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Representative video clips of echocardiography performed in unirradiated and irradiated mice 8 weeks post-RT.

Supplementary materials and figures

Supplemental Methods

Table S2. Genes that are upregulated in the S-PRT-treated bone revealed by RNASeq analysis

Figure S1 A) Gene Ontology (GO) enrichment analysis of the differentially expressed (upregulated) genes in the F-PRT-treated mouse skin. (B) Gene Ontology (GO) enrichment analysis of the differentially expressed (downregulated) genes in the S-PRT-treated mouse leg skin. (C) Gene Ontology (GO) enrichment analysis of the differentially expressed (downregulated) genes in the F-PRT-treated mouse skin. N=4 in all analyses.

In studies of electron and proton radiotherapy, ultrahigh dose rates of FLASH radiotherapy appear to produce fewer toxicities than standard dose rates while maintaining local tumor control. FLASH-proton radiotherapy (F-PRT) brings the spatial advantages of PRT to FLASH dose rates (>40 Gy/second), making it important to understand if and how F-PRT spares normal tissues while providing antitumor efficacy that is equivalent to standard-proton radiotherapy (S-PRT). Here we studied PRT damage to skin and mesenchymal tissues of muscle and bone and found that F-PRT of the C57BL/6 murine hind leg produced fewer severe toxicities leading to death or requiring euthanasia than S-PRT of the same dose. RNA-seq analyses of murine skin and bone revealed pathways upregulated by S-PRT yet unaltered by F-PRT, such as apoptosis signaling and keratinocyte differentiation in skin, as well as osteoclast differentiation and chondrocyte development in bone. Corroborating these findings, F-PRT reduced skin injury, stem cell depletion, and inflammation, mitigated late effects including lymphedema, and decreased histopathologically detected myofiber atrophy, bone resorption, hair follicle atrophy, and epidermal hyperplasia. F-PRT was equipotent to S-PRT in control of two murine sarcoma models, including at an orthotopic intramuscular site, thereby establishing its relevance to mesenchymal cancers. Finally, S-PRT produced greater increases in TGFβ1 in murine skin and the skin of canines enrolled in a phase I study of F-PRT versus S-PRT. Collectively, these data provide novel insights into F-PRT-mediated tissue sparing and support its ongoing investigation in applications that would benefit from this sparing of skin and mesenchymal tissues.Significance:These findings will spur investigation of FLASH radiotherapy in sarcoma and additional cancers where mesenchymal tissues are at risk, including head and neck cancer, breast cancer, and pelvic malignancies.

Supplemental Materials & Methods

PURPOSE: Radiation therapy delivered at ultrafast dose rates, known as FLASH RT, has been shown to provide a therapeutic advantage compared to conventional radiotherapy by selectively protecting normal tissues. Radiochemical depletion of oxygen has been proposed to underpin the FLASH effect, however experimental validation of this hypotheses has been lacking, in part due to the inability to measure oxygenation at rates compatible with FLASH. METHODS AND MATERIALS: We present a new variant of the phosphorescence quenching method for tracking oxygen dynamics with rates reaching up to ~3.3 kHz. Using soluble Oxyphor probes we were able to resolve, both in vitro and in vivo, oxygen dynamics during the time of delivery of proton FLASH. RESULTS: (1) In vitro in solutions containing bovine serum albumin (BSA) the O(2) depletion g-values (moles/L of O(2) depleted per radiation dose, e.g. μM/Gy) are higher for conventional irradiation (by ~13% at 75 μM [O(2)]) than for FLASH, and in the low-oxygen region (

