64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study

Abstract

BackgroundDiacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression.MethodsμPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels.ResultsIn vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages.ConclusionIn the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

Hypoxia imaging and theranostic potential of [64Cu][Cu(ATSM)] and ionic Cu(II) salts: a review of current evidence and discussion of the retention mechanisms

“…The same study also reported concerning stability issues of the complexing ligand in mouse blood serum and whole blood in vivo, despite being stable in mouse serum in vitro. Similarly, comparing two recent studies investigating the theranostic potentials of copper-64 with glioblastoma in similar setups but by two different groups, showed surprisingly similar results for [ 64 [68].…”

“…Accumulation of [ 64 Cu]CuCl 2 was elevated at pO 2 < 5%, but with no further significant increase at lower oxygen concentrations [68]. The authors concluded that while both [ 64 Cu][Cu(ATSM)] and [ 64 Cu]CuCl 2 could be considered as hypoxia-selective tracers, non-hypoxic accumulation in copper transporters should also be accounted for [68]. Interestingly, in an investigation of the possibility to use ] with enhanced efflux, while knockdown of MDR1 showed inverse effects, which further implies a more complex copper retention mechanism than what was previously proposed [69].…”

“…However, studies using ionic copper-64 have raised questions on the in vivo retention mechanism. As mentioned, a direct in vitro and in vivo comparison between [ 64 Cu][Cu(ATSM)] and [ 64 Cu]Cu-acetate has showed [ 68] essentially mirroring in vivo results of the copper-64 oxygen-dependent uptake and biodistribution, while the in vitro results showed significantly less hypoxia selectivity of [ 64 Cu]Cu-acetate [28]. The same study also reported concerning stability issues of the complexing ligand in mouse blood serum and whole blood in vivo, despite being stable in mouse serum in vitro.…”

“…[ 64 Cu][Cu(ATSM)] showed greater uptake in both healthy brain and malignant tumors than [ 64 Cu]CuCl 2 , while [ 64 Cu]CuCl 2 showed a greater tumor/brain ratio [68].…”

“…In vitro, elevated accumulation of [ 64 Cu][Cu(ATSM)] was detected in severe hypoxia (pO 2 < 0.5%), which further increased with decreasing oxygen concentration. Accumulation of [ 64 Cu]CuCl 2 was elevated at pO 2 < 5%, but with no further significant increase at lower oxygen concentrations [68]. The authors concluded that while both [ 64 Cu][Cu(ATSM)] and [ 64 Cu]CuCl 2 could be considered as hypoxia-selective tracers, non-hypoxic accumulation in copper transporters should also be accounted for [68].…”