Dynamic PD-L1 Imaging with a NIR-II Fluorescent Probe for Monitoring and Precision Radio-Immunotherapy in Triple-Negative Breast Cancer

Abstract Programmed death-ligand 1 (PD-L1) is a dynamic biomarker relevant to radiotherapy and immunotherapy, motivating the development of noninvasive approaches for longitudinal assessment. Here we report a PD-L1–targeted near-infrared II (NIR-II) fluorescent probe, PNF-ICG, generated by conjugating a cyclic PD-L1–binding peptide (PNF) with indocyanine green. PNF-ICG was characterized by HPLC, mass spectrometry, and UV–vis spectroscopy, and its PD-L1 binding was supported by molecular docking. In vitro, the probe exhibited PD-L1–dependent signal that could be competitively blocked and showed minimal cytotoxicity. In vivo, using a stably engineered PD-L1–high tumor model, PNF-ICG enabled time-resolved NIR-II imaging that distinguished tumors with different PD-L1 expression levels, and fractionated radiotherapy produced a dose-dependent increase in tumor NIR-II signal consistent with treatment-associated PD-L1 modulation. Finally, imaging-guided evaluation of radiotherapy combined with immune checkpoint blockade demonstrated improved tumor control and survival relative to monotherapy without evident systemic toxicity, supporting PNF-ICG as a peptide-based NIR-II probe for noninvasive visualization of PD-L1 dynamics and for facilitating imaging-guided combination therapy.