Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Significant efforts have been devoted to identify new biomarkers for molecular imaging and targeted therapy of HCC. Copper is a nutritional metal required for the function of numerous enzymatic molecules in the metabolic pathways of human cells. Emerging evidence suggests that copper plays a role in cell proliferation and angiogenesis. Increased accumulation of copper ions was detected in tissue samples of HCC and many other cancers in humans. Altered copper metabolism is a new biomarker for molecular cancer imaging with position emission tomography (PET) using radioactive copper as a tracer. It has been reported that extrahepatic mouse hepatoma or HCC xenografts can be localized with PET using copper-64 chloride as a tracer, suggesting that copper metabolism is a new biomarker for the detection of HCC metastasis in areas of low physiological copper uptake. In addition to copper modulation therapy with copper chelators, short-interference RNA specific for human copper transporter 1 (hCtr1) may be used to suppress growth of HCC by blocking increased copper uptake mediated by hCtr1. Furthermore, altered copper metabolism is a promising target for radionuclide therapy of HCC using therapeutic copper radionuclides. Copper metabolism has potential as a new theranostic biomarker for molecular imaging as well as targeted therapy of HCC.

Copper is a trace metal that is required for numerous metabolically important enzymes involved in various metabolic pathways of human physiology[1,2]. These include ceruloplasmin, superoxide dismutase, dopamine monooxygenase, lysyl oxidase, cytochrome c oxidase, factor V, and tyrosinase. These enzymes are used for a variety of purposes such as melatonin production, bone production, thrombosis and neurotransmitter synthesis. The amount of daily dietary copper required for an average adult is 1.0 to 1.6 mg according to the third National Health and Nutrition Survey[3]. Zero point nine mg per day of copper is the recommended daily allowance, and less than 10 mg per day is recommended by the National Academy of Sciences[4]. The adult human body contains about 75 mg of copper[5]. The liver and brain contain about one third of the overall quantity present, but copper is distributed throughout the human body and found in many organ systems, including the heart, kidneys, pancreas, spleen, bone and muscle[5].

The majority of daily copper intake is from vegetables and legumes, with other sources such as various meats. On average, vegetable sources of dietary copper require a more complex enzymatic process for absorption, compared to non-vegetable sources such as meat or milk. The variable amount of copper in food sources is dependent on the various amounts of copper in the soil as well as different food processing techniques[1,6]. When copper is ingested via food sources, dietary absorption of copper predominantly occurs in the stomach and small bowel, with only approximately 30%-40% of ingested copper being absorbed by those living in industrialized countries. However, depending on dietary intake of copper, the human body can theoretically absorb as much as 63%-67% in a copper deficient diet, or as little as 12% in those whose copper intake is very high. The high acidic environment in the stomach is believed to cause the release of copper from natural organic complexes. Subsequently, absorption in the small bowel is influenced by a change in the pH as well as pancreatic enzymes[6-8].

Metallothionein within the absorptive cells of the bowel are able to bind copper via mercaptide bonds and then release it in the plasma cell membrane on the serosal side. After being released from the intestinal mucosa, copper is bound to amino acids and albumin in the portal venous system. A small portion of the copper in the portal venous system is able to pass through to the systemic circulation, while the remainder is transferred into the cytosol of hepatocytes via cell membrane receptors. Within the liver, copper is bound to various proteins, but preferentially metallothionein[5,9].

