Alteration of Cellular Reduction Potential Will Change ⁶⁴Cu-ATSM Signal With or Without Hypoxia

Therapies targeting reductive/oxidative (redox) metabolism hold potential in cancers resistant to chemotherapy and radiation. A redox imaging marker would help identify cancers susceptible to redox-directed therapies. Copper(II)-diacetyl-bis(4-methylthiosemicarbazonato) (Cu-ATSM) is a PET tracer developed for hypoxia imaging that could potentially be used for this purpose. We aimed to demonstrate that Cu-ATSM signal is dependent on cellular redox state, irrespective of hypoxia. Methods: We investigated the relationship between ⁶⁴Cu-ATSM signal and redox state in human cervical and colon cancer cells. We altered redox state using drug strategies and single-gene mutations in isocitrate dehydrogenases (IDH1/2). Concentrations of reducing molecules were determined by spectrophotometry and liquid chromatography-mass spectrometry and compared with ⁶⁴Cu-ATSM signal in vitro. Mouse models of cervical cancer were used to evaluate the relationship between ⁶⁴Cu-ATSM signal and levels of reducing molecules in vivo, as well as to evaluate the change in ⁶⁴Cu-ATSM signal after redox-active drug treatment. Results: A correlation exists between baseline ⁶⁴Cu-ATSM signal and cellular concentration of glutathione, nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NADH). Altering NADH and NADPH metabolism using drug strategies and IDH1 mutations resulted in significant changes in ⁶⁴Cu-ATSM signal under normoxic conditions. Hypoxia likewise changed ⁶⁴Cu-ATSM signal, but treatment of hypoxic cells with redox-active drugs resulted in a more dramatic change than hypoxia alone. A significant difference in NADPH was seen between cervical tumor orthotopic implants in vivo, without a corresponding difference in ⁶⁴Cu-ATSM signal. After treatment with β-lapachone, there was a change in ⁶⁴Cu-ATSM signal in xenograft tumors smaller than 50 mg but not in larger tumors. Conclusion:⁶⁴Cu-ATSM signal reflects redox state, and altering redox state impacts ⁶⁴Cu-ATSM metabolism. Our animal data suggest there are other modulating factors in vivo. These findings have implications for the use of ⁶⁴Cu-ATSM as a predictive marker for redox therapies, though further in vivo work is needed.