64 Cu-diacetyl-bis(N 4 -methylthiosemicarbazonate), 64 Cu-ATSM, continues to be investigated clinically as a PET agent both for delineation of tumor hypoxia and as an effective indicator of patient prognosis, but there are still aspects of the mechanism of action that are not fully understood. Methods: The retention of radioactivity in tumors after administration of 64 Cu-ATSM in vivo is substantially higher for tumors with a significant hypoxic fraction. This hypoxia-dependent retention is believed to involve the reduction of Cu-ATSM, followed by the loss of copper to cellular copper processing. To shed light on a possible role of copper metabolism in hypoxia targeting, we have compared 64 Cu retention in vitro and in vivo in CaNT and EMT6 cells or cancers after the administration of 64 Cu-ATSM or 64 Cu-acetate. Results: In vivo in mice bearing CaNT or EMT6 tumors, biodistributions and dynamic PET data are broadly similar for 64 Cu-ATSM and 64 Cu-acetate. Copper retention in tumors at 15 min is higher after injection of 64 Cu-acetate than 64 Cu-ATSM, but similar values result at 2 and 16 h for both. Colocalization with hypoxia as measured by EF5 immunohistochemistry is evident for both at 16 h after administration but not at 15 min or 2 h. Interestingly, at 2 h tumor retention for 64 Cuacetate and 64 Cu-ATSM, although not colocalizing with hypoxia, is reduced by similar amounts by increased tumor oxygenation due to inhalation of increased O 2 . In vitro, substantially less uptake is observed for 64 Cu-acetate, although this uptake had some hypoxia selectivity. Although 64 Cu-ATSM is stable in mouse serum alone, there is rapid disappearance of intact complex from the blood in vivo and comparable amounts of serum bound activity for both 64 Cu-ATSM and 64 Cu-acetate. Conclusion: That in vivo, in the EMT6 and CaNT tumors studied, the distribution of radiocopper from 64 Cu-ATSM in tumors essentially mirrors that of 64 Cu-acetate suggests that copper metabolism may also play a role in the mechanism of selectivity of Cu-ATSM.

DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore-and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (gH2AX), which forms foci at sites of DNA double-strand breaks. Anti-gH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow 111 In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-gH2AX-Tat with gH2AX foci. In comparison with nonspecific antibody conjugates, 111 In-anti-gH2AX-Tat was retained longer in cells.Anti-gH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-gH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of gH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target gH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings. Cancer Res; 71(13); 4539-49. Ó2011 AACR.

Poly(ADP-ribose) polymerase (PARP) inhibitors are increasingly being studied as cancer drugs, as single agents, or as a part of combination therapies. Imaging of PARP using a radiolabeled inhibitor has been proposed for patient selection, outcome prediction, dose optimization, genotoxic therapy evaluation, and target engagement imaging of novel PARP-targeting agents. Methods: Here, via the copper-mediated 18 F-radiofluorination of aryl boronic esters, we accessed, for the first time (to our knowledge), the 18 F-radiolabeled isotopolog of the Food and Drug Administration–approved PARP inhibitor olaparib. The use of the 18 F-labeled equivalent of olaparib allows direct prediction of the distribution of olaparib, given its exact structural likeness to the native, nonradiolabeled drug. Results: 18 F-olaparib was taken up selectively in vitro in PARP-1–expressing cells. Irradiation increased PARP-1 expression and 18 F-olaparib uptake in a radiation-dose–dependent fashion. PET imaging in mice showed specific uptake of 18 F-olaparib in tumors expressing PARP-1 (3.2% ± 0.36% of the injected dose per gram of tissue in PSN-1 xenografts), correlating linearly with PARP-1 expression. Two hours after irradiation of the tumor (10 Gy), uptake of 18 F-olaparib increased by 70% ( P = 0.025). Conclusion: Taken together, we show that 18 F-olaparib has great potential for noninvasive tumor imaging and monitoring of radiation damage.