Correlation of hypoxia PET tracer uptake with hypoxic radioresistance in cancer cells: PET biomarkers of resistance to stereotactic radiation therapy?

Purpose: The pO₂ threshold of an ideal PET hypoxia tracer for radiotherapy planning in cancer would match those observed in clinically and biologically relevant processes such as radioresistance and HIF1α expression. To identify such tracers, we directly compared uptake in vitro of hypoxia PET tracers ([¹⁸F]FMISO, [⁶⁴Cu]CuATSM, and analogues [⁶⁴Cu]CuATS, [⁶⁴Cu]CuATSE, [⁶⁴Cu]CuCTS, [⁶⁴Cu]CuDTS, [⁶⁴Cu]CuDTSE, [⁶⁴Cu]CuDTSM) with levels of radioresistance and HIF1α expression in cultured cancer cells under identical hypoxic conditions ranging from extreme hypoxia to normoxia. Pimonidazole uptake was also compared as a marker of hypoxia.

Methods: A custom-built hypoxia apparatus enabled all experiments to be performed under identical hypoxic conditions with constant measurement of pO₂ in media using an OxyLab pO₂™ probe. HCT116 human colonic carcinoma and MCF-7 human Caucasian breast adenocarcinoma cells were irradiated using a cobalt teletherapy unit. Clonogenic assays were used to assess survival. HIF1α expression was determined by western blotting, tracer uptake by gamma counting and pimonidazole binding by flow cytometry.

Results: Radioresistance, pimonidazole binding and HIF1α expression increased gradually as pO₂ decreased between 25 mmHg and 0 mmHg. In contrast, all the PET hypoxia tracers showed a sharp increase in uptake only when pO₂ levels fell below 1 mmHg. Above this threshold, tracer uptake was not elevated above that in normoxic cells.

Conclusion: This study highlights an important mismatch in pO₂ thresholds between these PET tracers and other markers of hypoxia: tracer uptake only occurred at oxygen levels that were well below levels that induced radioresistance, pimonidazole uptake and HIF1α expression. Although their pO₂ thresholds do not match the threshold for resistance to conventionally fractionated radiotherapy (pO₂ 2.5-10 mmHg), their specificity for extreme hypoxia (pO₂ ≪ 1 mmHg) suggests these PET tracers may be of particular use to predict outcomes in stereotactic radiation therapy where these maximally resistant cells play a key role in determining the biological effect.