Pilot Study of 64Cu(I) for PET Imaging of Melanoma

At present, 64Cu(II) labeled tracers including 64CuCl2 have been widely applied in the research of molecular imaging and therapy. Human copper transporter 1 (hCTR1) is the major high affinity copper influx transporter in mammalian cells, and specially responsible for the transportation of Cu(I) not Cu(II). Thus, we investigated the feasible application of 64Cu(I) for PET imaging. 64Cu(II) was reduced to 64Cu(I) with the existence of sodium L-ascorbate, DL-Dithiothreitol or cysteine. Cell uptake and efflux assay was investigated using B16F10 and A375 cell lines, respectively. Small animal PET and biodistribution studies were performed in both B16F10 and A375 tumor-bearing mice. Compared with 64Cu(II), 64Cu(I) exhibited higher cellular uptake by melanoma, which testified CTR1 specially influx of Cu(I). However, due to oxidation reaction in vivo, no significant difference between 64Cu(I) and 64Cu(II) was observed through PET images and biodistribution. Additionally, radiation absorbed doses for major tissues of human were calculated based on the mouse biodistribution. Radiodosimetry calculations for 64/67Cu(I) and 64/67Cu(II) were similar, which suggested that although melanoma were with high radiation absorbed doses, high radioactivity accumulation by liver and kidney should be noticed for the further application. Thus, 64Cu(I) should be further studied to evaluate it as a PET imaging radiotracer.

Both 64 CuCl 2 and 64 CuCl displayed moderate to high tumor-to-blood and tumor-to-muscle ratios ( Table 2). For example, at 4 h after injection, the tumor-to-blood ratios of 64 CuCl 2 and 64 CuCl were 4.55 ± 0.02 and 4.48 ± 0.39 (P > 0.05), and the tumor-to-muscle ratios of 64 CuCl 2 and 64 CuCl were 11.75 ± 0.84 and 10.46 ± 3.26 (P > 0.05), respectively. Biodistribution Studies of Mice Bearing A375M Tumor. The in vivo biodistribution of 64 CuCl 2 and 64 CuCl in mice bearing A375 tumor was determined at 72 h after injection (Table 3). Similar to biodistribution data in mice bearing B16F10 tumor, 64 CuCl 2 and 64 CuCl also showed similar in vivo performance. A375M tumor accumulation of 64 CuCl 2 and 64 CuCl was 3.59 ± 0.36%ID/g and 3.44 ± 0.52%ID/g (P > 0.05), respectively, at 72 h p.i. The high accumulation of 64 CuCl 2 and 64 CuCl by the liver and kidney was observed, with values of 13.37 ± 1.32%ID/g and 13.29 ± 2.51%ID/g (P > 0.05), and 10.34 ± 0.53%ID/g and 8.87 ± 0.60%ID/g (P > 0.05), respectively. These data also demonstrated that in mice bearing A375M tumor models, both 64 CuCl 2 and 64 CuCl  Fig. 3A. For both probes, the tumors were clearly delineated at 1 h p.i., and persisted to 24 h after injection, which were no longer visible at 48 and 72 h p.i. High liver and kidney uptakes were observed at early time points and beyond, verifying the hepatobiliary and renal clearance route of the two probes. Moreover,other normal organs and tissues displayed relatively low accumulation of 64 CuCl 2 or 64 CuCl at the early time points, and the radioactivity was further decreased after 24 h p.i. Further quantification analysis ( Fig. 3B and C) showed that the tumor uptake of 64 CuCl 2 and 64 CuCl was 11.20 ± 2.18%ID/g and 11.35 ± 2.02%ID/g (P > 0.05), respectively, at 1 h p.i., of which values were 3.21 ± 1.72%ID/g and 3.69 ± 1.34%ID/g (P > 0.05), respectively, at 48 h p.i. The liver and kidney accumulation of 64 CuCl 2 was 35.96 ± 4.04%ID/g and 17.68 ± 3.02%ID/g, respectively, at 1 h p.i., and 11.96 ± 1.11%ID/g and 10.37 ± 1.21%ID/g, respectively, at 72 h p.i. The liver and kidney uptake of 64 CuCl was similar to that of 64 CuCl 2 at various time points, and there was nosignificant difference (P > 0.05). Both 64 CuCl 2 and 64 CuCl displayed low muscle uptake after the injection, which was 1.25 ± 0.15%ID/g and 1.05 ± 0.33%ID/g (P > 0.05), respectively, at 1 h p.i.  