Internal radiotherapy with copper-64-diacetyl-bis (N4-methylthiosemicarbazone) reduces CD133+ highly tumorigenic cells and metastatic ability of mouse colon carcinoma
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)
- et al.
Assessing tumor hypoxia in cervical cancer by positron emission tomography with 60Cu-ATSM: relationship to therapeutic response — a preliminary report
Int J Radiat Oncol Biol Phys
(2003) - et al.
Cell line-dependent differences in uptake and retention of the hypoxia-selective nuclear imaging agent Cu-ATSM
Nucl Med Biol
(2005) - et al.
Production of therapeutic quantities of 64Cu using a 12 MeV cyclotron
Nucl Med Biol
(2003) - et al.
Basic characterization of 64Cu-ATSM as a radiotherapy agent
Nucl Med Biol
(2005) - et al.
Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells
Biochem Biophys Res Commun
(2006) - et al.
Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma
Mol Cancer
(2006) - et al.
Copper-64-diacetyl-bis (N4-methylthiosemicarbazone) accumulates in rich regions of CD133+ highly tumorigenic cells in mouse colon carcinoma
Nucl Med Biol
(2010) The hypoxic cell: a target for selective cancer therapy — eighteenth Bruce F. Cain Memorial Award lecture
Cancer Res
(1999)- et al.
In vivo assessment of tumor hypoxia in lung cancer with 60Cu-ATSM
Eur J Nucl Med Mol Imaging
(2003) - et al.
Copper-62-ATSM: a new hypoxia imaging agent with high membrane permeability and low redox potential
J Nucl Med
(1997)
Evaluation of 64Cu-ATSM in vitro and in vivo in a hypoxic tumor model
J Nucl Med
Evaluation of 62Cu labeled diacetyl-bis(N4-methylthiosemicarbazone) as a hypoxic tissue tracer in patients with lung cancer
Ann Nucl Med
Tumor uptake of copper-diacetyl-bis(N4-methylthiosemicarbazone): effect of changes in tissue oxygenation
J Nucl Med
Comparison of molecular markers of hypoxia and imaging with 60Cu-ATSM in cancer of the uterine cervix
Mol Imaging Biol
Retention mechanism of hypoxia selective nuclear imaging/radiotherapeutic agent Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) in tumor cells
Ann Nucl Med
Copper bis(thiosemicarbazone) complexes as hypoxia imaging agents: structure–activity relationships
J Biol Inorg Chem
In vitro kinetic studies on the mechanism of oxygen-dependent cellular uptake of copper radiopharmaceuticals
Phys Med Biol
Assessing tumor hypoxia in cervical cancer by PET with 60Cu-labeled diacetyl-bis(N4-methylthiosemicarbazone)
J Nucl Med
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
Copper-64-diacetyl-bis(N4-methylthiosemicarbazone): an agent for radiotherapy
Proc Natl Acad Sci USA
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