Quantification of Oxygen Depletion During FLASH Irradiation In Vitro and In Vivo

Purpose

Delivery of radiation at ultrahigh dose rates (UHDRs), known as FLASH, has recently been shown to preferentially spare normal tissues from radiation damage compared with tumor tissues. However, the underlying mechanism of this phenomenon remains unknown, with one of the most widely considered hypotheses being that the effect is related to substantial oxygen depletion upon FLASH, thereby altering the radiochemical damage during irradiation, leading to different radiation responses of normal and tumor cells. Testing of this hypothesis would be advanced by direct measurement of tissue oxygen in vivo during and after FLASH irradiation.

Methods and Materials

Oxygen measurements were performed in vitro and in vivo using the phosphorescence quenching method and a water-soluble molecular probe Oxyphor 2P. The changes in oxygen per unit dose (G-values) were quantified in response to irradiation by 10 MeV electron beam at either UHDR reaching 300 Gy/s or conventional radiation therapy dose rates of 0.1 Gy/s.

Results

In vitro experiments with 5% bovine serum albumin solutions at 23°C resulted in G-values for oxygen consumption of 0.19 to 0.21 mm Hg/Gy (0.34-0.37 μM/Gy) for conventional irradiation and 0.16 to 0.17 mm Hg/Gy (0.28-0.30 μM/Gy) for UHDR irradiation. In vivo, the total decrease in oxygen after a single fraction of 20 Gy FLASH irradiation was 2.3 ± 0.3 mm Hg in normal tissue and 1.0 ± 0.2 mm Hg in tumor tissue (P < .00001), whereas no decrease in oxygen was observed from a single fraction of 20 Gy applied in conventional mode.

Conclusions

Our observations suggest that oxygen depletion to radiologically relevant levels of hypoxia is unlikely to occur in bulk tissue under FLASH irradiation. For the same dose, FLASH irradiation induces less oxygen consumption than conventional irradiation in vitro, which may be related to the FLASH sparing effect. However, the difference in oxygen depletion between FLASH and conventional irradiation could not be quantified in vivo because measurements of oxygen depletion under conventional irradiation are hampered by resupply of oxygen from the blood.

Introduction

It is now widely accepted that irradiation delivered at high dose rates (>40 Gy/s, termed a FLASH) results in a reduction of normal tissue radiation damage compared with conventional radiation therapy dose rates (~0.03Gy/s).¹ Although the respective area of research has advanced significantly in the last few years, most studies have been concerned with phenomenological observations using tissue-level functional assays rather than mechanistic measurements. There are several radiobiological hypotheses around the mechanisms underlying normal tissue sparing; however, to date, none are proven. Most proposed mechanisms are linked to oxygen depletion in tissues,^2,3 which is expected to occur rapidly under FLASH dose rates via oxygen-derived radicals and their subsequent consumption in chemical reactions.⁴ Although this is not the only existing hypothesis, few other mechanisms can alter the radiation damage as profoundly as reduction in the availability of oxygen.⁵ The overall data support the fact that high dose rates result in distinctly different radiobiological cell killing and/or repair, altering the survival curve for normal tissues but not tumor tissues. Studies have shown reduction in damage in normal brain,^6,7 colon,⁸ lung,^9,10 and skin^11,12 while still preserving the equivalent therapeutic effect in tumors.

One factor limiting the understanding of the radiobiological effect of FLASH has been the lack of accurate information regarding oxygen changes that occur in tissue as well as in in vitro model systems, which could be used to compare FLASH and conventional radiation with respect to various models of radiobiological damage. Oxygen is an efficient radiosensitizer, and well-oxygenated cells are more sensitive to ionizing radiation than fully hypoxic cells by approximately a factor of 3, known as the “oxygen enhancement ratio.” Radiolytic oxygen depletion and the induced sparing effect caused by radiochemical reactions during radiation at very high dose rates have been well demonstrated in bacteria^13,14 and mammalian cell lines.^15,16 Subsequently, several groups hypothesized that oxygen depletion might underpin the FLASH effect.^17,18 However, oxygen depletion has only been seen in cell cultures,19, 20, 21 and no reports have documented oxygen depletion under FLASH irradiation in vivo.

Oxygen measurements by phosphorescence quenching are based on the ability of dioxygen (O₂) to interact with a molecule of a phosphorescent probe in its excited triplet state, leading to shortening of the phosphorescence decay time. However, reactive oxygen species, such as superoxide anion or H₂O₂, potentially can also interact with triplet states and lead to their quenching via, for example, electron transfer reactions. The net effect of such reactions would also be shortening of the phosphorescence lifetime, making it difficult to distinguish which species, oxygen or another quencher molecule, led to quenching. Due to their slow response times, oxygen electrodes cannot be used for fast transient oxygen measurements; however, they may be instrumental for cross-validation of phosphorescence-based measurements under steady state conditions. In a conventional oxygen electrode system, the electro-active metal surface (usually platinum) is protected by an inert plastic membrane, through which neutral oxygen molecules can diffuse, whereas H₂O₂ and other polar molecules cannot, making the electrode selective for molecular oxygen. In this study, we used a commercial oxygen electrode system to cross-validate the phosphorescence decay measurements in vitro upon application of radiation.

Oxygen is transported by the hemoglobin in red blood cells with subsequent diffusion from the blood vessels to the surrounding tissue.^22,23 Consumption of oxygen in other processes, such as in production of oxygen radicals and their reactions, can limit oxygen available for respiration, and reoxygenation of tissue by diffusion from the blood capillaries could take seconds to minutes, depending on the extent of perfusion and intercapillary distances, which vary between tissues.²⁴ In this work, we measured oxygen concentration in solution samples in vitro as well as in tissues at the time of application of 10 MeV electron FLASH irradiation pulses. Oxygen was quantified optically using the phosphorescence quenching method²⁵ and oxygen probe Oxyphor 2P.²⁶ The oxygen levels were recorded to compare oxygen consumption by FLASH versus conventional radiation, using a range of solution samples, normal tissues, and xenograft tumors.