The liver is a critical organ in the systemic regulation of copper metabolism and the maintenance of copper homeostasis. Wilson’s disease (WD) is an inherited copper metabolism disorder caused by mutation of the ATP7B gene located on chromosome 13, for which numerous specific mutations have been identified[10-12]. Long-Evans Cinnamon rat, an animal model of WD, has a deletion in the copper transporting ATPase gene and develops hereditary hepatitis followed by spontaneous hepatocellular carcinoma (HCC)[13]. When these rats are treated with the copper chelating agent D-pencillamine, as commonly used in humans with WD, prevention of the onset of hepatitis and the inhibition of elevated serum transaminases were observed[14]. Togashi et al[14] therefore concluded that abnormal copper accumulation in the liver of Long-Evans Cinnamon rats was associated with the pathogenesis of hereditary hepatitis and subsequent development of HCC. Both low and high molecular weight copper binding species have been described. The high molecular weight species predominate in gallbladder bile, while low molecular weight species are more prevalent in hepatic bile. The low molecular weight species are thought to assist in the membrane transport of copper across the biliary canaliculus. The high molecular weight portion of copper binding species is principally related to copper homeostasis[9,15]. This is supported by the inability to adequately remove hepatic copper in the absence of the higher molecular weight copper binding species, in the setting of protein synthesis inhibitors[16]. Copper that is tightly bound to bile salts is unable to be absorbed in the gastrointestinal tract, and is lost in feces, which is the predominant route of excretion[5,6,9,17].

The plasma concentration of copper has been shown to increase throughout life, peaking around the age of 60, and then having a minimal decline[18]. This process is thought to be related to a progressive reduction in biliary clearance later in life, rather than an increase in gastrointestinal absorption[6,19]. Differences in the plasma concentration of copper have also been demonstrated due to gender, with females on average having higher concentrations than men. Women between the ages of 20 and 59 were shown to absorb more and have a quicker turnover of radiolabeled copper in a meal, when compared to men. Higher levels of ceruloplasmin are also present in females[18].

HCC is the fifth most common cancer worldwide. It is the third leading cause of cancer-related death worldwide. Overall, about 75%-80% of HCC occurs in patients with hepatitis B or C, with many other known risk factors including aflatoxin B1, obesity, alcohol usage, diabetes, and tobacco[20,21]. It was demonstrated that the copper content in hepatic parenchyma of patients with HCC was significantly higher than in those without HCC, with no significant difference in hepatic iron levels. In fact, the copper liver level was the only significant factor associated with the presence of HCC in the cohort of patients with hepatitis C and chronic liver disease[22]. There were reports of an increased incidence of HCC in patients diagnosed with WD[23,24]. The copper content and level of copper binding proteins in HCC has been shown to be higher than those seen in other liver malignancies such as cholangiocellular carcinoma and metastatic tumors[25,26]. In addition, the serum copper to zinc ratio was significantly higher in patients with HCC than matched controls[27].

There have also been reports of a decreased incidence of HCC in patients with copper metabolism disorders[28]. It has been proposed that WD patients treated with D-penicillamine have an elevated risk of developing HCC[29]. This may be secondary to the associated decrease in copper content in the liver, when on chelation therapy. This discrepancy could reflect either a carcinogenic or a protective role of copper in the pathogenesis of HCC, which remains to be elucidated in further studies.

Currently, the detection of liver masses is predominantly evaluated using anatomic imaging modalities[30], such as ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI). Molecular imaging is gaining momentum and is being used in various disease states[31]. Positron emitting fluorine-18-2-deoxy-gluocose (F-18 FDG) is a radioactive tracer used for the assessment of glucose metabolism in both benign and malignant tissues. After being delivered to the cells via blood flow, F-18 FDG is transported by GLUT transporters and then phosphorylated intracellularly. Typically, the FDG-6-phosphatase is trapped within the cells, unless there is a high level of phosphatase activity, as seen in the liver[32]. Secondary to the high level of phosphatase in the liver, the sensitivity for detecting well differentiated HCC is poor. However, there is usually high F-18 FDG uptake in moderately and poorly differentiated HCC. Positron emission tomography/computed tomography using F-18 FDG (F-18 FDG PET/CT) is also useful for the detection of recurrence and extrahepatic metastasis of HCC[33,34].