Purpose Recent studies suggest that ultrahigh-dose-rate, “FLASH,” electron radiation therapy (RT) decreases normal tissue damage while maintaining tumor response compared with conventional dose rate RT. Here, we describe a novel RT apparatus that delivers FLASH proton RT (PRT) using double scattered protons with computed tomography guidance and provide the first report of proton FLASH RT-mediated normal tissue radioprotection. Methods and Materials Absolute dose was measured at multiple depths in solid water and validated against an absolute integral charge measurement using a Faraday cup. Real-time dose rate was obtained using a NaI detector to measure prompt gamma rays. The effect of FLASH versus standard dose rate PRT on tumors and normal tissues was measured using pancreatic flank tumors (MH641905) derived from the KPC autochthonous PanCa model in syngeneic C57BL/6J mice with analysis of fibrosis and stem cell repopulation in small intestine after abdominal irradiation. Results The double scattering and collimation apparatus was dosimetrically validated with dose rates of 78 ± 9 Gy per second and 0.9 ± 0.08 Gy per second for the FLASH and standard PRT. Whole abdominal FLASH PRT at 15 Gy significantly reduced the loss of proliferating cells in intestinal crypts compared with standard PRT. Studies with local intestinal irradiation at 18 Gy revealed a reduction to near baseline levels of intestinal fibrosis for FLASH-PRT compared with standard PRT. Despite this difference, FLASH-PRT did not demonstrate tumor radioprotection in MH641905 pancreatic cancer flank tumors after 12 or 18 Gy irradiation. Conclusions We have designed and dosimetrically validated a FLASH-PRT system with accurate control of beam flux on a millisecond time scale and online monitoring of the integral and dose delivery time structure. Using this system, we found that FLASH-PRT decreases acute cell loss and late fibrosis after whole-abdomen and focal intestinal RT, whereas tumor growth inhibition is preserved between the 2 modalities.

FLASH is a high-dose-rate form of radiation therapy that has the reported ability, compared with conventional dose rates, to spare normal tissues while being equipotent in tumor control, thereby increasing the therapeutic ratio. The mechanism underlying this normal tissue sparing effect is currently unknown, however one possibility is radiochemical oxygen depletion (ROD) during dose delivery in tissue at FLASH dose rates. In order to investigate this possibility, we used the phosphorescence quenching method to measure oxygen partial pressure before, during and after proton radiation delivery in model solutions and in normal muscle and sarcoma tumors in mice, at both conventional (Conv) (~0.5 Gy/s) and FLASH (~100 Gy/s) dose rates. Radiation dosimetry was determined by Advanced Markus Chamber and EBT-XL film. For solutions contained in sealed glass vials, phosphorescent probe Oxyphor PtG4 (1 μM) was dissolved in a buffer (10 mM HEPES) containing glycerol (1 M), glucose (5 mM) and glutathione (5 mM), designed to mimic the reducing and free radical-scavenging nature of the intracellular environment. In vivo oxygen measurements were performed 24 h after injection of PtG4 into the interstitial space of either normal thigh muscle or subcutaneous sarcoma tumors in mice. The “g-value” for ROD is reported in mmHg/Gy, which represents a slight modification of the more standard chemical definition (μM/Gy). In solutions, proton irradiation at conventional dose rates resulted in a g-value for ROD of up to 0.55 mmHg/Gy, consistent with earlier studies using X or gamma rays. At FLASH dose rates, the g-value for ROD was ~25% lower, 0.37 mmHg/Gy. pO(2) levels were stable after each dose delivery. For normal muscle in vivo, oxygen depletion during irradiation was counterbalanced by resupply from the vasculature. This process was fast enough to maintain tissue pO(2) virtually unchanged at Conv dose rates. However, during FLASH irradiation there was a stepwise decrease in pO(2) (g-value ~0.28 mmHg/Gy), followed by a rebound to the initial level after ~8 s. The g-values were smaller and recovery times longer in tumor tissue when compared to muscle and may be related to the lower initial endogenous pO(2) levels in the former. Considering that the FLASH effect is seen in vivo even at doses as low as 10 Gy, it is difficult to reconcile the amount of protection seen by oxygen depletion alone. However, the phosphorescence probe in our experiments was confined to the extracellular space, and it remains possible that intracellular oxygen depletion was greater than observed herein. In cell-mimicking solutions the oxygen depletion g-vales were indeed significantly higher than observed in vivo.

