Pilot Study of 64CuCl2 for PET Imaging of Inflammation

Copper(II) ion (Cu2+) is the essential element for numerous pathophysiological processes in vivo. Copper transporter 1 (CTR1) is mainly responsible for maintaining Cu2+ accumulation in cells, which has been found to be over-expressed in inflammatory tissues. Therefore, we explored the potential application of 64CuCl2 for PET imaging of inflammation through targeting CTR1. The animal models of H2O2 induced muscle inflammation and lipopolysaccaharide induced lung inflammation were successfully established, then imaged by small animal PET (PET/CT) post-injection of 64CuCl2, and PET images were quantitatively analyzed. H&E and immunohistochemical (IHC) staining and western blot experiments were performed for evaluating CTR1 levels in the inflammatory and control tissues. Both inflammatory muscle and lungs can be clearly imaged by PET. PET image quantitative analysis revealed that the inflammatory muscle and lungs showed significantly higher 64Cu accumulation than the controls, respectively (p < 0.05). Furthermore, IHC staining and western blot analysis demonstrated that compared with the controls, CTR1 expression was increased in both the inflammatory muscle and lungs, which was consistent with the levels of 64Cu2+ accumulation in these tissues. 64CuCl2 can be used as a novel, simple, and highly promising PET tracer for CTR1 targeted imaging of inflammation.


Introduction
Copper(II) ion (Cu 2+ ) is an essential element for many pathophysiological processes in living subjects, such as respiration, iron transport, oxidative stress protection, peptide hormone production, pigmentation, blood clotting, normal cell growth, and so on [1][2][3][4]. Although Cu 2+ is not always abundant in the internal environment, Cu 2+ homeostasis is tightly regulated through a delicate and complex network of influx copper transporter, efflux copper transporters (ATP7A and ATP7B), copper chaperons (ATOX1, Cox17, CCS), and other copper binding molecules [3,5]. The copper transporter 1 (CTR 1) protein is a high-affinity Cu 2+ transporter, which mainly functions as regulation of copper accumulation in organisms with the range from yeast to mammals [6,7].
Importantly, CTR1 has been found to be over-expressed in Alzheimer s disease and a variety of cancers. It then has been explored as a novel target for tumor imaging and therapy in recent years [8][9][10][11][12][13][14]. For example, 64 CuCl 2 , as the substrate of CTR1, has been demonstrated as a potential positron emission tomography (PET) tracer for imaging animal models with melanoma, liver cancer, and prostate cancer [10,12,14,15]. Recently, Piccardo A et al. [16] showed that 64 CuCl 2 -PET/CT shows a significantly higher detection rate in human prostate cancer than 18 F-Choline-PET/CT. These studies have inspired us to explore more imaging applications of 64 CuCl 2 -PET in nuclear medicine. Inflammation is the response of the immune system that guards the body against several harmful stimuli in normal conditions. Inflammation is involved in various side effects such as gastrointestinal toxicity, mucositis, skin reactions, nervous system damage, pneumonitis, fibrosis, and so on. Therefore, early and accurate diagnosis of the inflammation is important for reducing the normal tissue injury and improving the therapeutic effect [17]. In the early 1980s, Milanino R et al. [18] and Conforti A et al. [19] found that acute and chronic inflammations featured with changes in the metabolism of Cu. Furthermore, Gomathy Narayanan I. et al. reported that CTR1 was significantly increased in Eales disease, which highlights the role of CTR1 in the pathogenesis of inflammation [20].
Therefore, in this work, we hypothesized that 64 CuCl 2 -PET can be a potential tool for inflammation imaging and further investigated the applications of 64 CuCl 2 in CTR1 targeted PET imaging of inflammation.

