Reinventing Radiobiology in the Light of FLASH Radiotherapy

Abstract

Ultrahigh-dose rate FLASH radiotherapy (FLASH-RT) is a potentially paradigm-shifting treatment modality that holds the promise of expanding the therapeutic index for nearly any cancer. At the heart of this exciting technology comes the capability to ameliorate major normal tissue complications without compromising the efficacy of tumor killing. This combination of benefits has now been termed the FLASH effect and relies on an in vivo validation to rigorously demonstrate the absence of normal tissue toxicity. The FLASH effect occurs when the overall irradiation time is extremely short (<500 ms), and in this review we attempt to understand how FLASH-RT can kill tumors but spare normal tissues-likely the single most pressing question confronting the field today.

Keywords: FLASH radiotherapy; conventional radiotherapy; isoefficient tumor killing; normal tissue sparing; ultrahigh dose rate.

Figure 1

Normal tissue sparing by FLASH radiotherapy. FLASH radiotherapy provides a unique opportunity to dose escalate while minimizing normal tissue toxicities throughout a variety of normal tissue beds. Reduced normal tissue complications have been found in nearly all normal tissues examined to date, using a variety of preclinical models. Figure adapted from images created with Biorender.com.

Figure 2

Blood volume as a target for FLASH radiotherapy (FLASH-RT). (Top) Under conventional radiotherapy (CONV-RT), standard dose rates have the potential to expose a significant fraction of the total blood volume as circulation moves blood continuously through the target volume during a prescribed treatment. In this scenario, prolonged exposure of blood cell constituents may maximize proinflammatory signaling or deplete factors important for prosurvival signaling. Certain blood cell constituents or other paracrine mediators such as secreted exosomes are primed to trigger more widespread pathogenic responses. (Bottom) The short irradiation times of FLASH-RT minimize the fraction of blood irradiated. Since perfusion transpires on a much shorter timescale, only blood residing at the target volume is exposed, but at a high dose. Resultant inflammatory signals are reduced, and the depletion of prosurvival factors is not as extensive. While this model may account for a certain level of normal tissue sparing after FLASH-RT, it fails to account for isoefficient tumor killing. Figure adapted from images created with Biorender.com.

Figure 3

U87 tumor cure after FLASH radiotherapy (FLASH-RT). Survival curves of U87 glioblastoma tumors orthotopically implanted in the striatum of female nude mice treated with 3 × 10 Gy whole-breast irradiation in 48-h intervals delivered with FLASH-RT or conventional radiotherapy (CONV-RT). There were N = 12 animals per group, and p-values were derived from the logrank test (**** p < 0.0001 for the FLASH-RT group compared with the CONV-RT group).

Figure 4

Quantification of select lipids in the hippocampus of mice after CONV-RT and FLASH-RT. Endocannabinoids and endogenous lipids were analyzed four months after irradiation. The hippocampi of control (0 Gy), CONV-RT (10 Gy), and FLASH-RT (10 Gy) mice (n = 5–6/group) were homogenized in methanol. Lipids were extracted with chloroform and lipid levels were quantified using liquid chromatography tandem mass spectrometry (Agilent 6410 system). Compared to the control group, p-values indicated by asterisks were * p < 0.05 for 2-AG and OEA and *** p < 0.001 for PEA, and between CONV-RT and FLASH-RT, *** p < 0.001 by two-tailed t-test or one-way ANOVA (analysis of variance). Abbreviations: 2-AG, arachidonoylglycerol; CONV-RT, conventional radiotherapy; FLASH-RT, FLASH radiotherapy; OEA, oleoylethanolamide; PEA, palmitoylethanolamide.

Figure 5

Reverse electron flow and the FLASH effect. (Top) In normal situations mitochondrial OXPHOS mobilizes electrons through a series of electron donor and acceptor subunits embedded in ETCs I–IV. Resultant ATP production is relatively high with a minimal of ROS leakage into the mitochondrial matrix and intermembrane space. (Middle) CONV-RT compromises efficient electron transfer through the ETC by a variety of mechanisms, leading to compromised ATP production and elevated ROS production. In this scenario, forward electron flow is maintained with oxygen serving as the principal terminal electron acceptor. (Bottom) FLASH-RT saturates the intracellular and mitochondrial milieu with electrons, possibly favoring reverse electron flow where fumarate can act as a terminal electron acceptor from complex II. In this scenario, ATP is also relatively lower, but the production of toxic ROS may be minimized. Tissue hypoxia may promote reverse electron flow, but if or how this possibility might differ between normal tissues and tumors or between different FLASH modalities remains to be elucidated. Figure adapted from images created with Biorender.com. Abbreviations: Cyt c, cytochrome complex; CONV-RT, conventional radiotherapy; ETC, electron transport complex; FLASH-RT, FLASH radiotherapy; NAD⁺, nicotinamide adenine dinucleotide; NADH, NAD + hydrogen; O₂^•−, superoxide; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; SDH, succinate dehydrogenase.

Figure 6

Metabolic hibernation and the FLASH effect. Normal cells and tumors rely on oxidative phosphorylation (OXPHOS) and glycolysis at different levels to meet energy demands. (a) Following conventional radiotherapy (CONV-RT), OXPHOS is uniformly disrupted, leading to an increase in toxic reactive oxygen species (ROS) and an upregulation of glycolysis in tumors. Over the long term, normal tissue toxicity results and tumors succumb to suboptimal energy production and oxidative injury. (b) The situation differs following FLASH radiotherapy (FLASH-RT), where tumor cells change little, but for normal tissue reduced OXPHOS activity yields lower levels of ATP but also lower ROS accumulation and reduced oxidative injury. The result is a quiescent state of metabolic hibernation that can be tolerated over protracted times and minimizes late normal tissue toxicities. Figure adapted from images created with Biorender.com.

Figure 7

Mechanistic summary: Why does FLASH kill tumors? (Top) Plausible mechanisms that can account for the FLASH effect include stem cell niche preservation, differential lipid peroxidation and Fenton chemistry, structural predeterminants in specific protein classes, and changes in mitochondrial metabolism such as reverse electron flow or metabolic hibernation. (Bottom) Implausible mechanisms include those involving radical-radical recombination, genetic predisposition, DNA damage and repair, partial blood volume irradiation, oxygen depletion, and adaptive immunity (not exclusive of innate immunity). Experimental evidence for and against these specific hypotheses varies tremendously, and while certain hypotheses may substantiate normal tissue sparing (i.e., oxygen depletion), few can fully account for both normal tissue sparing and isoeffective tumor killing. As the field advances and evidence accumulates, so too will the evolution of more hypotheses requiring experimental validation. Figure adapted from images created with Biorender.com; succinate dehydrogenate structure is from the Protein Data Bank (PDB ID: 6VAX; https://doi.org/10.2210/pdb6VAX/pdb), rendered with Biorender.com. Abbreviations: OER, oxygen enhancement ratio; ROS, reactive oxygen species.