Review of Oxygen Measurement and Relevance to the Mechanisms of FLASH Radiotherapy

FLASH radiotherapy (FLASH-RT) is the phenomenon of relative sparing of normal tissue when ultra-high dose rates (UHDR) are used compared with conventional dose rates (CDR) as clinically used. Despite extensive investigation, the underlying mechanisms remain unexplained. Among the proposed hypotheses, tissue oxygen has consistently been a central theme because oxygen is the most dominant factor known to modulate radiation-induced damage. The factors implicated in FLASH sparing include the baseline partial pressure of oxygen (pO2), transient radiolytic oxygen consumption (ROC), and oxygen-dependent changes in the chemistry of reactive oxygen species (ROS) that vary with dose rate. This review synthesizes current evidence on in vivo oxygen measurement techniques, highlighting their capabilities and limitations in capturing the spatial and temporal heterogeneity of tissue oxygenation. Key experimental studies in skin are summarized and interpreted by modulating oxygen levels via changes in inspired oxygen gas and vascular clamping interventions, demonstrating that the FLASH effect occurs only at intermediate baseline pO2 (normoxic or slightly hypoxic) values, but not at hypoxia or hyperoxia. Direct measurement of oxygen consumption during UHDR irradiation is possible, providing one of the first in situ measurements of radiation chemistry in patients. In parallel, recent advances in fast in vitro radiation chemistry assays indicate that UHDR irradiation alters radical yields, favoring increased production of solvated electrons and reduced hydroxyl radical-mediated damage. Taken together, the available data suggest that the FLASH sparing effect arises from an interplay among the delivered dose and dose rate, local oxygen availability, and radiation chemistry, with tissue-specific variation in scavenging, leading to altered biological responses across the CDR-to-UHDR shift. This more complex interpretation seems more likely than the simpler interpretation of broad-area radiolytic oxygen depletion alone. However, it must be acknowledged that we have partial data on all aspects of this hypothesis, and further improvements in oxygen sampling are very likely to help in understanding ROS and scavenging effects in vivo. Key challenges in quantifying oxygen dynamics in vivo are highlighted, and the conclusions are used to identify critical areas for future research to enable mechanistic understanding and clinical translation of FLASH-RT.