Small Animal PET/CT of Inflammatory Muscle
Representative coronal small animal PET and PET/CT images of animal models of H 2 O 2 induced muscle inflammation at 1 h and 6 h after injection of 64 CuCl 2 were displayed in Figure 1A. The radioactivity accumulated in the inflammatory muscle of the right hind leg was observed at early time point of 1 h and following 6 h post-injection (p.i.). High liver and kidney accumulation were also observed, which verified the hepatobiliary and renal clearance route of radioactive 64 Cu 2+ . Moreover, other normal organs and tissues showed relatively low radioactivity accumulation.
The quantification analysis ( Figure 1B) demonstrated that radioactivity uptake of the inflammatory muscle was 3.90 ± 0.83%ID/g and 4.46 ± 0.49%ID/g at 1 and 6 h p.i., respectively. Meanwhile, the control muscle uptake of radioactivity was the background level, which was 0.91 ± 0.38%ID/g and 1.02 ± 0.27%ID/g at 1 h and 6 h p.i., respectively. There was obviously difference between the radioactivity accumulation of the inflammatory muscle and the control muscle (* p = 0.021, # p = 0.036). The average ratio of the inflammatory muscle to the control was 4.29 ± 0.32 and 4.37 ± 0.38 at 1 and 6 h p.i., respectively ( Figure 1C). The liver and kidney accumulation of radioactive 64 Cu 2+ was 32.01 ± 7.94%ID/g and 22.83 ± 5.51%ID/g at 1 h p.i., respectively, and 35.87 ± 8.53%ID/g and 24.46 ± 4.19%ID/g at 6 h p.i., respectively, which was consistent with the results of PET/CT imaging ( Figure 1B).

Small Animal PET of Inflammatory Lungs
Representative coronal small animal PET images of the animal models of lipopolysaccaharide (LPS) induced lung inflammation at the time point of 1 h and 6 h after injection of 64 CuCl2 were displayed in Figure 2A. Compared with the control group, much more radioactivity was observed in the inflammatory lungs than in the lungs of normal mice. High liver and kidney accumulation were also observed, and other normal tissues and organs showed relatively low accumulation of 64 Cu 2+ .
Further quantification analysis ( Figure 2B) showed that 64 Cu 2+ uptake of inflammatory and control lungs was 4.39 ± 0.64%ID/g and 2.85 ± 0.09%ID/g at 1 h p.i., and 5.46 ± 0.71%ID/g and 2.98 ± 0.49%ID/g at 6 h p.i., respectively. There was significant difference in the 64 Cu 2+ accumulation between the inflammatory group and control group (* p = 0.027, # p = 0.009). The mean ratio of the inflammatory lungs to the control was 1.54 ± 0.70 and 1.83 ± 0.55 at 1 and 6 h p.i., respectively ( Figure 2C). For the group of mice with inflammatory lungs, the liver and kidney accumulation of radioactivity was 35.37 ± 5.13%ID/g and 19.53 ± 5.79%ID/g at 1 h p.i., and 28.55 ± 4.68%ID/g and 21.56 ± 3.58%ID/g at 6 h p.i., respectively. Moreover, the muscle uptake was 1.09 ± 0.05%ID/g and 0.95 ± 0.25%ID/g at 1 h and 6 h p.i., respectively ( Figure 2B). The quantification analysis ( Figure 1B) demonstrated that radioactivity uptake of the inflammatory muscle was 3.90 ± 0.83%ID/g and 4.46 ± 0.49%ID/g at 1 and 6 h p.i., respectively. Meanwhile, the control muscle uptake of radioactivity was the background level, which was 0.91 ± 0.38%ID/g and 1.02 ± 0.27%ID/g at 1 h and 6 h p.i., respectively. There was obviously difference between the radioactivity accumulation of the inflammatory muscle and the control muscle (* p = 0.021, # p = 0.036). The average ratio of the inflammatory muscle to the control was 4.29 ± 0.32 and 4.37 ± 0.38 at 1 and 6 h p.i., respectively ( Figure 1C). The liver and kidney accumulation of radioactive 64 Cu 2+ was 32.01 ± 7.94%ID/g and 22.83 ± 5.51%ID/g at 1 h p.i., respectively, and 35.87 ± 8.53%ID/g and 24.46 ± 4.19%ID/g at 6 h p.i., respectively, which was consistent with the results of PET/CT imaging ( Figure 1B).

Small Animal PET of Inflammatory Lungs
Representative coronal small animal PET images of the animal models of lipopolysaccaharide (LPS) induced lung inflammation at the time point of 1 h and 6 h after injection of 64 CuCl 2 were displayed in Figure 2A. Compared with the control group, much more radioactivity was observed in the inflammatory lungs than in the lungs of normal mice. High liver and kidney accumulation were also observed, and other normal tissues and organs showed relatively low accumulation of 64 Cu 2+ .

