Detection of Increased Cu-64 Uptake by Human Copper Transporter 1 Gene Overexpression Using PET with (CuCl2)-Cu-64 in Human Breast Cancer Xenograft Model

Abstract

Copper is an essential cofactor for a variety of biochemical processes including oxidative phosphorylation, cellular antioxidant activity, and elimination of free radicals. The copper transporter 1 is known to be involved in cellular uptake of copper ions. In this study, we evaluated the utility of human copper transporter 1 (hCTR1) gene as a new reporter gene for Cu-64 PET imaging. Methods: Human breast cancer cells (MDA-MB-231) were infected with a lentiviral vector constitutively expressing the hCTR1 gene under super cytomegalovirus promoter, and positive clones (MDA-MB-231-hCTR1) were selected. The expression of hCTR1 gene in MDA-MB-231-hCTR1 cells was measured by reverse transcription polymerase chain reaction, Western blot, and Cu-64 uptake assay. To evaluate the cytotoxic effects induced by hCTR1 expression, the dose-dependent cell survival rate after treatment with cisplatin (Cis-diaminedichloroplatinum (II) [CDDP]) was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion. Small-animal PET images were acquired in tumor-bearing mice from 2 to 48 h after an intravenous injection of Cu-64. Results: The hCTR1 gene expression in MDA-MB-231-hCTR1 cells was confirmed at the RNA and protein expression and the cellular Cu-64 uptake level. MTT assay and trypan blue dye exclusion showed that the cell viability of MDA-MB-231-hCTR1 cells decreased more rapidly than that of MDA-MB-231 cells after treatment with CDDP for 96 or 72 h, respectively. Small-animal PET imaging revealed a higher accumulation of Cu-64 in MDA-MB-231-hCTR1 tumors than in MDA-MB-231 tumors. With respect to the biodistribution data, the percentage injected dose per gram of Cu-64 in the MDA-MB-231 tumors and MDA-MB-231-hCTR1 tumors at 48 h after Cu-64 injection was 2.581 +/- 0.254 and 5.373 +/- 1.098, respectively. Conclusion: An increase in Cu-64 uptake induced by the expression of hCTR1 gene was demonstrated in vivo and in vitro, suggesting the potential use of hCTR1 gene as a new imaging reporter gene for PET with (CuCl2)-Cu-64.