Radio-copper-labeled Cu-ATSM: an indicator of quiescent but clonogenic cells under mild hypoxia in a Lewis lung carcinoma model

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Abstract

The purpose of this study is to reveal characteristics of 64Cu-labeled diacetyl-bis(N4-methylthiosemicarbazone) ([64Cu]Cu-ATSM) during cell proliferation and hypoxia by autoradiography imaging and immunohistochemical staining.

Methods

The intratumoral distributions of [64Cu]Cu-ATSM and [18F]-2-fluoro-2-deoxy-D-gloucose ([18F]FDG) in mice implanted with Lewis lung carcinoma (LLC1) tumor cells according to dual autoradiography were compared with the immunohistochemical staining patterns of proliferating markers [Ki-67 and 5-bromo-2′-deoxyuridine (BrdU)] and a hypoxic marker (pimonidazole). A clonogenic assay was performed using the cells of LLC1 tumor-implanted mice, and it was compared with the distribution of [64Cu]Cu-ATSM.

Results

[64Cu]Cu-ATSM mainly accumulated at the edge of tumors, whereas [18F]FDG was distributed inside the tumor and inside the [64Cu]Cu-ATSM accumulation. The number of Ki-67-positive cells/area tended to increase with [18F]FDG accumulation and decrease with [64Cu]Cu-ATSM accumulation. On the other hand, the number of BrdU-positive cells/area was negatively correlated with [18F]FDG accumulation and positively correlated with [64Cu]Cu-ATSM accumulation. High [64Cu]Cu-ATSM accumulation was found outside the high-[18F]FDG-accumulation and pimonidazole-positive regions. Colony formation ability was significantly higher in the tumor cells obtained from high-[64Cu]Cu-ATSM-accumulation regions than the cells from the intermediate- and the low-accumulation regions.

Conclusion

[64Cu]Cu-ATSM accumulation regions in tumor cells indicate quiescent but clonogenic tumor cells under mild hypoxia. [64Cu]Cu-ATSM could play an important role in planning appropriate tumor radiotherapy.

Introduction

Hypoxia is a key microenvironmental factor for tumor development; not only does it stimulate angiogenesis and glycolysis for tumor expansion, but it also induces cell cycle arrest and genetic instability with tumor progression [1]. In addition, hypoxic regions in solid tumors are known to be resistant to radiotherapy as well as chemotherapy [2]. Thus, precise detection of hypoxic regions in tumors is of importance to predict tumor malignancy and therapeutic outcome.

Radio-copper-labeled Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) has been developed as a positron emission tomography (PET) agent for hypoxia imaging [3], [4], [5], [6] as well as an internal radiotherapy agent that allows selective delivery of β-emitting Cu nuclides [7], [8]. Clinical study has indicated the usefulness of radio-copper-labeled Cu-ATSM for predicting the prognosis of radiotherapy in several types of cancer [9], [10]. A basic comparative study of [64Cu]Cu-ATSM and immunohistochemical staining revealed that high-[64Cu]Cu-ATSM regions demonstrate fewer Ki-67-positive “proliferating” cells and lower vascularity, but a slight increase in apoptotic cells (although this was less than 1%), when compared with low-[64Cu]Cu-ATSM regions [11]. These findings are consistent with the known characteristics of hypoxic tumor masses.

However, we also found that intratumor [18F]-2-fluoro-2-deoxy-D-gloucose ([18F]FDG) uptake, an indication of glycolysis, was not positively correlated with “hypoxia” as shown by [64Cu]Cu-ATSM uptake [11], [12]. In addition, the intratumor distribution of [64Cu]Cu-ATSM is reported to be different from that of F-18-fluoromisonidazole ([18F]F-MISO), a traditional hypoxia marker [13], [14]. Considering these differing findings, radio-copper-labeled Cu-ATSM might visualize different aspects of hypoxia than traditional nitroimidazole compounds such as [18F]F-MISO.

Supply of oxygen as well as nutrition is limited to the range of 100 to 200 μm from the vessel, and outside this range, it should become necrotic. Positron emission tomography with 3- to 5-mm resolution cannot visualize such microenvironment; hence, the hypoxic region in the PET image should be evaluated as a mixture or an average of heterogenous phenotypes.

In the present study, we compared the intratumor distribution of [64Cu]Cu-ATSM with [18F]FDG, a marker of glycolysis that is known to be enhanced under hypoxic conditions, macroscopically. Pimonidazole staining was also performed as a “low-oxygen, tension-specific” probe, at macroscopic as well as microscopic levels. The intratumor distribution of the three “hypoxia”-seeking probes with different aspects was compared with immunohistochemical staining of Ki-67 and 5-bromo-2′-deoxyuridine (BrdU) to elucidate the regional proliferation status, which is considered as an important feature of tumor cells. Regional clonogenicity was also examined as another aspect of tumor cells. Based on these results, possible interpretation of PET images obtained with radiolabeled Cu-ATSM, FDG and nitroimidazole probes was discussed.

Section snippets

Radiopharmaceutical synthesis

64Cu was produced in a small biomedical cyclotron at the Biomedical Imaging Research Center at the University of Fukui, Japan, according to a published method [12]. [64Cu]Cu-ATSM was synthesized by mixing 200 mM of glycine buffer containing 64Cu and H2ATSM in dimethyl sulfoxide (1:100 by mole ratio), as described previously [6]. The radiochemical purity of synthesized [64Cu]Cu-ATSM was >99%, as evaluated by high-performance liquid chromatography (LC-10ADVP; Shimadzu, Kyoto, Japan) using a

Intratumor distribution of [18F]FDG and [64Cu]Cu-ATSM

To analyze the intratumor distributions of [64Cu]Cu-ATSM and [18F]FDG, we performed dual autoradiography with mouse-implanted LLC1 tumors. Representative images are shown in Fig. 1. [64Cu]Cu-ATSM mainly accumulated at the edge of the tumors, and no accumulation was seen in the center where the cells were necrotic. On the other hand, the highest uptake region of [18F]FDG was seen inside that of [64Cu]Cu-ATSM. The most intense regions of [18F]FDG and [64Cu]Cu-ATSM staining, which were colored

Discussion

Copper-ATSM is a stable Cu(II) complex with high membrane permeability and is able to pass through the cell membrane between the blood and tissues instantly. Under abnormally high-NADH conditions such as hypoxia, however, Cu(II) in the Cu(II)-ATSM complex can be easily reduced to Cu(I) by a mitochondrial electron transport enzyme (NADH dehydrogenase) in an NADH-dependent manner. Hypoxia-selective reduction occurs in tumor cells also [4], but reduction in tumors is mediated by microsome

Conclusion

We have demonstrated that regions of accumulation of [64Cu]Cu-ATSM indicate quiescence in tumor cells and clonogenic tumor cells under mild hypoxia in an LLC1-bearing animal model. [64Cu]Cu-ATSM would be a tool to provide information on disease prognosis and radiotherapy planning, though further basic as well as clinical studies would be needed to clarify its precise usefulness.

Acknowledgments

The authors thank Shingo Kasamatsu, Satonao Nakakoji and the rest of the staff of the Biomedical Imaging Research Center and Nobuo Takimoto of the Department of Pathology at the University of Fukui, Japan. This study was partly funded by a Grant-in-Aid in Scientific Research from the Japan Society for the Promotion of Science (20791185) and the 21st Century COE Program (Medical Science).

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