Intracellular oxygen transient quantification in vivo during Ultra-High Dose Rate (UHDR) FLASH radiotherapy

Purpose Large, rapid extracellular oxygen transients (ΔpO2) have been measured in vivo during Ultra-High Dose Rate (UHDR) radiotherapy; however, it has been unclear if this matches intracellular oxygen levels. Here, the endogenously produced Protoporphyrin IX (PpIX) delayed fluorescence (DF) signal was measured as an intracellular in vivo oxygen sensor to quantify these transients, with direct comparison to extracellular pO2. Intracellular ΔpO2 is closer to the cellular DNA, the site of major radiobiological damage and therefore should help elucidate radiochemical mechanisms of the FLASH effect and potentially be translated to human tissue measurement. Methods & Materials Protoporphyrin IX (PpIX) was induced in mouse skin through intraperitoneal injection of 250mg/kg of aminolevulinic acid. The animals were also administered a 50 µL intradermal injection of 10 µM Oxyphor G4 (PdG4) for phosphorescence lifetime pO2 measurement. Paired oxygen transients were quantified on leg or flank tissues, while delivering 10 MeV electrons in 3 µs pulses at 360 Hz for a total dose of 10-28Gy. Results Transient reductions in pO2 were quantifiable in both PpIX delayed fluorescence and Oxyphor phosphorescence, corresponding to intracellular and extracellular pO2 values, respectively. Reponses were quantified for 10, 22, and 28 Gy doses, with ΔpO2 being proportional to dose on average. The ΔpO2 values were dependent upon initial pO2 in a logistic function. The average and standard deviations in ΔpO2 per dose were 0.56±0.18 mmHg/Gy and 0.43±0.06 mmHg/Gy for PpIX and Oxyphor, respectively, for initial pO2>20 mmHg. While there was large variability in the individual animal measurements of ΔpO2, the average values demonstrated a direct and proportional correlation between intracellular and extracellular pO2 changes, following a linear 1:1 relationship. Conclusions A fundamentally new approach to measure intracellular oxygen depletion in living tissue showed that ΔpO2 transients seen during UHDR-RT matched those taken using extracellular oxygen measurement. This approach could be translated to humans to quantify intracellular ΔpO2. The measurement of these transients could potentially allow estimation of intracellular reactive oxygen species production.