<i>In Vivo</i> Evaluation of a Self-Excitatory Near-Infrared ImmunoSCIFI Probe

Secondary Cerenkov-induced fluorescence imaging (SCIFI) utilizes blue-weighted Cerenkov luminescence from radioactive decay to excite proximal fluorophores that emit near-infrared light with optimal penetrance through biological tissues and offers potential utility in clinical imaging applications, including guidance of surgical resection. Recently, we developed a self-excitatory immunoSCIFI probe based on an antibody modified with the Cerenkov luminescence generating radioisotope zirconium-89 and a near-infrared boron-dipyrromethene dye (BOD665) and observed an immunoSCIFI signal in <i>in vitro</i> cell-based experiments. In this study, we have evaluated the <i>in vivo</i> application of immunoSCIFI using a clinically relevant orthotopic mouse model of dedifferentiated chondrosarcoma as a reproducible, high contrast setting in which to challenge the optical method under bone and soft tissue attenuation. Herein, we report the synthesis, characterization, preclinical imaging, and <i>ex vivo</i> biodistribution analysis of a novel immunoSCIFI probe, [⁸⁹Zr]Zr-DFO-MT1-MMP-BOD665, based on a murine monoclonal immunoglobulin G (IgG) with high binding specificity for the sarcoma biomarker MT1-MMP. Both <i>in vivo</i> imaging and <i>ex vivo</i> data indicated significantly higher total uptake and femur-to-muscle ratios in the inoculated femurs with high MT1-MMP expression relative to contralateral femurs. These preliminary findings establish that antibody-mediated SCIFI can operate <i>in vivo</i> with favorable signal-to-background performance under physiologically relevant photon attenuation. The study therefore provides a methodological foundation for future SCIFI probes, for which rigorous specificity testing and broader biomarker panels will be pursued separately.