The sensitivity of FDG PET/CT in the detection of HCC was found to be approximately 50%, compared to the sensitivity of CT (75%)[35]. However, Wang et al[36] showed improved performance in the detection of HCC when an early dynamic F-18 FDG PET/CT was performed 240 s after tracer injection. Even better detection rates were obtained when early dynamic and conventional delayed whole body information was used in combination. The detection rates improved from 56.7% to 91.9% when using whole body delay versus the combination of early dynamic and whole body scans, respectively. In patients who were scheduled to undergo liver transplantation, F-18 FDG PET/CT was found to be useful for predicting microvascular invasion by HCC. The presence of microvascular invasion by HCC was predicted when the ratio of maximum standardized uptake value (SUV) of HCC to mean SUV of normal liver parenchyma was 1.2 or greater[37].

C-11 acetate, a tracer that evaluates free fatty acid synthesis, may have better sensitivity than that of F-18 FDG[38]. According to a study performed by Ho et al[39], the sensitivity of HCC detection in patients with less than 3 lesions was 87% for C-11 acetate and 47% for F-18 FDG. When this was correlated with histologic findings, it appears that well differentiated tumors were better detected by C-11 acetate, while the poorly differentiated tumors were better detected by F-18 FDG. None of the non-HCC tumors demonstrated abnormal C-11 acetate uptake. The use of dual phase C-11 acetate, using the change in uptake values in early and conventional imaging, correctly differentiated between small, 1-3 cm, well differentiated HCC from focal nodular hyperplasia and hemangiomas[40].

The tracer C-11 choline is used to evaluate the metabolism of phospholipids subsequently used as constituents of the cell membrane. F-18 FDG negative HCC showed elevated uptake of C-11 choline, which was predominantly seen in the moderately differentiated group[41]. F-18 fluorocholine has also been shown to perform better than F-18 FDG for well differentiated HCC, with a combination of both tracers appearing to be the best option[42]. Compared to a single modality, a combination of imaging modalities, including F-18 FDG PET, CT, MRI and ultrasound, currently has higher sensitivity, with minimal effects on specificity[38].

Continuous efforts are being made to develop new tracers for molecular imaging of HCC. Radioactive copper has been used for the assessment of copper metabolism disorders in patients diagnosed with WD using nuclear imaging for at least 45 years[43-46]. When exploring copper metabolism as a biomarker for molecular imaging of HCC, Peng et al[47], for the first time, demonstrated that mouse extrahepatic hepatoma could be visualized by PET using copper-64 chloride (⁶⁴CuCl₂) as a tracer, based on increased copper uptake mediated by mouse copper transporter 1 (mCtr1). There was relatively less ⁶⁴Cu uptake in the hepatoma compared to the liver, which was thought to be related to several factors: less mCtr1 in the tumor relative to the liver, the possibility that endogenous mCtr1 may be less active on the tumor, other copper transporters in normal hepatocytes not expressed on the tumor, and more rapid efflux of copper in tumor cells than in normal hepatocytes[48]. More recently, human HCC xenografts in athymic mice were also visualized by PET after intravenous injection of ⁶⁴CuCl₂ as a tracer[49]. There was abundant physiologic distribution of ⁶⁴Cu in the liver, which resulted in limited evaluation of primary HCC. Given the normal intense uptake of FDG by cortical brain tissue and low physiological cerebral uptake of ⁶⁴Cu[47-53], ⁶⁴CuCl₂-PET is a promising technique for non-invasive assessment of intracranial and other extrahepatic metastasis of HCC located in areas with low physiological copper uptake (Figure 1). The prognosis for patients with intracranial HCC metastasis is poor as they are often resistant to systemic chemotherapy. The use of ⁶⁴CuCl₂-PET/CT for early detection of HCC intracranial metastasis is significant for improving the prognosis of patients with metastatic HCC. On the other hand, ⁶⁴CuCl₂-PET is expected to be useful for excluding extra-hepatic metastases in pre-transplant work up of patients who are considered candidates for liver transplantation. Positron emitting ⁶⁴Cu radionuclide has a half-life of 12.7 h, making it possible to ship it to an imaging facility distant from the production site of this radiotracer. Preclinical radiation dosimetry of ⁶⁴CuCl₂ using the Atp7b^-/- knockout mouse model of WD was comparable to that of F-18 FDG[50], supporting the use of ⁶⁴CuCl₂ as a radiotracer for PET of HCC metastasis, with the exception of the metastatic lesions in the abdomen due to excreted ⁶⁴Cu in the intestinal tract.