In studies of electron and proton radiotherapy, ultrahigh dose rates of FLASH radiotherapy appear to produce fewer toxicities than standard dose rates while maintaining local tumor control. FLASH-proton radiotherapy (F-PRT) brings the spatial advantages of PRT to FLASH dose rates (>40 Gy/second), making it important to understand if and how F-PRT spares normal tissues while providing antitumor efficacy that is equivalent to standard-proton radiotherapy (S-PRT). Here we studied PRT damage to skin and mesenchymal tissues of muscle and bone and found that F-PRT of the C57BL/6 murine hind leg produced fewer severe toxicities leading to death or requiring euthanasia than S-PRT of the same dose. RNA-seq analyses of murine skin and bone revealed pathways upregulated by S-PRT yet unaltered by F-PRT, such as apoptosis signaling and keratinocyte differentiation in skin, as well as osteoclast differentiation and chondrocyte development in bone. Corroborating these findings, F-PRT reduced skin injury, stem cell depletion, and inflammation, mitigated late effects including lymphedema, and decreased histopathologically detected myofiber atrophy, bone resorption, hair follicle atrophy, and epidermal hyperplasia. F-PRT was equipotent to S-PRT in control of two murine sarcoma models, including at an orthotopic intramuscular site, thereby establishing its relevance to mesenchymal cancers. Finally, S-PRT produced greater increases in TGFβ1 in murine skin and the skin of canines enrolled in a phase I study of F-PRT versus S-PRT. Collectively, these data provide novel insights into F-PRT-mediated tissue sparing and support its ongoing investigation in applications that would benefit from this sparing of skin and mesenchymal tissues. Significance: These findings will spur investigation of FLASH radiotherapy in sarcoma and additional cancers where mesenchymal tissues are at risk, including head and neck cancer, breast cancer, and pelvic malignancies.

Purpose/Objective(s) FLASH is a promising form of radiation therapy due to its reported ability to spare normal tissues while being equipotent in tumor control. The mechanism underlying this tissue sparing effect is currently unknown, however the depletion of tissue oxygen at FLASH dose rates has been proposed as a potential source. To date, the oxygen depletion hypothesis has only been supported by analytical or numerical modeling studies, primarily due to the difficulty of measuring oxygen concentration on millisecond time scales during irradiation. In this project, we use the phosphorescence quenching method to probe oxygen depletion during proton delivery at both conventional (∼1Gy/sec) and FLASH (∼100 Gy/sec) dose rates. Materials/Methods Oxygen concentration was measured at rates of up to 3kHz using a phosphorescent probe, Oxyphor PtG4, and a time-domain OxyLED phosphorometer modified for ultra-fast operation. Sealed glass vials, containing the experimental solution and probe, were placed such that they were exposed to a uniform dose rate where the proton beam entered the vial. Radiation was delivered at conventional (conv) (∼1 Gy/s) and FLASH (∼100 Gy/s) dose rates. The g-value of oxygen depletion was measured as a function of dose rate, total dose, oxygen concentration, biomolecule concentration, and in the presence or absence of oxygen scavengers. Biomolecule relevant experiments were carried out using either 20mM phosphate buffer containing 1, 2, and 5% bovine serum albumin (BSA) or a solution designed to emulate the intracellular environment, containing 5mM glucose, 1M glycerol, 5mM glutathione, and 10 mM HEPES (Buffer 3G). Oxygen concentration was continuously monitored before, during, and after irradiation for all experiments. Results We found that FLASH dose rates resulted in lower g-values for oxygen depletion when compared to conventional dose rates by ∼15% (range: 7.2 to 22.5%) for each of the solutions tested. For example, in Buffer 3G, for a 30Gy dose, the g-value was 16% lower at FLASH dose rates (gconv = 0.72 ± 0.02 μM/Gy, gFLASH = 0.63 ± 0.02 μM/Gy) (all values listed are mean ± S.E.M.). The g-value increased with increasing BSA concentration (values for 1% and 5% BSA listed) at both conventional and FLASH dose rates (gconv = 0.45 ± 0.05 and 0.58 ± 0.03 μM/Gy respectively, gFLASH = 0.37 ± 0.01 and 0.62 ± 0.01 μM/Gy respectively). The g-value was higher for Buffer 3G than for the BSA solutions (gconv = 0.72 ± 0.02 and 0.58 ± 0.03 μM/Gy respectively, gFLASH = 0.63 ± 0.02 and 0.54 ± 0.01 μM/Gy respectively). An “after FLASH” period of gradual oxygen depletion which persisted for ∼0.5s and depleted an additional 32.1% oxygen was observed for Buffer 3G. Conclusion We have demonstrated a method for ultra-fast measurements of oxygen concentration during the delivery of proton radiation. Furthermore, we found that the g-value of oxygen depletion at FLASH dose rates is lower than that of conventional dose rates by approximately 15%.

