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 Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry.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.
Highlights
ResultsIn vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors
Diacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia
In vivo uptake of Cu-ATSM and Cu-Cl2 in a GB model In order to investigate the uptake of 64Cu-ATSM in GB and its specificity for hypoxia, C6 glioma cells were selected based on our previous studies showing that this preclinical model presents a pronounced hypoxic component [10, 28]
Summary
In vivo uptake of Cu-ATSM and Cu-Cl2 in a GB model In order to investigate the uptake of 64Cu-ATSM in GB and its specificity for hypoxia, C6 glioma cells were selected based on our previous studies showing that this preclinical model presents a pronounced hypoxic component [10, 28]. Concerning 64Cu-Cl2, in the normal healthy brain, SUV was 0.31 ± 0.05 g/mL and 0.29 ± 0.06 g/mL at 3 h and 24 h respectively These values were significantly lower than those observed for 64Cu-ASTM at 3 h and 24 h (Fig. 1b). As a result of the low uptake observed in the healthy tissue, the tumor/contralateral ratio was greater for 64Cu-Cl2 than 64Cu-ATSM at both 3 h (p < 0.05) and 24 h (p < 0.01) (Fig. 1c). A large spatial overlap was observed between 18F-FMISO and 64Cu-ATSM by comparing 60% maximum signal or 5% signal abnormality At this late time point, an elevated uptake of the tracer was observed with SUV values larger than those of 18F-FMISO. The results sustain that if a reperfusion occurs in case of transient hypoxia in the tumor, copper transporter expression rapidly return to basal level
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