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Figure 1 Metabolic imaging of metastasis of hepatocellular carcinoma with ⁶⁴CuCl₂-position emission tomography and computed tomography. A: Preclinical metabolic imaging of HCC xenografts in mice injected with ⁶⁴CuCl₂ as a tracer. ⁶⁴Cu bound to copper binding molecules in the blood immediately after intravenous injection of ⁶⁴CuCl₂. PET/CT images were then obtained that show the expected biodistribution of ⁶⁴CuCl₂ in the liver and intestinal tracts, with low uptake in the brain and muscle tissues. The HCC xenografts implanted on the shoulder showed increased ⁶⁴Cu uptake on PET/CT images; B: Schematic showing the clinical perspective of metabolic imaging of HCC in humans. Human patients may be injected with ⁶⁴CuCl₂ and subjected to PET/CT for detection of HCC metastasis in areas of low physiological ⁶⁴Cu uptake, such as brain and musculoskeletal tissues. HCC: Hepatocellular carcinoma; PET/CT: Hybrid positron emission tomography and computed tomography.

Early detection and treatment are most critical for reducing mortality in patients with HCC[54,55]. The use of conventional transarterial chemoembolization (TACE) for the treatment of unresectable HCC has been found to improve the overall survival of patients compared to available supportive care[56]. The use of cisplatin or doxorubicin in a large review comparing chemoembolization showed a significant benefit compared to embolization alone[57]. A major limitation in the literature regarding TACE is the lack of consistent methods between various investigators[56]. The use of TACE with drug eluting beads (DEB-TACE), primarily using doxorubicin allows for slow drug release and lower levels of systemic chemotherapeutic agents when compared to TACE using lipiodol[58]. Although no survival benefit was shown, Malagari et al[59] were also able to show longer times to progression, less recurrence, and a better local response when using doxorubicin-eluting beads compared to bland embolization. Despite additional studies not showing a difference in radiographic response, survival or adverse events[60], Sieghart et al[56], still recommend that any future trials should include drug eluting bead TACE secondary to lower systemic levels of doxorubicin and then a possible reduction in drug-drug interactions.

The ability to bridge a patient to liver transplant has been achieved using several types of neo-adjuvant therapies including TACE, radiofrequency ablation, trans-arterial radioembolization (TARE), external beam radiotherapy and surgical resection. Bridging has been shown to decrease waiting list dropout, reduce HCC recurrence, and improve post-transplant survival with the goal of obtaining similar post-transplant outcomes to non-HCC patients[61].