Purpose: To investigate the transcriptomic changes induced by FLASH proton radiotherapy (F-PRT) that could be responsible for the protection of normal epithelial tissues by radiation-induced toxicities as have been previously shown by us and others. Methods: C57BL/6J mice received 30 Gy of F-PRT or S-PRT to the hind leg at respective dose rates of 69-124 Gy/sec or 0.39-0.65 Gy/sec. RNA sequencing was performed using full-thickness leg skin at 5 days after radiation revealing major pathways regulated by F-PRT and S-PRT. In an endeavor to identify the full repertoire of cells and gene expression profiles that are involved in the sparing effects of FLASH PRT, we expanded our studies to include single-cell RNA sequencing (sc-RNA seq) and examined additional time points such as Day 2 and Day 10 after radiation. Single-cell transcriptome libraries were generated on a 10X Genomics Chromium system. Datasets were acquired from cell samples derived and sequenced from pooled skin samples of three mice per group. Skin from the sequenced mice was also embedded for spatial analysis of gene expression. Results: RNA sequencing revealed that F-PRT uniquely upregulates almost four times more genes compared to S-PRT (F-PRT-uniquely upregulated 489 genes vs S-PRT-uniquely upregulated 129 genes). Also, F-PRT uniquely downregulated 178 genes, compared to the 125 genes uniquely downregulated by S-PRT. GO analysis demonstrates that the keratinization and apoptosis pathways are uniquely upregulated by S-PRT, whereas F-PRT uniquely upregulates genes involved in vascular development pathway. During submission of the abstract, analysis of sc-RNA seq samples was pending. Conclusion: Our comprehensive studies inform on the transcriptomic profiling of skin cell populations that are affected by F-PRT vs S-PRT; this insight will further spur discoveries on the biology of FLASH radiotherapy effects. Citation Format: Anastasia Velalopoulou, Ilias V. Karagounis, Giorgos Skoufos, Ioannis I. Verginadis, Michele Kim, Khayrullo Shoniyozov, Artemis G. Hatzigeorgiou, Eric Diffenderfer, Lei Dong, James Metz, Constantinos Koumenis, Keith A. Cengel, Amit Maity, Theresa M. Busch. Gene expression profiling of full-thickness skin after FLASH proton radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3304.

