Spread-out Bragg peak FLASH: quantifying normal tissue toxicity in a murine model.

Objective: A favorable effect of ultra-high dose rate (FLASH) radiation on normal tissue-sparing has been indicated in several preclinical studies. In these studies, the adverse effects of radiation damage were reduced without compromising tumor control. Most studies of proton FLASH investigate these effects within the entrance of a proton beam. However, the real advantage of proton therapy lies in the Spread-out Bragg Peak (SOBP), which allows for giving a high dose to a target with a limited dose to healthy tissue at the entrance of the beam. Therefore, a clinically relevant investigation of the FLASH effect would be of healthy tissues within a SOBP. Our study quantified the tissue-sparing effect of FLASH radiation on acute and late toxicity within an SOBP in a murine model. Material/Methods: Radiation-induced damage was assessed for acute and late toxicity in the same mice following irradiation with FLASH (Field dose rate of 60 Gy/s) or conventional (CONV, 0.34 Gy/s) dose rates. The right hindleg of unanesthetized female CDF1 mice was irradiated with single-fraction doses between 19.9-49.7 Gy for CONV and 30.4-65.9 Gy for FLASH with 5-8 mice per dose. The leg was placed in the middle of a 5 cm SOBP generated from a mono-energetic beam using a 2D range modulator. Acute skin toxicity quantified by hair loss, moist desquamation and toe separation was monitored daily within 29 days post-treatment. Late toxicity of fibrotic development measured by leg extendibility was monitored biweekly until 30 weeks post-treatment. Results: Comparison of acute skin toxicity following radiation indicated a tissue-sparing effect of FLASH compared to conventional single-fraction radiation with a mean protection ratio of 1.40 (1.35-1.46). Fibrotic development similarly indicated normal tissue sparing with a 1.18 (1.17-1.18) protection ratio. The acute skin toxicity tissue sparing was similar to data from entrance-beam irradiations of Sørensen et al. (4). Conclusion: Full dose-response curves for acute and late toxicity after CONV and FLASH radiation were obtained. Radiation within the SOBP retains the normal-tissue-sparing effect of FLASH with a dose-modifying factor of 40% for acute skin damage and 18% for fibrotic development.

Human enteroids as a tool to study conventional and ultra-high dose rate radiation.

Radiation therapy, one of the most effective therapies to treat cancer, is highly toxic to healthy tissue. The delivery of radiation at ultra-high dose rates, FLASH radiation therapy (FLASH), has been shown to maintain therapeutic anti-tumor efficacy while sparing normal tissues compared to conventional dose rate irradiation (CONV). Though promising, these studies have been limited mainly to murine models. Here, we leveraged enteroids, three-dimensional cell clusters that mimic the intestine, to study human-specific tissue response to radiation. We observed enteroids have a greater colony growth potential following FLASH compared with CONV. In addition, the enteroids that reformed following FLASH more frequently exhibited proper intestinal polarity. While we did not observe differences in enteroid damage across groups, we did see distinct transcriptomic changes. Specifically, the FLASH enteroids upregulated the expression of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response. These studies validate human enteroids as a model to investigate FLASH and provide further evidence supporting clinical study of this therapy. Insight Box Promising work has been done to demonstrate the potential of ultra-high dose rate radiation (FLASH) to ablate cancerous tissue, while preserving healthy tissue. While encouraging, these findings have been primarily observed using pre-clinical murine and traditional two-dimensional cell culture. This study validates the use of human enteroids as a tool to investigate human-specific tissue response to FLASH. Specifically, the work described demonstrates the ability of enteroids to recapitulate previous in vivo findings, while also providing a lens through which to probe cellular and molecular-level responses to FLASH. The human enteroids described herein offer a powerful model that can be used to probe the underlying mechanisms of FLASH in future studies.

Synergetic effect of high dose rate radiations (10× FFF/2400 MU/min/10 MV x-rays) and paclitaxel selectively eliminates melanoma cells.

