Internal radiotherapy with copper-64-diacetyl-bis (N4-methylthiosemicarbazone) reduces CD133+ highly tumorigenic cells and metastatic ability of mouse colon carcinoma

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Abstract

Introduction

64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) is an imaging agent for positron emission tomography (PET) that targets hypoxic tumors. 64Cu-ATSM is also reported to be a potential agent for internal radiotherapy. In a mouse colon carcinoma (Colon-26) model, we have shown that 64Cu-ATSM preferentially localizes in intratumoral regions with a high density of CD133+ cells, which show characteristics of cancer stem cells or cancer stem cell-like cells (collectively referred here as CSCs). In this study, we evaluated the therapeutic effect of 64Cu-ATSM in relation to CD133 expression using this model.

Methods

Systemic administration of 37 MBq 64Cu-ATSM or saline was conducted twice within a 1-week interval to mice bearing 1-week-old Colon-26 tumors (days 0–7). At day 19, tumor size measurement, flow cytometry analysis and experimental lung metastatic assay were performed. The therapeutic effect of 64Cu-ATSM on sorted CD133+ and CD133 Colon-26 cells was also examined in vitro.

Results

In vivo studies showed that 64Cu-ATSM treatment inhibited tumor growth. The percentage of CD133+ cells and metastatic ability in 64Cu-ATSM treated tumors was decreased compared with that in control animals. In vitro studies demonstrated that 64Cu-ATSM accumulated in cells under hypoxic conditions and incorporation of 64Cu-ATSM under hypoxia caused cell death in both CD133+ and CD133 cells in a similar extent.

Conclusions

64Cu-ATSM administration reduced tumor volume as well as the percentage of CD133+ cells and the metastatic ability of Colon-26 tumors. Together with our data, it is suggested that 64Cu-ATSM accumulates in regions high in CD133+ highly tumorigenic cells and kills such regions by radiation, resulting in a decrease of the percentage of CD133+ cells.

Introduction

Hypoxic tumor is related to the malignant behaviors of tumors, such as poor outcome in radiotherapy/chemotherapy and metastasis [1]. Thus, it is of great significance to develop methods for diagnosis and therapy of hypoxic tumors.

Radiolabeled Cu-diacetyl-bis (N4-methylthiosemicarbazone) (Cu-ATSM) has been developed as an imaging agent targeting hypoxic tumors for use with positron emission tomography (PET) [2], [3], [4], [5], [6], [7], [8]. Previous studies have shown that Cu-ATSM uptake is dependent upon the level of hypoxia, in vitro and in vivo. The mechanism of Cu-ATSM accumulation in hypoxic regions has been reported [4], [9], [10], [11], [12]: under highly reduced intracellular conditions such as hypoxia, Cu(II) in Cu-ATSM is reduced to Cu(I), instantly released from the ATSM ligand and trapped in the cells [4], [9], [10], [11]. It has been also shown that Cu-ATSM uptake is associated with a tumor's therapeutic resistance and metastatic potential in several tumors including cervical carcinoma and rectal carcinoma [2], [8], [13], [14].

Among the Cu radioisotopes available (60Cu, 61Cu, 62Cu, 64Cu and 67Cu), 64Cu has several advantages, including the fact that it can be readily produced with an in-hospital small cyclotron and can be used not only for imaging but also internal radiation therapy [15], [16], [17], [18]. 64Cu decays by β+ decay (0.66 MeV, 19%), β decay (0.58 MeV, 40%) and electron capture (44%), which means that this nuclide can be detected by PET and can also act as a therapeutic nuclide due to β particle emission and Auger electron emission. In addition, the half-life of 64Cu (t1/2=12.7 h) is appropriate for both diagnostic and therapeutic use. Copper-64 has been reported to have similar lethality to 67Cu for tumor treatment [19]. The therapeutic effect of 64Cu-ATSM has been demonstrated in both in vitro [17] and in vivo studies [16], [20]. In these studies, Obata et al. [17] showed that 64Cu-ATSM reduced the clonogenic survival rate of tumor cells and caused postmitotic apoptosis in vitro. Lewis et al. [16] showed that treatment with 64Cu-ATSM increased the survival time of tumor-bearing hamsters. Thus, 64Cu-ATSM is a potential agent for internal radiotherapy of tumors.

Recently, the concept of “cancer stem cells” has been developed and provides a new paradigm to understand tumor biology [21], [22]. Based on published data, it is shown that a certain type of cancer cells called cancer stem cells or cancer stem cell-like cells (collectively referred here as CSCs) are present in small numbers within tumors but contribute to difficulties in eradicating tumors by possessing high therapeutic resistance and metastatic ability [21], [22]. CD133 (Prominin-1) is a frequently used marker to identify the CSCs in various human and murine cancers, such as brain, prostate, breast, liver and colon [23], [24], [25], [26], [27], [28], [29]. It has also been reported that CD133+ cancer cells, rather than CD133 cancer cells, show high colony-forming ability and high tumor-initiating ability and possess survival advantage under hypoxia [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33]. CD133+ cells are also recognized as possessing resistance to radiotherapy/chemotherapy and metastatic potential of tumors [34], [35]. Therefore, it is considered that CD133+ cells contribute to difficulties in eradicating tumors due to their therapeutic resistance, although the CD133+ cells are present in a small population within tumors. In a previous study [34], using a mouse colon carcinoma (Colon-26) model, we demonstrated through immunohistochemistry and autoradiography analyses that 64Cu-ATSM preferentially accumulated in intratumoral regions with a relatively high density of CD133+ cells: in the study, we classified tumor sections into several areas by 64Cu-ATSM uptake and examined the density of CD133+ cells in each area. Also, our previous study showed that the CD133+ Colon-26 cells possess the typical characteristics of CSCs, such as high colony-forming ability, high tumor-initiating ability and increased survival under hypoxia. Namely, since the CD133+ cells are in a small population but possess malignant characteristics within tumors, it is important in tumor treatment to decrease the number of CD133+ cells present within tumors. In this study, we have developed a therapeutic technique that decreases the number of CD133+ cells present within the tumor. We hypothesized that internal radiotherapy with 64Cu-ATSM will reduce the number of CD133+ cells present in tumors by accumulating in CD133+ cell-rich regions. In this study, we examined the therapeutic effect of 64Cu-ATSM in relation to CD133 expression, with Colon-26 model in vivo and in vitro.