Small Animal PET of Inflammatory Lungs
Representative coronal small animal PET images of the animal models of lipopolysaccaharide (LPS) induced lung inflammation at the time point of 1 h and 6 h after injection of 64 CuCl 2 were displayed in Figure 2A. Compared with the control group, much more radioactivity was observed in the inflammatory lungs than in the lungs of normal mice. High liver and kidney accumulation were also observed, and other normal tissues and organs showed relatively low accumulation of 64 Cu 2+ .

Pathological Results
As shown in Figure 3 left, hematoxylin and eosin (H&E, ×100) staining showed that muscle tissues were injured with infiltrating inflammatory cells after the induction of H2O2. Compared with almost no CTR1 expression of the control muscle, CTR1 levels were obviously increased in the inflammatory muscle based on immunohistochemical (IHC, ×100) staining, which were mainly presented in both injured muscle tissues and infiltrating inflammatory cells. The H&E image of Figure 3 right demonstrated that lung tissues were obviously injured with infiltrating inflammatory cells after the induction of LPS. Different from the control muscle, CTR1 was observed to be expressed in normal lung tissues, which was consistent with previous findings [21], but it was not as much as the inflammatory lung induced by LPS. Further quantification analysis ( Figure 2B) showed that 64 Cu 2+ uptake of inflammatory and control lungs was 4.39 ± 0.64%ID/g and 2.85 ± 0.09%ID/g at 1 h p.i., and 5.46 ± 0.71%ID/g and 2.98 ± 0.49%ID/g at 6 h p.i., respectively. There was significant difference in the 64 Cu 2+ accumulation between the inflammatory group and control group (* p = 0.027, # p = 0.009). The mean ratio of the inflammatory lungs to the control was 1.54 ± 0.70 and 1.83 ± 0.55 at 1 and 6 h p.i., respectively ( Figure 2C). For the group of mice with inflammatory lungs, the liver and kidney accumulation of radioactivity was 35.37 ± 5.13%ID/g and 19.53 ± 5.79%ID/g at 1 h p.i., and 28.55 ± 4.68%ID/g and 21.56 ± 3.58%ID/g at 6 h p.i., respectively. Moreover, the muscle uptake was 1.09 ± 0.05%ID/g and 0.95 ± 0.25%ID/g at 1 h and 6 h p.i., respectively ( Figure 2B).

Pathological Results
As shown in Figure 3 left, hematoxylin and eosin (H&E, ×100) staining showed that muscle tissues were injured with infiltrating inflammatory cells after the induction of H 2 O 2 . Compared with almost no CTR1 expression of the control muscle, CTR1 levels were obviously increased in the inflammatory muscle based on immunohistochemical (IHC, ×100) staining, which were mainly presented in both injured muscle tissues and infiltrating inflammatory cells. The H&E image of Figure 3 right demonstrated that lung tissues were obviously injured with infiltrating inflammatory cells after the induction of LPS. Different from the control muscle, CTR1 was observed to be expressed in normal lung tissues, which was consistent with previous findings [21], but it was not as much as the inflammatory lung induced by LPS.

Discussion
A variety of copper radionuclides ( 64 Cu, 67 Cu, etc.) have been used in the field of nuclear medicine, and they offer versatile choices for applications in radionuclide imaging and therapy. Particularly, 64 Cu has an intermediate half-life of 12.7 h and unique decay profile (β+: 18%, β−: 38%, and electron capture: 44%), which make it a favorable option for radiolabeling nanoparticles, antibodies, antibody fragments, peptides, and small molecules for PET imaging and radionuclide