Purpose: Radiation-induced cardiotoxicity is a significant concern in thoracic oncology patients. However, the basis for this disease pathology is not well characterized. We developed a novel mouse model of radiation-induced cardiotoxicity to investigate pathophysiologic mechanisms and identify clinically targetable biomarkers of cardiac injury. Experimental Design: Single radiation doses of 20, 40, or 60 Gy were delivered to the cardiac apex of female C57BL/6 mice ages 9–11 weeks, with or without adjacent lung tissue, using conformal radiotherapy. Cardiac tissue was harvested up to 24 weeks post-radiotherapy for histologic analysis. Echocardiography and Technetium-99m sestamibi single photon emission computed tomography (SPECT) at 8 and 16 weeks post-radiotherapy were implemented to evaluate myocardial function and perfusion. Mouse cardiac tissue and mouse and human plasma were harvested for biochemical studies. Results: Histopathologically, radiotherapy resulted in perivascular fibrosis 8 and 24 (P < 0.05) weeks post-radiotherapy. Apical perfusion deficits on SPECT and systolic and diastolic dysfunction on echocardiography 8 and 16 weeks post-radiotherapy were also observed (P < 0.05). Irradiated cardiac tissue and plasma showed significant increases in placental growth factor (PlGF), IL6, and TNFα compared with nonradiated matched controls, with greater increases in cardiac cytokine levels when radiotherapy involved lung. Human plasma showed increased PlGF (P = 0.021) and TNFα (P = 0.036) levels after thoracic radiotherapy. PlGF levels demonstrated a strong correlation (r = 0.89, P = 0.0001) with mean heart dose. Conclusions: We developed and characterized a pathophysiologically relevant mouse model of radiation-induced cardiotoxicity involving in situ irradiation of the cardiac apex. The model can be used to integrate radiomic and biochemical markers of cardiotoxicity to inform early therapeutic intervention and human translational studies.

Purpose: There is growing interest in the use of modern 3D printing technology to implement intensity-modulated radiation therapy (IMRT) on the preclinical scale which is analogous to clinical IMRT. However, current 3D-printed IMRT methods suffer from complex modulation patterns leading to long delivery times, excess filament usage, and inaccurate compensator fabrication. In this work, we have developed a total variation regularization (TVR) approach to address these issues. Methods: TVR-IMRT, a technique designed to minimize the intensity difference between neighboring beamlets, was used to optimize the beamlet intensity map, which was then converted to corresponding compensator thicknesses in copper-doped PLA filament. IMRT and TVR-IMRT plans using five beams were generated to treat a mouse heart while sparing lung tissue. The individual field doses and composite dose were delivered to film and compared to the corresponding planned doses using gamma analysis. Results: TVR-IMRT reduced the total variation of both the beamlet intensities and compensator thicknesses by around 50% when compared to standard 3D printed compensator IMRT. The total mass of compensator material consumed and radiation beam-on time were reduced by 20-30%, while DVHs remained comparable. Gamma analysis passing rate with 3%/0.3mm criterion was 89.07% for IMRT and 95.37% for TVR-IMRT. Conclusion: TVR can be applied to small animal IMRT beamlet intensities in order to produce fluence maps and subsequent 3D-printed compensator patterns with less total variation, simplifying 3D printing and reducing the amount of filament required. The TVR-IMRT plan required less beam-on time while maintaining the dose conformity when compared to a traditional IMRT plan.

Simple Summary FLASH radiotherapy is a treatment technique of interest that involves radiation delivered at ultra-high dose rates >100 times faster than traditional radiation therapy, which has been shown to spare radiation damage to normal tissue but maintain tumor control capabilities. Proton therapy uses spread-out proton Bragg peaks to reduce radiation dose to normal tissue by directing the highest dose of radiation to the tumor volume. In this study, irradiation of the whole abdomen of mice was performed with proton beams at FLASH dose rates in order to investigate the normal tissue sparing capabilities of the spread-out Bragg peak compared to the entrance region of the proton depth dose curve. Abstract 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.

