Detection of increased 64Cu uptake by human copper transporter 1 gene overexpression using PET with 64CuCl2 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 (64)Cu 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 (64)Cu 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-2-yl)-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 (64)Cu.

Results: The hCTR1 gene expression in MDA-MB-231-hCTR1 cells was confirmed at the RNA and protein expression and the cellular (64)Cu 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 (64)Cu 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 (64)Cu in the MDA-MB-231 tumors and MDA-MB-231-hCTR1 tumors at 48 h after (64)Cu injection was 2.581 ± 0.254 and 5.373 ± 1.098, respectively.

Conclusion: An increase in (64)Cu 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 (64)CuCl2.

Keywords: 64Cu; PET; cisplatin; hCTR1; reporter gene.