Browsing by Subject "FLASH"

Purpose: An interest in FLASH radiotherapy has been reawakened due to its noted ability to spare normal tissue, equal tumor control compared to conventional irradiation methods and technological advancement allowing for ultra-high dose rates required for FLASH radiotherapy to be more accessible compared to previous decades. The underlying biological mechanism of the FLASH effect are unknown and developing an in vitro model to study it has proven difficult. This work aims to combine two unique technologies, an organotypic rat brain slice model which models the in-vivo micro-environment in an in vitro setting and a linear accelerator capable of delivering variable FLASH pulses to design experiments which will facility the study of the FLASH effect.Methods: The experiments utilize a 35 MeV electron beam provided by Triangle Universities Nuclear Laboratory’s (TUNL) High Intensity Gamma-ray Source linac (HIGS). The beam can supply electron pulses with a temporal width of 1.2 s or 100 ns and work was performed with Gafchromic EBT3 and EBT-XD film to accurately determine the dose and dose rates of each pulse. Experiments were performed over 5 sessions to establish the use and effectiveness of the HIGS linac and biological rat brain model. A 2D translational stage was developed and targeting procedures were developed to ensure accurate targeting of each well containing an organotypic rat brain slice in a 12 well plate. Each rat brain was shot with a yellow fluorescent protein marker and seeded with 4T1 cancer cells tagged with mCherry and firefly luciferase. An imaging analysis workflow was developed to effectively capture and segment mCherry signal and determine the 4T1 proliferation four to five days after irradiation. These were compared to a final firefly luciferase readout. Each experiment was followed by a conventional irradiation as a control group. Monte Carlo model using TOPAS was created to simulate the HIGS linac dose profiles. Results: The HIGS linac can provide a mean dose rate up to 100 Gy/s and an instantaneous dose rate up to 100 MGy/s. The repeatability of the pulse dose was found to be within 4-5% of the average dose for a given experiment. Targeting was repeatable and dose superposition was confirmed. Well targeting quality assurance procedures of the translational stage allowed for consistent targeting of the pulse to each well. Yellow fluorescent protein bleed through in the mCherry signal was effectively filtered out and mCherry analysis reflects the end readout of firefly Luciferase. A gamma analysis between simulated and measured dose demonstrates a passing rate of 99.4% when using a criteria of 2%/2mm and threshold of 10%. Conclusions: FLASH capable dose rates can be supplied by the HIGS linac and is amongst the highest instantaneous dose rates currently available. The HIGS linac and organotypic rat brain model can be combined to irradiate and measure radiation effects to 4T1 cancer cell growth. There is qualitative data to support the observation of the FLASH effect in the rat brain model and the mCherry and firefly luciferase analysis agreement demonstrates the capabilities of the model to measure radiation effects to cancer cells in the 1-10 Gy range. Future work will be to quantitively measure the neuron health of the brain slices and DNA damage differences between FLASH and conventional irradiation.

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.