Development of a Novel Approach for <i>In Vivo</i> Tumor Hypoxia PET Imaging with Nitroreductase-Specific Responsive Radiotracers

Tumor hypoxia is a major contributor to therapeutic resistance, including radio-, chemo-, and immunotherapy, and is closely linked to unfavorable clinical outcomes. PET imaging has shown promise in guiding the clinical treatment of hypoxic tumors. Here, we developed an NTR-responsive PET strategy for <i>in vivo</i> tumor hypoxia imaging using radiolabeled nitrogen mustard analogues (¹⁸F-NTRP and ¹⁸F-NCRP). Cell uptake studies with A549 cells showed that ¹⁸F-NTRP and ¹⁸F-NCRP exhibited approximately two fold higher uptake and good retention under hypoxic conditions. MicroPET imaging of A549 tumor-bearing mice revealed pronounced tumor accumulation of both ¹⁸F-NTRP and ¹⁸F-NCRP, with uptake values of 1.95 ± 0.37 and 2.86 ± 0.49 %ID/g, respectively, and a rapid clearance rate in normal organs. At 45 min postinjection, the tumor/muscle (T/M) ratios of ¹⁸F-NTRP and ¹⁸F-NCRP were 2.62 and 3.44, respectively, both substantially higher than that of ¹⁸F-FMISO (1.40, <i>p</i> < 0.05). Following dicoumarin-mediated inhibition of tumor NTR, the uptake and T/M ratio of ¹⁸F-NCRP were decreased to 1.25 ± 0.32 %ID/g and 1.26 (<i>p</i> < 0.001), respectively. Moreover, T/M ratios correlated well with HIF-1α (r² = 0.71) and NTR (r² = 0.66) levels in the A549 tumor. Collectively, PET imaging demonstrated that ¹⁸F-NCRP specifically targets tumor NTR and can potentially discriminate between varying degrees of hypoxia.