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Positron Emission Tomography of Human Hepatocellular Carcinoma Xenografts in Mice Using Copper (II)-64 Chloride as a Tracer

Posted Nov 30, 2011 Updated Apr 10, 2023
By Haiyuan Zhang PhD , Huawei Cai PhD , Xin Lu MD , Otto Muzik PhD , Fangyu Peng MD PhD
10 min read

Rationale and Objectives

The aim of this study was to assess copper metabolism of human hepatocellular carcinoma (HCC) with positron emission tomographic (PET) imaging using copper (II)-64 chloride ( 64 CuCl 2 ) as a tracer.

Materials and Methods

PET imaging of athymic mice ( n = 5) bearing extrahepatic HCC xenografts was performed 24 hours after the intravenous injection of 64 CuCl 2 , followed by ex vivo tissue radioactivity assay. Expression of human copper transporter 1 (hCTR1) in HCC cells and tissues was examined by real-time reverse transcription polymerase chain reaction and immunohistochemistry analysis, respectively.

Results

The extrahepatic HCC xenografts in mice with increased uptake of 64 Cu radionuclide were visualized on the micro-PET images obtained 24 hours after the intravenous injection of 64 CuCl 2 . PET quantitative analysis revealed increased 64 Cu radioactivity in tumor tissues (2.7 ± 0.6 %ID/g) compared to that in the soft tissue of the left shoulder opposite to the tumor site (0.6 ± 0.2 %ID/g) and the brain (0.7 ± 0.1 %ID/g) but lower than that of the liver (16.6 ± 1.3 %ID/g). Expression of hCTR1 in the HCC cells and xenograft tumor tissues was demonstrated by real-time reverse transcription polymerase chain reaction and immunohistochemistry analysis, respectively. The expression level of hCTR1 in the Hep3B HCC xenograft tissues was lower than that detected in the normal hepatic tissues and the tissue samples of well-differentiated primary HCC. Variable expression of hCTR1 was detected in the tissue samples of moderately differentiated primary HCC.

Conclusions

Extrahepatic human HCC xenografts in mice could be localized with 64 CuCl 2 PET imaging, which might be useful for the localization and quantitative assessment of copper metabolism in extrahepatic metastases of HCC in humans.

Hepatocellular carcinoma (HCC) is a threat to the health of the global population, particularly to people living in Asia and sub-Saharan Africa . Significant advances have been made in the treatment of patients diagnosed with the early stages of HCC, but the prognosis for patients with extrahepatic HCC metastases is poor, because they are often resistant to systemic chemotherapy . Moreover, the prognosis of patients with intracranial metastases of HCC is dismal . Liver transplantation is a potentially curative treatment for those patients with HCC localized within the liver . In the pretransplantation workup, however, it is essential to exclude extrahepatic metastases in those patients who are considered candidates for liver transplantation. Positron emission tomographic (PET) imaging using 2-deoxy-2-[ 18 F]-fluoro- d -glucose ( 18 F-FDG) is clinically well accepted for the staging of many human cancers and has been found to be highly sensitive for the detection of the extrahepatic spread of hypermetabolic HCC . However, 18 F-FDG PET imaging is limited for the detection of intracranial HCC metastases because of the high background of FDG uptake by the normal brain tissue. Thus, it is necessary to establish an alternative tracer that may be used for the evaluation of intracranial metastases of HCC under circumstances in which the use of 18 F FDG PET imaging is limited.

Copper is an essential nutrient in mammals, and intracellular copper homeostasis in humans is regulated by a delicate network of copper transporters , which include human copper transporter 1 (hCTR1), ATP7A, ATP7B, as well as copper chaperons antioxidant protein 1, cytochrome c oxidase 17, and copper chaperone for superoxide dismutase . Copper is required for cell proliferation and tumor growth, and high concentrations of copper have been observed in many of human tumor tissues . Extrahepatic mouse hepatoma grafts and human prostate xenografts in mice could be localized with PET imaging using 64 CuCl 2 as a tracer . In this study, PET imaging of athymic mice bearing human HCC xenografts was conducted to determine whether extrahepatic human HCC metastases could be localized with 64 CuCl 2 PET imaging. To study the molecular mechanism of copper hypermetabolism in HCC, expression of hCTR1 in HCC cells and tumor tissues was examined by quantitative real-time reverse transcription polymerase chain reaction (RC-PCR) and immunohistochemistry analysis, respectively.