BACKGROUND: Melanoma is one of the most aggressive cancers, with 1.6% of total cancer deaths in the United States. In recent years treatment options for metastatic melanoma have been improved by the FDA approval of new therapeutic agents. However, these inhibitors-based therapies are non-specific and have severe toxicities, including hyperkeratosis, photosensitivity, hepatitis, arthralgia, and fatigue. AIMS: The aim of this study is to determine the synthetic lethal effect (paclitaxel and radiations) on melanoma cells and reduce the total radiation doses by increasing the dose rates up to 2400 MU/min. METHODS AND RESULTS: We previously reported a radiation treatment (10 MV x-rays, 10X-FFF, dose rate 2400MU/min, low total dose 0.5 Gy) that kills melanoma cells with 80% survival of normal HEM in vitro. In this study, we extended the radiation cycle up to four and included paclitaxel treatment to study the synthetic lethal effect on melanoma and two other normal primary cells, HDF and HEK. Cells were treated with paclitaxel prior to the radiation at a dose rate of 400 and 2400 MU/min with a total radiation dose of only 0.5 Gy. Mitochondrial respiration assay, DNA damage assay, and colony formation assays were performed to study apoptosis and cell death induction. Four days of consequent radiation treatment with paclitaxel significantly reduces the survival of melanoma cells by inducing apoptosis and mitochondrial damage. After treatment, excessive DNA damage in melanoma cells leads to an increase in the expression of pro-apoptotic genes (Caspase-3) and a decrease in the expression of DNA repair gene (PARP1) and anti-apoptotic gene (Bcl-2) to activate the apoptosis pathway. The combination of paclitaxel and radiation reduces the survival of melanoma cells colonies compared to radiation alone. CONCLUSION: Our study indicates that radiations with paclitaxel have a potential synthetic lethal effect on melanoma cells and can be developed as a melanoma therapy without toxicities or harmful effects on normal primary skin cells.

The Therapeutic Potential of FLASH-RT for Pancreatic Cancer.

Recent preclinical evidence has shown that ionizing radiation given at an ultra-high dose rate (UHDR), also known as FLASH radiation therapy (FLASH-RT), can selectively reduce radiation injury to normal tissue while remaining isoeffective to conventional radiation therapy (CONV-RT) with respect to tumor killing. Unresectable pancreatic cancer is challenging to control without ablative doses of radiation, but this is difficult to achieve without significant gastrointestinal toxicity. In this review article, we explore the propsed mechanisms of FLASH-RT and its tissue-sparing effect, as well as its relevance and suitability for the treatment of pancreatic cancer. We also briefly discuss the challenges with regard to dosimetry, dose rate, and fractionation for using FLASH-RT to treat this disease.

Heat management of a compact x-ray source for microbeam radiotherapy and FLASH treatments.

BACKGROUND: Microbeam and x-ray FLASH radiation therapy are innovative concepts that promise reduced normal tissue toxicity in radiation oncology without compromising tumor control. However, currently only large third-generation synchrotrons deliver acceptable x-ray beam qualities and there is a need for compact, hospital-based radiation sources to facilitate clinical translation of these novel treatment strategies. PURPOSE: We are currently setting up the first prototype of a line-focus x-ray tube (LFxT), a promising technology that may deliver ultra-high dose rates (UHDRs) of more than 100 Gy/s from a table-top source. The operation of the source in the heat capacity limit allows very high dose rates with micrometer-sized focal spot widths. Here, we investigate concepts of effective heat management for the LFxT, a prerequisite for the performance of the source. METHODS: For different focal spot widths, we investigated the temperature increase numerically with Monte Carlo simulations and finite element analysis (FEA). We benchmarked the temperature and thermal stresses at the focal spot against a commercial x-ray tube with similar power characteristics. We assessed thermal loads at the vacuum chamber housing caused by scattering electrons in Monte Carlo simulations and FEA. Further, we discuss active cooling strategies and present a design of the rotating target. RESULTS: Conventional focal spot widths led to a temperature increase dominated by heat conduction, while very narrow focal spots led to a temperature increase dominated by the heat capacity of the target material. Due to operation in the heat capacity limit, the temperature increase at the focal spot was lower than for the investigated commercial x-ray tube. Hence, the thermal stress at the focal spot of the LFxT was considered uncritical. The target shaft and the vacuum chamber housing require active cooling to withstand the high heat loads. CONCLUSIONS: The heat capacity limit allows very high power densities at the focal spot of the LFxT and thus facilitates very high dose rates. Numerical simulations demonstrated that the heat load imparted by scattering electrons requires active cooling.