The quantification results of small animal PET image analysis were shown in Fig. 4B and C. The tumor uptake of 64 CuCl 2 and 64 CuCl was 6.12 ± 1.38%ID/g and 6.51 ± 1.62%ID/g (P > 0.05), respectively, at 1 h p.i., and 3.15 ± 1.22%ID/g and 3.13 ± 1.13%ID/g (P > 0.05), respectively, at 48 h p.i. The liver and kidney accumulation of 64 CuCl 2 was 32.59 ± 4.20%ID/g and 16.68 ± 2.95%ID/g,respectively, at 1 h p.i., and 12.16 ± 2.01%ID/g and 10.03 ± 1.67%ID/g, respectively, at 72 h p.i. The liver and kidney uptake of 64 CuCl was similar to that of 64 CuCl 2 at 1, 2, 4, 24, 48 and 72 h, and there was no significant difference (P > 0.05). Moreover, both 64 CuCl 2 and 64 CuCl displayed low and similarmuscle uptake after the injection, which was around 1%ID/g at 1 h p.i.

Radiation Absorbed Dose Distribution in Human.
The calculated radiation absorbed dose distributions in major organs of a human adult male are shown in Table 4. For both 64 CuCl 2 and 64 CuCl probe, the liver and kidneys showed the highest theoretical radiation absorbed doses; thus, the liver and kidneys would be the dose-limiting organ to carry out cancer-targeted radionuclide therapy. 64 CuCl 2 and 64 CuCl had the similar tumor radiation absorbed dose (1.621 vs. 1.808 ID/g·h). They were anticipated to be promising agents for radionuclide therapy of tumors with CTR1 overexpression. Compared with 64 Cu based probes, 67 Cu-probes showed higher tumor doses, e.g. 67 CuCl 2 (2.695 ID/g·h) and 67 CuCl (2.971 ID/g·h). However, the 67 Cu-probes radiodose in normal organs and tissues were also higher than those of 64 Cu based probes. For example: the radiation absorbed dose by liver for 67 CuCl 2 and 67 CuCl was 1.03 and 0.947 cGy/mCi, respectively, whereas for 64 CuCl 2 and 64 CuCl, it was 0.514 and 0.466 cGy/mCi, respectively.

Discussioin
Copper has many important biological roles in vivo, such as electron transfer, catalysis, and structural shaping 2 . Many copper-containing compounds are biologically active, and have anti-inflammatory and anti-proliferative properties 24,25 . On the other hand, Cu can be toxic because of its ability to generate reactive dioxygen species by cycling between Cu(I) and Cu(II) under physiological conditions 26 . Therefore, Cu homeostasis is tightly regulated by the delicate and complex network in vivo of influx copper transporter (CTR1), efflux copper transporters (ATP7A and ATP7B), copper chaperons (ATOX1, Cox17, CCS), and other copper binding molecules 27 . 64 Cu is a cyclotron-produced radionuclide with an intermediate half-life that decays by both β + and βemission, which makes it suitable for both PET imaging and radionuclide therapy of cancer 28,29 . Traditionally, 64 Cu(II) has been widely applied in radiolabeling small molecules, peptides, proteins, antibodies and nanoparticles through various biofunctional chelators, such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N, N′, N″, N′″-tetraacetic acid (DOTA) and Triethylenetetramine (TETA), and AmBaSar, which have been proven to display in vivo good metabolism in animal models. Some of 64 Cu(II) labeled probes have been translated into clinical applications, such as successfully using 64 Cu-DOTATATE for imaging of human neuroendocrine tumors 30 . Recently, with the better understanding the role of CTR1 as a new biomarker for tumor, 64 CuCl 2 has been reported to be a novel and promising PET probe for imaging several types of cancers including melanoma, human head and neck cancer, and prostate cancer 12,22,31,32 . However, previous reports indicate that CTR1 is the specific influx copper transporter for Cu(I) 3,23 . In our previous study, melanoma cell lines B16F10 as well as A375M displayed high level of CTR1 expression, which could be clearly visible by 64 CuCl 2 PET imaging 12 . Thus, in this study, both B16F10 and A375M cell lines were continued to use for evaluation of 64 Cu(I) probe.
It is reported that with the existence of antioxidants VitC or DTT, 64 Cu(II) can be reduced to 64 Cu(I) [33][34][35][36] . Compared with 64 Cu(II) uptake by melanoma cell lines, both B16F10 and A375M showed significantly higher 64 Cu(I) uptake (P < 0.05). Wang C et al. reported 64 Cu(II) was also reduced to 64 Cu(I) under the existence of both DTT and cysteine, which could help the cell uptake of 64 Cu via CTR1 34 . Interestingly, obvious decreasing cell uptake of copper radioactivity was observed in our study, which may be caused by different tumor cell lines used in different studies. The cell efflux of radioactive copper was further studied in our study. There was small difference of cellular retention of 64 Cu between 64 Cu(II) and 64 Cu(I), and this observation could be attributed to that the cellular efflux of copper was mainly mediated by copper transporters (ATPases) 27,37,38 . ATPases, ATP7A and ATP7B, translocate to the cell membrane and function as efflux pumps to excrete copper from cytosol.
B16F10 tumor-bearing mice were well visualized by small animal PET at 1 h after the intravenous administration of 64 Cu(II) or 64 Cu(I) via tail vein. Although 64 Cu was existed in the form of 64 Cu(I) under the conditions mixed with VitC in vitro, the quick changing from unstable Cu(I) to stable Cu(II) could happen because of the dilution of VitC, and next oxidation reaction under physiological conditions in vivo 2, 5 . Therefore, overall, the results of PET image quantitative analysis of 64 Cu radioactivity in tumor and other normal tissues of 64 Cu(I) were highly similar to those of 64 Cu(II). In addition, the biodistribution data for both 64 Cu(II) and 64 Cu(I), in general, agreed well with the small animal PET quantification results. Moreover, for A375M tumor-bearing mice, the PET images and biodistribution results of 64 Cu(II) and 64 Cu(I) were similar.
Considering the combination therapy with imaging, the radiation absorbed dose distribution of 64 Cu(I/II) and 67 Cu(I/II) in a human adult male was analyzed in this study. For 64/67 Cu(II/I), theoretical radiation absorbed dose distributions in major organs of a human adult male further suggest that the melanoma have high radiation absorbed dose. However, because of the high accumulation of copper in normal organs, such as liver and kidney, and high costs of this kind of radionuclide, 64/67 Cu could not be recommended for melanoma therapy use unless  Table 4. Estimated radiation absorbed doses of 67/64 Cu(II/I) in major organs of an adult male patient based on the biodistribution data obtained from B16F10 tumor-bearing mice.
targeted therapy can be great potentials if with good ideas. It also should be noted that this dose calculation does not mean the exactly same dose distribution results in patient studies. The real patient-specific dosimetry needs to be performed in patient studies.
In conclusion, compared with 64 Cu(II), 64 Cu(I) exhibited higher cellular uptake by melanoma, which further testified CTR1 specially influx of Cu(I). However, due to oxidation reaction in vivo, no significant difference between 64 Cu(I) and 64 Cu(II) was observed through PET images and biodistribution. The in vivo stability of 64 Cu(I) should be further studied to evaluate it as a PET imaging radiotracer.

Materials and Methods
Reagents and Cell Culture. 64  B16F10 murine melanoma cells and A375M human melanoma cells were obtained from American Type Culture Collection (Manassas, VA), and cultured in Dulbecco's modified Eagle's high-glucose medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). The cells were maintained at 37 °C in a humidified 95% air and 5% CO 2 incubator.
In Vitro Cell uptake and Efflux Studies. Cell uptake and efflux studies were performed on B16F10 and A375M cells, respectively. Briefly, melanoma cells (0.3 × 10 6 per well, triplicate for each group) were plated in 12-well plates and incubated at 37 °C overnight. The cells were then incubated for various times (0  (20 mM)] in serum-free medium, respectively. Due to much excess of VitC, DTT or cysteine, Cu(II) could be instantaneously reduced to Cu(I) at the moment of the mixed together and be maintained the reduction statement. Moreover, non-radioactive Cu(II/I) (20 nmol/mL, 0.5 mL per well) was added to block the uptake of 64 Cu radioactivity in the blocking groups. At designated time points, radioactive medium was aspirated and cells were washed 3 times with ice-cold PBS and lysed with 0.1 M NaOH for 5 min at room temperature. The radioactivity of the cell lysates was counted by a Wallac 1480 automated γ-counter (PerkinElmer, Waltham, MA, USA).
For efflux studies, cells were initially incubated with 64 CuCl 2 or 64 CuCl (prepared by reducing 64 CuCl 2 with VitC) for 2 h under the conditions described above, respectively. Then radioactive medium was aspirated and the cells were washed 3 times with PBS buffer. Fresh medium was added and cells were maintained at 37 °C. At different time points (0.5, 1, 2, 4, 16 and 24 h), the supernatant and cell lysate were collected separately and their radioactivity was counted. Cellular retention was calculated by dividing the radioactivity of the cells by the total radioactivity added into the cells at 0 h.

Subcutaneous Tumor Model.
Female C57BL/6 mice and female athymic nude mice (nu/nu) were purchased from Charles River Laboratories (Boston, MA, USA) at 5-6 weeks old and kept under sterile conditions. About 3 × 10 6 B16F10 and 1 × 10 7 A375M cells suspended in 100 μL of PBS were implanted subcutaneously into the right shoulders of C57BL/6 mice and nude mice, respectively. Tumors were grown to a size of 0.5-1 cm in diameter (2-4 weeks). All animal experiments were performed under the approval of Stanford University's Administrative Panel on Laboratory Animal Care (APLAC). All methods were carried out in accordance with relevant guidelines and regulations.
Small Animal PET Imaging. PET of tumor-bearing mice was performed using a small animal PET scanner (Siemens Invenon). B16F10 tumor-bearing mice (n = 4 for each group) were injected via the tail vein with 2.96-3.33 MBq (80-90 μCi) 64 CuCl 2 and 2.96-3.33 MBq (80-90 μCi) 64 CuCl (prepared by 64 CuCl 2 with VitC (2.5 mM)], respectively. At 1, 2, 4, 24, 48 and 72 h post-injection (p.i.), mice were anesthetized with 2% isoflurane (5% for induction and 2% for maintenance in 100% O 2 ) for imaging experiments. With the help of a laser beam attached to the scanner, the mice were placed in the prone position and near the center of the field of view of the scanner. Static scans at 24, 48 and 72 h after injection (scanning time, 10 min) and at other time points (scanning time, 5 min) were obtained. The images were reconstructed with two-dimensional ordered-subset expectation maximization (OSEM 2D) algorithm. The method for quantification analysis of small-animal PET images was the same as previously reported 39 . Small Animal PET Imaging and quantification analysis of mice bearing A375M tumors was similar to that of B16F10. Biodistribution Studies. Anesthetized B16F10 tumor-bearing mice (n = 4 for each group) were injected with approximately 64 CuCl 2 (2.96-3.33 MBq [80-90 μCi]) and 64 CuCl (prepared by adding 2.96-3.33 MBq [80-90 μCi] 64 CuCl 2 with VitC (2.5 mM)], respectively, via the tail vein and sacrificed at different time points from 1 to 72 h p.i. Tumor and normal tissues of interest (blood, muscle, heart, liver, lungs, kidneys, spleen, brain, intestine, skin, stomach, pancreas and so on) were removed and weighed, and their radioactivity levels were measured with a γ-counter. The radioactivity uptake in the tumor and normal tissues was expressed as a percentage of the injected radioactive dose per gram of tissue (%ID/g).
Similar to the biodistribution in B16F10 tumor-bearing mice, A375M tumor-bearing mice (n = 4 for each group) were injected with approximately 64 CuCl 2 (