<i>In vivo</i> [⁶⁴Cu]CuCl₂ PET imaging reveals activity of Dextran-Catechin on tumor copper homeostasis

Abstract

Given the strong clinical evidence that copper levels are significantly elevated in a wide spectrum of tumors, copper homeostasis is considered as an emerging target for anticancer drug design. Monitoring copper levels <i>in vivo</i> is therefore of paramount importance when assessing the efficacy of copper-targeting drugs. Herein, we investigated the activity of the copper-targeting compound Dextran-Catechin by developing a [⁶⁴Cu]CuCl₂ PET imaging protocol to monitor its effect on copper homeostasis in tumors. <b>Methods:</b> Protein expression of copper transporter 1 (CTR1) in tissue microarrays representing 90 neuroblastoma patient tumors was assessed by immunohistochemistry. Western blotting analysis was used to study the effect of Dextran-Catechin on the expression of CTR1 in neuroblastoma cell lines and in tumors. A preclinical human neuroblastoma xenograft model was used to study anticancer activity of Dextran-Catechin <i>in vivo</i> and its effect on tumor copper homeostasis. PET imaging with [⁶⁴Cu]CuCl₂ was performed in such preclinical neuroblastoma model to monitor alteration of copper levels in tumors during treatment. <b>Results:</b> CTR1 protein was found to be highly expressed in patient neuroblastoma tumors by immunohistochemistry. Treatment of neuroblastoma cell lines with Dextran-Catechin resulted in decreased levels of glutathione and in downregulation of CTR1 expression, which caused a significant decrease of intracellular copper. No changes in CTR1 expression was observed in normal human astrocytes after Dextran-Catechin treatment. <i>In vivo</i> studies and PET imaging analysis using the neuroblastoma preclinical model revealed elevated [⁶⁴Cu]CuCl₂ retention in the tumor mass. Following treatment with Dextran-Catechin, there was a significant reduction in radioactive uptake, as well as reduced tumor growth. <i>Ex vivo</i> analysis of tumors collected from Dextran-Catechin treated mice confirmed the reduced levels of CTR1. Interestingly, copper levels in blood were not affected by treatment, demonstrating potential tumor specificity of Dextran-Catechin activity. <b>Conclusion:</b> Dextran-Catechin mediates its activity by lowering CTR1 and intracellular copper levels in tumors. This finding further reveals a potential therapeutic strategy for targeting copper-dependent cancers and presents a novel PET imaging method to assess patient response to copper-targeting anticancer treatments.