Washington University in St. Louis

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Metabolomics to elucidate novel biochemical mechanisms of disease
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Altering Cellular Reducing Potential Changes 64Cu-ATSM Signal with or without Hypoxia

Floberg JM, Wang L, Bandara N, Rashmi R, Mpoy C, Garbow JR, Rogers BE, Patti GJ, and Schwarz JK
Altering Cellular Reducing Potential Changes 64Cu-ATSM Signal with or without Hypoxia
Journal of Nuclear Medicine, in press, 2019

Background: 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 positron emission tomography (PET) tracer developed for hypoxia imaging that could potentially be used for this purpose. We aimed to demonstrate that Cu-ATSM signal is dependent upon 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 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, and 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, 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. There was a change in 64Cu-ATSM signal in xenograft tumors <50mg after treatment with β-lapachone, 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.

Washington University, Departments of Chemistry, Genetics, and Medicine. Saint Louis, Missouri 63110 USA