Comparison of FLASH Proton Entrance and the Spread-Out Bragg Peak Dose Regions in the Sparing of Mouse Intestinal Crypts and in a Pancreatic Tumor Model.

Ultra-high dose rate FLASH proton radiotherapy (F-PRT) has been shown to reduce normal tissue toxicity compared to standard dose rate proton radiotherapy (S-PRT) in experiments using the entrance portion of the proton depth dose profile, while proton therapy uses a spread-out Bragg peak (SOBP) with unknown effects on FLASH toxicity sparing. To investigate, the biological effects of F-PRT using an SOBP and the entrance region were compared to S-PRT in mouse intestine. In this study, 8-10-week-old C57BL/6J mice underwent 15 Gy (absorbed dose) whole abdomen irradiation in four groups: (1) SOBP F-PRT, (2) SOBP S-PRT, (3) entrance F-PRT, and (4) entrance S-PRT. Mice were injected with EdU 3.5 days after irradiation, and jejunum segments were harvested and preserved. EdU-positive proliferating cells and regenerated intestinal crypts were quantified. The SOBP had a modulation (width) of 2.5 cm from the proximal to distal 90%. Dose rates with a SOBP for F-PRT or S-PRT were 108.2 ± 8.3 Gy/s or 0.82 ± 0.14 Gy/s, respectively. In the entrance region, dose rates were 107.1 ± 15.2 Gy/s and 0.83 ± 0.19 Gy/s, respectively. Both entrance and SOBP F-PRT preserved a significantly higher number of EdU + /crypt cells and percentage of regenerated crypts compared to S-PRT. Moreover, tumor growth studies showed no difference between SOBP and entrance for either of the treatment modalities.

Spatial and temporal dosimetry of individual electron FLASH beam pulses using radioluminescence imaging.

Purpose.In this study, spatio-temporal beam profiling for electron ultra-high dose rate (UHDR; >40 Gy s-1) radiation via Cherenkov emission and radioluminescence imaging was investigated using intensified complementary metal-oxide-semiconductor cameras.Methods.The cameras, gated to FLASH optimized linear accelerator pulses, imaged radioluminescence and Cherenkov emission incited by single pulses of a UHDR (>40 Gy s-1) 10 MeV electron beam delivered to the isocenter. Surface dosimetry was investigated via imaging Cherenkov emission or scintillation from a solid water phantom or Gd2O2S:Tb screen positioned on top of the phantom, respectively. Projected depth-dose profiles were imaged from a tank filled with water (Cherenkov emission) and a 1 g l-1quinine sulfate solution (scintillation). These optical results were compared with projected lateral dose profiles measured by Gafchromic film at different depths, including the surface.Results.The per-pulse beam output from Cherenkov imaging agreed with the photomultiplier tube Cherenkov output to within 3% after about the first five to seven ramp-up pulses. Cherenkov emission and scintillation were linear with dose (R2 = 0.987 and 0.995, respectively) and independent of dose rate from ∼50 to 300 Gy s-1(0.18-0.91 Gy/pulse). The surface dose distribution from film agreed better with scintillation than with Cherenkov emission imaging (3%/3 mm gamma pass rates of 98.9% and 88.8%, respectively). Using a 450 nm bandpass filter, the quinine sulfate-based water imaging of the projected depth optical profiles agreed with the projected film dose to within 5%.Conclusion.The agreement of surface dosimetry using scintillation screen imaging and Gafchromic film suggests it can verify the consistency of daily beam quality assurance parameters with an accuracy of around 2% or 2 mm. Cherenkov-based surface dosimetry was affected by the target's optical properties, prompting additional calibration. In projected depth-dose profiling, scintillation imaging via spectral suppression of Cherenkov emission provided the best match to film. Both camera-based imaging modalities resolved dose from single UHDR beam pulses of up to 60 Hz repetition rate and 1 mm spatial resolution.