Palliation for patients with end-stage or terminal HCC includes various options, with the primary goal of improving patient symptoms rather than definitive treatment[62]. Average survival for patients with end-stage or terminal HCC is 3-4 mo, and includes about 15%-20% of all HCC patients at presentation. The treatment options for end-stage disease are opiates, acetaminophen and corticosteroids[62]. HCC can be difficult to treat despite significant efforts devoted to the development of effective therapies for the treatment of this devastating disease[55]. Continuous efforts are being made to identify new targets for the treatment of HCC. Angiogenesis is an important pathway in tumor growth and copper is an important angiogenic factor for tumor growth[63]. Copper has been shown to be a cofactor in several mediators of angiogenesis including angiogenin, matrix metalloproteinase and fibroblast growth factor[64-66]. Moriguchi et al[67] demonstrated the antiangiogenic effects of the copper chelator, trientine dihydrochloride, on HCC in a rat model. Other groups have also shown that the copper chelator, pencillamine, together with diet modification can lower copper levels and microvascular density in cerebral rabbit models. Brem et al[68] also concluded that using pharmacologic withdrawal and dietary depletion of copper suppressed intracerebral tumor angiogenesis. However, prolonged anti-copper cancer therapy with copper chelators or long-term use of D-pencillamine for anti-inflammatory treatment in rheumatoid arthritis has been shown to cause toxicity such as bone marrow suppression, rash and neurologic symptoms[69,70]. Significant advancement has been made in understanding the molecular biology of copper transporters and chaperons regulating cellular copper homeostasis[71]. Recent advances in understanding the role of copper in the signal transduction pathway of cellular proliferation[53,72-76] support further study of copper metabolism as a target for molecular therapy of HCC. The selection of patients with copper hypermetabolic, metastatic HCC using ⁶⁴CuCl₂-PET/CT may be helpful for improving the efficacy of anti-copper therapy of HCC. Human copper transporter 1 (hCtr1) is a high affinity copper transporter which mediates cellular copper uptake in humans[77]. To overcome the side effects of anti-copper therapy with long-term or high-dose use of copper chelators, RNAi-mediated knockdown of hCtr1[53] may be a promising approach for targeted anti-copper therapy of HCC.

The use of external beam radiation for HCC has been limited as the liver is considered a radiosensitive organ, which may have led to early under-dosing of patients[78]. This limitation can be compounded when HCC occurs in the setting of an already diseased liver as seen with hepatitis C. Radiation-induced liver disease in patients subjected to external beam radiation can cause endothelial damage, platelet activation, fibrin thrombus and venous occlusion. These changes can lead to subsequent hepatic fibrosis. However, there may be a role for radiotherapy in patients with tumors that are in challenging locations, for palliative purposes, a bridge to transplant, or in combination with other treatment options[79]. External beam radiation as well as percutaneous cementoplasty has been used for palliative purposes with successful management of symptoms[80,81].

The targeted delivery of radionuclide therapy has been carried out by intra-arterial delivery of various conjugates radiolabeled with therapeutic radioisotopes including yttrium-90, iodine-131, holmium-166 and rhenium-188[82,83]. Yttrium-90 labeled microspheres are used for interventional radionuclide therapy of HCC[84]. Currently, there are both glass- and resin-based particles available for radioembolization of HCC. The glass-based form has a smaller size with a reduced embolic effect and lower incidence of post-embolization syndrome. One limitation of TACE is possible decompensation of the liver after use in patients with hepatic artery and portal thrombus. The use of Y-90 glass microspheres in patients with HCC and branch or lobar portal vein thrombosis showed favorable tumor response rates and was safe in a trial which included 108 patients[85]. Y-90 does not emit gamma rays and is therefore not optimal for imaging. In contrast, Rhenium-188 is a therapeutic radionuclide with a physical half-life of 16.9 h and emits both beta and gamma rays. The use of intra-arterial Rhenium-188-conjugates for radioembolization of HCC has been shown to inhibit tumor growth[86]. Attempts were also made to develop I-131 radiogene therapy of HCC based on tumor-specific expression of the human sodium/iodide symporter (hNIS) under control of the alpha fetoprotein promoter and enhancer[87-89]. Tumor-specific expression of the hNIS in HCC cells was achieved by transfection of HCC cells with a vector encoding the hNIS gene driven by an alpha fetoprotein promoter/enhancer. Increased uptake of I-131 by the cells expressing hNIS was detected by gamma counting in vitro and by imaging with a gamma camera in vivo. Growth of extrahepatic tumor xenografts derived from cells expressing hNIS was inhibited, secondary to radiation effects of ¹³¹I accumulated in the transfected HCC cells expressing hNIS[89]. The development of technologies to allow safe and efficient delivery of the vector encoding the hNIS gene is critical for the clinical application of I-131 radiogene therapy of HCC, based on the findings in preclinical studies.

Multiple copper isotopes are available for cancer imaging and therapy[90-93]. Copper-64 emits both β⁺ and β^- particles and has potential as a theranostic copper radionuclide for both cancer imaging and therapy. Apelgot et al[94] demonstrated that ⁶⁴Cu had a lethal effect in mammalian cells similar to that of ⁶⁷Cu radionuclide. Significant efforts have been made to develop ⁶⁴Cu-radiolabeled conjugates for cancer imaging and therapy[95-99]. Based on its simplicity and increased tumor uptake of ⁶⁴Cu demonstrated by PET[47-49,52,53,100,101], ionic ⁶⁴CuCl₂ has potential as a therapeutic radiopharmaceutical for the treatment of tumors expressing high levels of hCtr1. Recently, it was reported that growth of malignant melanoma overexpressing hCtr1 was suppressed in mice treated with ⁶⁴CuCl₂[102]. In addition to its potential as a reporter gene for tracking gene delivery with PET, targeted overexpression of hCtr1 may be used for copper radiogene therapy of tumors expressing low levels of endogenous hCtr1[103]. The findings from preclinical studies support further investigation of ionic ⁶⁴CuCl₂ as a radiopharmaceutical for targeted radionuclide therapy of HCC, in addition to copper modulation therapy with copper chelators (Figure 2).

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Figure 2 Perspective on targeting copper metabolism for the treatment of hepatocellular carcinoma. A: Copper metabolism in hepatocytes. Copper metabolism in hepatocytes is regulated by a network of copper transporters and chaperons. Following copper uptake mediated by the influx copper transporter, hCtr1, copper is transported intracellularly by copper chaperons and copper homeostasis is maintained by the outflow of copper mediated by the efflux copper transporter, ATP7B; B: Targeting copper metabolism for the treatment of hepatocellular carcinoma (HCC). Copper is required for cell proliferation and may play a role in the signaling transduction pathway regulating proliferation of HCC cells. Targeting copper metabolism with copper chelators has been tested for anti-copper therapy of HCC, with variable response. RNAi-mediated knockdown of hCtr1 and/or other copper chaperons is a potential new approach for targeted anti-copper gene therapy of HCC. Furthermore, ionic ⁶⁴CuCl₂ or ⁶⁷CuCl₂ have potential as new radiopharmaceuticals for systemic radionuclide therapy of HCC, based on increased ⁶⁴Cu uptake of HCC visualized on preclinical PET/CT images. hCtr1: Human copper transporter 1; ATOX-1: Antioxidant 1; Cox 17: Cytochrome c oxidase 17; CCS: Copper chaperone for superoxide dismutase; SOD1: Superoxide dismutase 1; GSH: Glutathione; MT: Metallothionein; ATP7A: Copper-transporting ATPase 1; ATP7B: Copper-transporting ATPase 2; PET/CT: Hybrid position emission tomography and computed tomography.

Copper is a transitional metal required for the regulation of cell proliferation and angiogenesis. The exact role of copper in the development of HCC is still poorly understood, as demonstrated by the paradoxical suppression or increase of HCC in patients with copper metabolic disorders such as WD. The findings of increased uptake of radioactive copper by extrahepatic HCC xenografts in mice invite clinical exploration of altered copper metabolism as a new imaging biomarker for metabolic imaging of HCC metastasis with PET using ⁶⁴CuCl₂ as a radioactive tracer. In addition, copper metabolism has potential as a target for copper modulation gene therapy of HCC based on RNAi-mediated knockdown of hCtr1 followed by administration of copper chelators. Furthermore, ⁶⁴CuCl₂ or ⁶⁷CuCl₂ may be used as radiopharmaceuticals for radionuclide therapy of HCC and ablation of extrahepatic HCC metastasis.