Materials and methods

Cells and Animal Model

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PET Imaging

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PET Quantitative Analyses of Tracer Concentration

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Radioactivity Assay of Tissue Radiotracer Concentration

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Quantitative Real-time RT-PCR

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IHC Analysis

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Statistical Analysis

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Results

Visualization of Hep3B HCC Xenografts in Mice with 64 CuCl 2 PET Imaging

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Figure 1, Micro–positron emission tomographic (PET) imaging of human hepatocellular carcinoma (HCC) xenografts in mice with 64 CuCl 2 . The HCC xenograft with increased 64 Cu radioactivity was well visualized ( arrow ) on a representative micro-PET image of a tumor-bearing mouse injected with 64 CuCl 2 intravenously. Color scale indicates tracer concentrations in percentage of injected dose per gram of tissue (%ID/g).

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Biodistribution of 64 Cu Radioactivity by PET Quantitative Analysis

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Biodistribution of 64 Cu Radioactivity by Ex Vivo Tissue Radioactivity Assay

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Figure 2, Distribution of tissue 64 Cu radioactivity as determined by ex vivo tissue radioactivity assay in athymic mice bearing human hepatocellular carcinoma xenografts. Postmortem tissues were harvested from the tumor-bearing mice ( n = 5) 24 hours after the intravenous injection of 64 CuCl 2 . The tracer concentration of the tumor was significantly higher than that of the muscular tissue and the brain but much less than that measured from the liver. The error bars represent the standard deviation. %ID/g, percentage of injected dose per gram of tissue.

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High-level Transcription of hCTR1 in Hep3B Cells by Real-time RT-PCR

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Figure 3, Transcriptional activity of human copper transporter 1 (hCTR1) in Hep3B hepatocellular carcinoma (HCC) cells by real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real-time RT-PCR analysis demonstrated that hCTR1 messenger ribonucleic acid (mRNA) levels in the Hep3B HCC cells and the PC-3 prostate cancer cells were significantly higher than that in the nonmalignant PZ-HPV-7 prostate epithelial cells. Abundant hCTR1 mRNA molecules were also detected in the control HEK293 human embryonic kidney cells. Transcriptional activity of hCTR1 determined by quantitative real-time RT-PCR was graphed as relative values against that in the control PZ-HPV-7 cells. The error bars represent the standard deviation.

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Detection of hCTR1 Expression in Hep3B and Primary HCC Tissues by IHC Analysis

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Figure 4, Expression of human copper transporter 1 (hCTR1) in Hep3B xenograft tumor tissues by immunohistochemistry analysis. Nonhomogeneous hCTR1 immunoreactivity ( brown color shows horseradish peroxidase product) was detected in the tissue sections of postmortem Hep3B hepatocellular carcinoma xenografts (a) , compared to homogeneous hCTR1 immunoreactivity visualized on the normal hepatic tissues (b) . The tissue sections were counterstained with 4′, 6-diamidino-2-phenylindole to show cell nuclei in blue. Scale bar = 50 μm.

Table 1

Expression of hCTR1 in Malignant and Nonmalignant Hepatic Tissues by IHC Analysis

Subject Histopathologic Diagnosis hCTR1 Immunoreactivity by IHC ∗ 1 HCC, well differentiated +++ 2 HCC, well differentiated +++ 3 HCC, moderately differentiated +++ 4 HCC, moderately differentiated +++ 5 HCC, moderately differentiated +++ 6 HCC, moderately differentiated + 7 HCC, moderately differentiated + 8 HCC, moderately differentiated ++ 9 HCC, moderately differentiated + 10 Normal liver parenchyma +++ 11 Normal liver parenchyma +++ 12 Mucinous cystadenoma with steatosis ++ 13 Benign liver hemangioma with mild portal inflammation ++ 14 Cirrhotic liver with steatosis and portal inflammation + 15 Benign liver parenchyma ++

HCC, hepatocellular carcinoma; hCTR1, human copper transporter 1; IHC, immunohistochemistry.

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Figure 5, Expression of human copper transporter 1 (hCTR1) in primary hepatocellular carcinoma (HCC) tissues by immunohistochemistry analysis. Intense hCTR1 immunoreactivity ( brown color ) shows horse radish peroxidase product) was detected in the tissue sample of a moderately differentiated HCC (a) , compared to that observed in the tissue of a well-differentiated HCC (c) and normal hepatic tissue (d) . In contrast, only mild hCTR1 immunoreactivity was detected in the tissue sample of another moderately differentiated HCC (b) . The tissue sections were counterstained with 4′, 6-diamidino-2-phenylindole to show cell nuclei in blue. Scale bar = 50 μm.

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Discussion

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Acknowledgments

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Academic Radiology, Volume 18, Issue 12
Journals General Radiology
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