Oxygen depletion in FLASH ultra-high-dose-rate radiotherapy: A molecular dynamics simulation.

PURPOSE: We present a first-principles molecular dynamics (MD) simulation and expound upon a mechanism of oxygen depletion hypothesis to explain the mitigation of normal tissue injury observed in ultra-high-dose-rate (UHDR) FLASH radiotherapy. METHODS: We simulated damage to a segment of DNA (also representing other biomolecules such as RNA and proteins) in a simulation box filled with H 2 O and O 2 molecules. Attoseconds physical interactions (ionizations, electronic, and vibrational excitations) were simulated by using the Monte Carlo track structure code Geant4-DNA. Immediately after ionization, ab initio Car-Parrinello molecular dynamics (CPMD) simulation was used to identify which H 2 O and O 2 molecules surrounding the DNA molecule were converted into reactive oxygen species (ROS). Subsequently, the femto- to nanosecond reactions of ROS were simulated by using MD with reactive force field (ReaxFF), to illustrate ROS merging into new types of non-reactive oxygen species (NROS) due to strong coupling among ROS. A coarse-grained model was constructed to describe the relevant collective phenomenon at the macroscopic level on ROS aggregation and formation of NROS agglomerates consistent with the underlying microscopic pathways obtained from MD simulations. RESULTS: Time-dependent molecular simulations revealed the formation of metastable and transient spaghetti-like complexes among ROS generated at UHDR. At the higher ROS densities produced under UHDR, stranded chains (i.e., NROS) are produced, mediated through attractive electric polarity forces, hydrogen bonds, and magnetic dipole-dipole interactions among hydroxyl ( . OH ) radicals. NROS tend to be less mobile than cellular biomolecules as opposed to the isolated and sparsely dense ROS generated at conventional dose rates (CDR). We attribute this effect to the suppression of biomolecular damage induced per particle track. At a given oxygen level, as the dose rate increases, the size and number of NROS chains increase, and correspondingly the population of toxic ROS components decreases. Similarly, at a given high dose rate, as the oxygen level increases, so do the size and number of NROS chains until an optimum level of oxygen is reached. Beyond that level, the amount of oxygen present may be sufficient to saturate the production of NROS chains, thereby reversing the sparing effects of UHDRs. CONCLUSIONS: We showed that oxygen depletion, hypothesized to lead to lower normal-tissue toxicity at FLASH dose rates, takes place within femto- to nanoseconds after irradiation. The mechanism is governed by the slow dynamics of chains of ROS complexes (NROS). Under physoxic (≈ 4-5% oxygen) conditions (i.e., in normal tissues), NROS are more abundant than in hypoxic conditions (e.g., <0.3% in parts of tumors), suggesting that biomolecular damage would be reduced in an environment with physoxic oxygen levels. Hence irradiation at UHDRs would be more effective for sparing physoxic normal tissues but not tumors containing regions of hypoxia. At much higher levels of oxygen (e.g., >10-15%), oxygen depletion by UHDRs may not be sufficient for tissue sparing.

An integrated physico-chemical approach for explaining the differential impact of FLASH versus conventional dose rate irradiation on cancer and normal tissue responses.

For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1-0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors.