficacy, achieved by sensing GzmA-induced apurinic/apyrimidinic endonuclease 1 (APE1) inactivation. This biosensor integrates an APE1-responsive molecular beacon (MB) with a spatially organized CHA amplification circuit on a rigid DNA nanowire scaffold. In tumor cells with high endogenous APE1 activity, cleavage of the MB initiates a confined CHA reaction, yielding a strong fluorescence readout. Upon NK cell attack, delivered GzmA specifically inactivates intracellular APE1, thereby suppressing CHA signal amplification and producing a distinct fluorescence attenuation that dynamically visualizes NK cell cytotoxicity. Owing to the spatial confinement effect of the DNA nanowire, the biosensor achieved a 6.44-fold improvement in sensitivity over conventional free-diffusion CHA system. This platform enabled real-time visualization of NK cell-tumor cell interactions, and accurately reported NK cell cytotoxicity through GzmA-mediated APE1 inactivation. In vivo experiments further demonstrated its capability in evaluating immunotherapy efficacy. Collectively, this DNA nanowire-based biosensor provides a highly sensitive and reliable in situ imaging toolkit for evaluating cancer immunotherapy in real time, offering a promising translational approach for preclinical drug screening and personalized immunotherapeutic monitoring.