The FLASH effect depends on oxygen concentration

Abstract

Objective: Recent in vivo results have shown prominent tissue sparing effect of radiotherapy with ultra-high dose rates (FLASH) compared to conventional dose rates (CONV). Oxygen depletion has been proposed as the underlying mechanism, but in vitro data to support this have been lacking. The aim of the current study was to compare FLASH to CONV irradiation under different oxygen concentrations in vitro.

Methods: Prostate cancer cells were irradiated at different oxygen concentrations (relative partial pressure ranging between 1.6 and 20%) with a 10 MeV electron beam at a dose rate of either 600 Gy/s (FLASH) or 14 Gy/min (CONV), using a modified clinical linear accelerator. We evaluated the surviving fraction of cells using clonogenic assays after irradiation with doses ranging from 0 to 25 Gy.

Results: Under normoxic conditions, no differences between FLASH and CONV irradiation were found. For hypoxic cells (1.6%), the radiation response was similar up to a dose of about 5-10 Gy, above which increased survival was shown for FLASH compared to CONV irradiation. The increased survival was shown to be significant at 18 Gy, and the effect was shown to depend on oxygen concentration.

Conclusion: The in vitro FLASH effect depends on oxygen concentration. Further studies to characterize and optimize the use of FLASH in order to widen the therapeutic window are indicated.

Advances in knowledge: This paper shows in vitro evidence for the role of oxygen concentration underlying the difference between FLASH and CONV irradiation.

Figure 1.

Surviving fraction of cells at different doses, under normoxic (20% oxygen concentration) and hypoxic (1.6% oxygen concentration) conditions, for FLASH [circles (normoxia) and triangles (hypoxia)] and conventional dose rates [CONV, squares (normoxia) and diamonds (hypoxia)], determined by clonogenic assays. Bold markers denote the average surviving fraction, with error bars representing one standard deviation, plotted against the average of the actual dose for each dose group (nominally multiples of 3 Gy). Curves represent data fitted to the LQ-model as described under Results, with corresponding R²—values shown in the legend. Data from 10 separate experiments, in total 393 flasks with individually determined irradiation dose. ***p < 0.001 using Wilcoxon rank sum-test comparing FLASH vs CONV for data points within 18 ± 0.5 Gy, and *p < 0.05 at 21 ± 0.5 Gy. LQ, linear–quadratic.

Figure 2.

Surviving fraction of cells under different relative partial oxygen pressure after irradiation with 18 Gy, with either FLASH or conventional dose rates (CONV). Panels show the results from three separate experiments (box: quartiles; band: median; whiskers: highest/lowest point if ≤1.5 IQR; outlier:>1.5 IQR). Results from statistical comparison (Wilcoxon rank-sum test) between FLASH and CONV revealed highly significant (***, p < 0.001) difference for relative partial oxygen pressure of 1.6%, significant (*, p < 0.05) for 2.7 and 4.4%, and no significant differences for either 8.3% or 20%. IQR, interquartile range.

Figure 3.

The surviving fraction of cells irradiated with 18 Gy as a function of relative partial oxygen pressure for FLASH (circles) and CONV (squares) irradiations, together with the linear quadratic fit (solid lines). Error bars indicate the range of data from the three separate experiments, bold markers the average surviving fraction, and asterisks indicate data below our detection limit. The relative sparing effect of FLASH (i.e. the ratio between the surviving fractions for FLASH and CONV), as calculated from the fitted curves, is also shown, with its lines becoming dashed where the model extrapolates the data.

Figure 4.

Summary of all experimental data for the different doses and oxygen levels (i.e. from Figures 1 and 2) together with the linear-quadratic fit, both for FLASH (triangles, circles and dashed lines) and CONV (diamonds, squares, and solid lines), including the model’s prediction of the anoxic limit at a relative partial oxygen pressure of 0% (top solid line), as well as the prediction at 0.01 and 0.1% relative partial oxygen pressures. The assumption of an anoxic limit is based on previously published data. Markers denote the average surviving fraction, plotted against the average of the actual dose for each dose group (nominally multiples of 3 Gy).