Sensitive simultaneous measurements of oxygenation and extracellular pH by EPR using a stable monophosphonated trityl radical and lithium phthalocyanine.

ABSTRACT

The monitoring of acidosis and hypoxia is crucial because both factors promote cancer progression and impact the efficacy of anti-cancer treatments. A phosphonated tetrathiatriarylmethyl (pTAM) has been previously described to monitor both parameters simultaneously, but the sensitivity to tackle subtle changes in oxygenation was limited. Here, we describe an innovative approach combining the pTAM radical and lithium phthalocyanine (LiPc) crystals to provide sensitive simultaneous measurements of extracellular pH (pHe) and pO2. Both parameters can be measured simultaneously as both EPR spectra do not overlap, with a gain in sensitivity to pO2 variations by a factor of 10. This procedure was applied to characterize the impact of carbogen breathing in a breast cancer 4T1 model as a proof-of-concept. No significant change in pHe and pO2 was observed using pTAM alone, while LiPc detected a significant increase in tumor oxygenation. Interestingly, we observed that pTAM systematically overestimated the pO2 compared to LiPc. In addition, we analyzed the impact of an inhibitor (UK-5099) of the mitochondrial pyruvate carrier (MPC) on the tumor microenvironment. In vitro, the exposure of 4T1 cells to UK-5099 for 24 h induced a decrease in pHe and oxygen consumption rate (OCR). In vivo, a significant decrease in tumor pHe was observed in UK-5099-treated mice, while there was no change for mice treated with the vehicle. Despite the change observed in OCR, no significant change in tumor oxygenation was observed after the UK-5099 treatment. This approach is promising for assessing in vivo the effect of treatments targeting tumor metabolism.