Investigation of Normal Tissue Response to FLASH Irradiation Using the HIGS Linear Accelerator at TUNL

Abstract

Purpose: FLASH irradiation shows strong potential for clinical applications, offering tumor control comparable to conventional irradiation with lower levels of normal tissue toxicity. This combination of effects, the FLASH effect, could widen the therapeutic gap and improve the effectiveness of radiation therapy treatments of cancer and other diseases. While the FLASH effect is typically seen at mean dose rates (MDRs) above 40 Gy/s, the exact conditions for it are unknown. Furthermore, the underlying mechanisms are unclear, with current theories suggesting that FLASH irradiation induces transient hypoxia in normal tissue and causes reduced DNA damage. Duke University is in a unique position to investigate both the conditions and mechanisms behind the FLASH effect through the High Intensity Gamma-ray Source (HIGS) linear accelerator at the Triangle Universities Nuclear Laboratory (TUNL). This FLASH source has been combined with a unique ex vivo rat brain slice organotypic model to create a novel FLASH experimental platform. This platform has been demonstrated to reduce tumor burden, a key component of the FLASH effect. However, the normal tissue sparing portion of the FLASH effect has not been explored. The purpose of this work is to assess independent methods for measuring normal tissue health in the rat brain slice organotypic model and determine if a normal tissue sparing effect is present in this experimental setup. Methods: Two main experiments were conducted: an experiment using the HIGS linac, and an experiment using a Varian 2100EX clinical linac to replicate the HIGS experiment at a conventional clinical dose rate. For the HIGS irradiation, nine well-plates, each with eight 350-micron thick rat brain slices, were divided into one unirradiated (No IR) and two experimental arms. Plates for each of the experimental arms were irradiated with 4 pulses of 35 MeV electrons from the HIGS linac. One experimental arm, the FLASH arm, was irradiated with 0.15 seconds in-between pulses; the other arm, the non-FLASH arm, was irradiated with 10 seconds in-between pulses. EBT-XD film was scanned using an EPSON 11000XL scanner to determine the dose delivered to each slice. Each treatment arm had a plate dedicated to one of three independent normal tissue health assays: yellow fluorescent protein (YFP), immunofluorescence, or cytokines. Depending on the assay, the slices in the plate either underwent YFP transfection of neurons pre-irradiation, were fixed and underwent immunofluorescence staining three days post-irradiation, or were fixed and underwent cytokine collection three days post-irradiation. Stereoscope images of YFP slices were taken over five days post-irradiation. Healthy neurons were manually tracked to determine surviving fractions for each arm, and YFP intensity was measured for each arm. Confocal images of immunofluorescent slices were taken, with microglia morphology and intensity measurements made with a custom CellProfiler pipeline. Morphology measurements of microglia included: area, perimeter, circularity, eccentricity, mean radius, median radius, major and minor axis lengths, and maximum and minimum Feret diameters. Intensity measurements included integrated intensity, mean intensity, and median intensity. The mean for each measurement was determined for each arm, and ANOVA tests were used to determine statistically significant differences between treatment arm means. Astrocyte branches were also segmented using a separate CellProfiler pipeline to measure total intensity, total intensity per unit area, mean intensity, and mean intensity per unit area for the segmented regions. ANOVA tests were performed to determine statistical significance between treatment arm means. Cytokine profiles were analyzed by Eve Technologies (Alberta, CA), and statistical significance determined using ANOVA tests. The conventional experiment replicated the dose delivered to the FLASH arm for two cytokine plates and an immunofluorescent plate and followed similar analysis methods.Results: Both the YFP-based surviving fraction measurements and the YFP signal intensity measurements could not effectively distinguish between different treatment arms. Both FLASH and CONV irradiation resulted in an increase in microglia size between the irradiated and non-irradiated arms based on area, perimeter, major and minor axis lengths, and maximum and minimum Feret diameter measurements (p < 0.0001). Microglia from the FLASH arm were larger compared with all other irradiated arms based off area, perimeter, major and minor axis lengths, and maximum and minimum Feret diameters (p < 0.05 to p < 0.0001), suggesting increased activation. This is further supported by a higher integrated intensity compared with the non-FLASH and HIGS No IR arms (p < 0.0001). No statistically significant difference was determined between treatment arms with astrocyte analysis. Increased levels of TNF-alpha in the FLASH arm compared with all other arms suggested activation of microglia into a pro-inflammatory M1 state (p < 0.01 to p < 0.0001). Increased levels of fractalkine in the FLASH arm compared with all other arms suggested the transition of microglia from the pro-inflammatory M1 state into an anti-inflammatory, restorative M2 state (p < 0.01 to p < 0.0001). Conclusions: Any differences present in the YFP assay were below the sensitivity detection threshold of the assay. Microglia immunofluorescence and cytokine profile assays proved effective in detecting differences between treatment arms. Astrocyte analysis was not sensitive enough to distinguish between the different treatment arms. The increased size of microglia, TNF-alpha levels, and fractalkine levels in the FLASH arm compared with other arms suggest a stronger transition from a short-term pro-inflammatory state to an anti-inflammatory state compared with other treatment arms. Together, these results indicate differences in normal tissue response between treatment arms and suggests the possible presence of a normal tissue sparing effect.