Discussion
A variety of copper radionuclides ( 64 Cu, 67 Cu, etc.) have been used in the field of nuclear medicine, and they offer versatile choices for applications in radionuclide imaging and therapy. Particularly, 64 Cu has an intermediate half-life of 12.7 h and unique decay profile (β+: 18%, β− : 38%, and electron capture: 44%), which make it a favorable option for radiolabeling nanoparticles, antibodies, antibody fragments, peptides, and small molecules for PET imaging and radionuclide therapy [22,23]. Furthermore, 64 CuCl 2 has been reported as a promising PET probe for imaging animal models with tumors through targeting of CTR1 [10,12,14,15]. More importantly, some studies have shown that 64 CuCl 2 -PET/CT has been used in human study, which prove the quick translation of 64 CuCl 2 to the clinic over other tracers [16,24]. In this study, considering the previous publications that revealed the important role of Cu 2+ and CTR1 in the pathogenesis of inflammation [18][19][20], we explore 64 CuCl 2 as a novel radiotracer for PET imaging of inflammation and further investigate the relationship between 64 Cu 2+ accumulation and CTR1 levels in inflammatory tissues in mice models.
Two representative inflammatory animal models have thus been selected for testing our hypothesis, which are muscle inflammation during the development of muscle injury after the local intramuscular injection of H 2 O 2 and lung inflammation during the development of acute lung injury (ALI) after inhaled LPS. As expected, compared with the control muscle, the inflammatory muscle showed about four-fold elevated 64 Cu 2+ uptake based on the quantitative analysis of PET images. Consistent with PET finding, it is also notable that compared with the controls, CTR1 expression of the inflammatory muscle has been significantly increased. Therefore, our data clearly indicate that Molecules 2018, 23, 502 6 of 9 64 Cu 2+ accumulation is associated with increased CTR1 expression, and 64 CuCl 2 can successfully image inflammatory muscle through CTR1 targeted PET imaging. C57BL/6 mice develop a lung inflammatory response to inhaled LPS, which has been verified to closely replicate many physiological and biological features of human acute lung injury [25]. Kim ES et al. [21] reported that CTR1 expression was found in normal pulmonary epithelial tissues, which is similar to our study. Although CTR1 expression has been found in the normal lungs, approximately two-fold increased expression in the inflammatory lungs has been observed in our study. The results of PET images are consistent with IHC and western blot analysis. Our study clearly indicates that the degree of copper accumulation is correlative with CTR1 levels. The higher CTR1 levels, the more radioactive copper was uptaken. Therefore, 64 CuCl 2 may serve as a valuable tool to image the change of CTR1 expression of lung tissues after the induction of LPS.
Peng F et al. [26] and Xie F et al. [27] once evaluated 64 CuCl 2 uptake by muscular injury and traumatic brain injury with PET/CT, respectively. Their studies demonstrated that 64 CuCl 2 had potential to be a new radiotracer for the assessment of injury, but the molecular mechanism was not clarified. Moreover, Xie F et al. [27] showed that, compared with significantly increased 64 Cu 2+ uptake by the electroporation-injured muscular tissue, only minimal increase 64 Cu 2+ uptake by the muscle with LPS-induced inflammation. However, in the present study, IHC result showed CTR1 was highly expressed in both injured muscle tissues and infiltrating inflammatory cells. 64 Cu 2+ accumulation is associated with increased CTR1 expression, therefore 64 CuCl 2 should be uptaken by both injured muscle and inflammatory cells. Moreover, consistent with previous studies, our work also shows that 64 CuCl 2 displays high accumulation in the liver and kidney, which suggests that 64 CuCl 2 is mainly metabolized through hepatobiliary and kidney systems. These results are consistent with previous findings that 64 Cu can bind with superoxide disumutase, which is widely distributed in the cytosol of eukaryotic cells and abundant in the liver and kidney [28].
Inflammation imaging has been challenging over the past decades, and the quest continues to find an ideal imaging agent. Recent studies reported that 68 Ga-Citrate could be as a possible agent for PET imaging of inflammation based on the transferrin receptor. Radionuclide 68 Ga is good for early time imaging, generator-based, and easy for clinical use. However, high background activity of 68 Ga-Citrate in the thorax and upper abdomen may interfere with detecting lesions in these regions. Moreover, the half-life of 68 Ga is short (68 min), which may be advantageous from low dosimetry to the patients, but disadvantageous for longer periods of study [29].
With the expected growth of PET/CT examination to be an essential strategy in clinical cancer and other benign diseases management, there is a recognized need for new PET probes to address clinical challenges. In this endeavor, CTR1 becomes a new target of inflammation imaging, and 64 Cu has an intermediate half-life of 12.7 h, and the need for imaging for longer periods could be warranted. The high background of 64 Cu focuses on the upper abdomen, which shows relatively smaller field than 68 Ga-Citrate. The prospect of using 64 Cu in the form of simple Cu 2+ ions as a PET probe is not only a cost-effective proposition but also seems poised to broaden the palette of molecular imaging probes in the foreseeable future [30]. Therefore, based on our study, 64 CuCl 2 shows high potential to be used as not only cancers but also a novel PET inflammation imaging probe through targeting CTR1 expression of inflammatory diseases.