Section snippets

Cell lines and growth

We used a mouse colon carcinoma cell line, Colon-26 (TKG 0518; Cell Resource Center for Biomedical Research, Tohoku University, Sendai, Japan). The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C. Dulbecco's modified Eagle's medium (DMEM 11995-065; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and antibiotics were used for cell growth. Exponentially growing cells were used for the study. The cells were trypsinized to detach them from the plates and

Tumor volume after 64Cu-ATSM treatment in vivo

To investigate the effectiveness of the 64Cu-ATSM therapy, therapeutic doses of 64Cu-ATSM were injected into mice bearing a Colon-26 tumor. In this study, 1 mCi of 64Cu-ATSM or saline was administered twice within a 1-week interval to 1-week-old tumor-bearing mice from day 0 (n=6). Images of tumors treated by 64Cu-ATSM and control tumors on day 19 were shown in Fig. 1A. The volume of tumors treated by 64Cu-ATSM was 14-fold smaller than that of control tumors (P<.02) (Fig. 1B). There was no

Discussion

In this study, we found that 64Cu-ATSM treatment reduced not only tumor size but also the proportion of CD133+ cells and the metastatic potential in Colon-26 tumors. Our previous study demonstrated that, in Colon-26 tumors, 64Cu-ATSM accumulates in low-vascular regions with relatively high density of CD133+ cells and the CD133+ Colon-26 cells possess distinctive characteristics of CSCs, such as high colony-forming ability, high tumor-initiating ability and increased survival under hypoxic

Conclusion

This study shows that 64Cu-ATSM treatment can reduce not only the tumor volume, but also the proportion of CD133+ cells and the metastatic potential in Colon-26 tumors. Our in vitro study also showed that 64Cu-ATSM treatment was effective on both CD133+ and CD133 Colon-26 cells under hypoxia. Our previous data demonstrated that 64Cu-ATSM accumulates into low vascular–hypoxic regions with a high density of CD133+ cells in Colon-26 tumors and the CD133+ Colon-26 cells show typical

Acknowledgments

We thank the members of the Biomedical Imaging Research Centre of the University of Fukui, Japan, for discussions and S. Nakakoji, J. Yamamoto, H. Takagi and H. Maeda for technical help. This work was supported in part by the Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science, Japan (JSPS) (to Y.Y.); the Joint Research Grant from the Wakasa Wan Energy Research Center, Japan (to Y.F. and Y.Y.); the Research for Promoting Technological Seeds from Japan

References (40)

  • LewisJ.S. et al.

    Evaluation of 64Cu-ATSM in vitro and in vivo in a hypoxic tumor model

    J Nucl Med

    (1999)
  • TakahashiN. et al.

    Evaluation of 62Cu labeled diacetyl-bis(N4-methylthiosemicarbazone) as a hypoxic tissue tracer in patients with lung cancer

    Ann Nucl Med

    (2000)
  • LewisJ.S. et al.

    Tumor uptake of copper-diacetyl-bis(N4-methylthiosemicarbazone): effect of changes in tissue oxygenation

    J Nucl Med

    (2001)
  • GrigsbyP.W. et al.

    Comparison of molecular markers of hypoxia and imaging with 60Cu-ATSM in cancer of the uterine cervix

    Mol Imaging Biol

    (2007)
  • ObataA. et al.

    Retention mechanism of hypoxia selective nuclear imaging/radiotherapeutic agent Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells

    Ann Nucl Med

    (2001)
  • DearlingJ.L. et al.

    Copper bis(thiosemicarbazone) complexes as hypoxia imaging agents: structure–activity relationships

    J Biol Inorg Chem

    (2002)
  • HollandJ.P. et al.

    In vitro kinetic studies on the mechanism of oxygen-dependent cellular uptake of copper radiopharmaceuticals

    Phys Med Biol

    (2009)
  • DehdashtiF. et al.

    Assessing tumor hypoxia in cervical cancer by PET with 60Cu-labeled diacetyl-bis(N4-methylthiosemicarbazone)

    J Nucl Med

    (2008)
  • DietzD.W. et al.

    Tumor hypoxia detected by positron emission tomography with 60Cu-ATSM as a predictor of response and survival in patients undergoing neoadjuvant chemoradiotherapy for rectal carcinoma: a pilot study

    Dis Colon Rectum

    (2008)
  • LewisJ. et al.

    Copper-64-diacetyl-bis(N4-methylthiosemicarbazone): an agent for radiotherapy

    Proc Natl Acad Sci USA

    (2001)
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