Intracellular Oxygen Transient Quantification in Vivo During Ultra-High Dose Rate FLASH Radiation Therapy.

Abstract

Large, rapid extracellular oxygen transients (ΔpO₂) have been measured in vivo during ultra-high dose rate radiation therapy; however, it has been unclear if they match intracellular oxygen levels. Here, the endogenously produced protoporphyrin IX (PpIX) delayed fluorescence 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. PpIX was induced in mouse skin through intraperitoneal injection of 250 mg/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 in leg or flank tissues while delivering 10 MeV electrons in 3 µs pulses at 360 Hz for a total dose of 10 to 28 Gy. 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 found to be proportional to the dose on average. The ΔpO₂ values were dependent on initial pO₂ in a logistic function. The average and standard deviations in ΔpO₂ per dose were 0.56 ± 0.18 mm Hg/Gy and 0.43 ± 0.06 mm Hg/Gy for PpIX and oxyphor, respectively, for initial pO₂ > 20 mm Hg. Although 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. A fundamentally new approach to measuring intracellular oxygen depletion in living tissue showed that ΔpO₂ transients seen during ultra-high dose rate radiation therapy matched those quantified using extracellular oxygen measurement. This approach could be translated to humans to quantify intracellular ΔpO₂. The measurement of these transients could potentially allow the estimation of intracellular reactive oxygen species production.