General
64 CuCl 2 was purchased from the Department of Medical Physics, University of Wisconsin at Madison (Madison, WI, USA). The pH was adjusted to 7.0 and 64 CuCl 2 solution was diluted with phosphate-buffered saline (PBS, 0.01 M, pH 7.4) buffer, which was obtained from Gibco/Invitrogen (Carlsbad, CA, USA). All other chemicals were purchased from Sigma unless otherwise specified.

Animal Models of H 2 O 2 Induced Muscle Inflammation
All procedures were approved by the Administrative Panel on Laboratory Animal Care (APALC 9547) at Stanford University. The six-to-seven-week-old male BALB/C mice (Charles River Laboratories, Boston, MA, USA) were anesthetized by isoflurane inhalation, and 20 µM H 2 O 2 was then administered intra-muscularly into the right hind leg of the mice. The contralateral muscle of left hind leg which received no treatment was used as the control. Three days after the induction of inflammation by H 2 O 2 , the animal models were used for small animal PET/computed tomography (CT) imaging study.

Animal Models of LPS Induced Lung Inflammation
The six-to-seven-week-old male C57BL/6 mice (Charles River Laboratories, Boston, MA, USA) were anesthetized by isoflurane inhalation, and 50 µg of LPS (from Escherichia coli serotype 0127: B8, Sigma, St. Louis, MO, USA) in 20 µL of saline (0.9% NaCl) was then administered via intranasal instillation [25]. The control group was consisted of normal mice. Three days after LPS administration, the mice were imaged by small animal PET.

Small Animal PET/CT Imaging of Inflammatory Muscle
PET/CT imaging of animal models of H 2 O 2 induced muscle inflammation was performed using a small animal PET/CT scanner (Siemens Inveon). Mice (n = 4 for each group) were injected with approximately 3.7-5.55 MBq (100-150 µCi) of 64 CuCl 2 via the tail vein. At 1 and 6 h p.i., mice were anesthetized with 2% isoflurane (5% isoflurane for induction and 2% for maintenance in 100% O 2 ) for imaging experiments. The images were reconstructed with two-dimensional ordered-subset expectation maximization algorithm with CT-based attenuation correction. Image files were analyzed using the vendor-supplied software Inveon Research Workspace (Preclinical Solutions, Siemens Healthcare Molecular Imaging). For each small-animal PET scan, three-dimensional (3D) regions of interest (ROIs) were drawn over the organs and tissues on decay-corrected whole-body images. The average radioactivity concentration in the ROI was obtained from the mean pixel values within the ROI volume. These data were converted to counts per milliliter per minute by using a predetermined conversion factor. The results were divided by the injected dose to obtain an image ROI derived percent injected dose per gram of tissue [14].

Small Animal PET Imaging of Inflammatory Lungs
Similar to PET scan of animal models of H 2 O 2 induced muscle inflammation, animal models of LPS induced lung inflammation and the controls (n = 4 for each group) were also injected with approximately 3.7-5.55 MBq (100-150 µCi) of 64 CuCl 2 through the tail vein. The mice were then anesthetized and imaged at 1 and 6 h p.i., respectively.

Pathology
The mice were sacrificed after imaging studies, and muscle and lung samples (with or without inflammation) were obtained. The slides of paraffin-embedded muscle and lung samples were used for common H&E staining. To determine whether 64 Cu 2+ accumulation in the inflammatory muscle and lungs was associated with the levels of CTR1 expression, IHC staining was performed. The antibody for CTR1 (Novus Biologicals, Littleton, CO, USA) was diluted at 1:500 and incubated on tissue sections overnight at 4 • C.

Western Blot for CTR1 Levels
Proteins from muscle and lung tissues were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred (400 mA, 1 h) in a Mighty Small Transphor Transfer Tank (R&D System) to activated polyvinylidene flouride membranes in buffer containing 20%