Multiple studies indicate that ultra-high dose rate, “FLASH” Radiotherapy (F-RT), augments the therapeutic index compared to Standard RT (S-RT) by sparing normal tissue from damage while maintaining tumor responses. We previously reported the first studies using FLASH proton RT (F-PRT), demonstrating that compared to S-PRT, F-PRT spares mice from intestinal epithelium fibrosis and leads to improved overall survival at single doses from 13-16 Gy. Moreover, F-PRT spares a higher percentage of proliferating crypt cells and better maintains the villi architecture 3.5 days post-RT. Importantly, F-PRT was equipotent with S-PRT in controlling syngeneic pancreatic tumors in the same mouse strain. Here, we present new data into potential cellular mechanisms leading to the sparing effect. Using single-cell transcriptomics, we find that F-PRT leads to activation of RIG-I-mediated signaling in the immune cell compartment, which has been linked to normal tissue radioprotection. F-PRT led to higher levels of proliferation genes (Myc, mTOR) in the stem/progenitor cell compartment, consistent with higher proliferative rates. Using a model of epithelial-specific knockout of p53, we find that mice treated with 13.5 Gy S-PRT quickly succumb to acute radiation syndrome while F-PRT significantly improves overall survival. This suggests that F-PRT may preserve a higher proportion of progenitor epithelial cells, leading to reduced fibrosis and long-term toxicity. We also expanded studies to include normal skin damage and control of fibrosarcoma tumors. F-PRT comprehensively decreased toxicity relative to S-PRT, as measured by survival endpoints of mortality/compulsory euthanasia. F-PRT decreased perivascular inflammation, dermal inflammatory infiltrates, and dermal fibrosis on histopathology. Critically, the effect of F-PRT on control of syngeneic sarcomas grown in the leg was indistinguishable from that of S-PRT. Based on these results, we initiated a pilot trial for canine osteosarcoma. To date, we have treated 20 canine patients with a single pre-op fraction of S-PRT or F-PRT. Early results following RNAseq from normal and tumor bone tissue revealed that F-PRT and S-PRT differentially affect myogenesis, adipogenesis, EMT, and inflammatory response. While multiple studies with electron and proton F-RT have demonstrated its normal tissue sparing effects, the mechanism at the molecular level remains elusive. Several models have been proposed, including depletion of oxygen in the vicinity of the DNA, ROS recombination, etc. We will present data from studies of hypoxia and its impact on the FLASH effect and discuss this and alternative models for preferential radioprotection. Our results support the notion that F-PRT significantly augments in the therapeutic window of radiotherapy. Several key questions concerning the mechanism of normal tissue sparing, impact of S-RT vs. F-RT on different tissues and biophysical parameters such as fractionation and optimal dose rates will need to be addressed prior to full clinical implementation of this new modality. Citation Format: Ioannis Verginadis, Anastasia Velalopoulou, Sarah Hagan, Denisa Goia, Khayrullo Shoniyozov, Michele Kim, Eric Diffenderfer, Lei Dong, James Metz, Keith Cengel, Theresa Busch, Andy Minn, Amit Maity, Constantinos Koumenis. Preclinical studies with proton FLASH radiotherapy in mice and canines: Biological effects, biophysical considerations and potential mechanisms [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-019.

Purpose: To investigate the potential normal tissue sparing effects of proton FLASH radiation on skin injury, leg contraction, and soft tissue inflammation while maintaining tumor cell killing efficacy for orthotopic murine fibrosarcoma tumor. Materials and Methods: C3H/HeJ and C57BL/6J mice (n= 6) received 45 Gy of proton FLASH (>85Gy/sec) or conventional radiation (CONV) (1Gy/sec), at the right hind leg. Normal tissue responses were evaluated by recording the skin reaction and the leg contraction over time as acute and chronic macroscopic observations of radiation-induced damage. The skin reactions were recorded using a scoring system of 10 grades, ranging from 0.5 to 3.5, with breakdown of irradiated skin area with moist exudate to be defined as 3. Leg contraction assay examined the extensibility of the leg using a jig and leg contracture was defined as the difference in length between the irradiated and non-irradiated hind leg. Neutrophil infiltration, measured by myeloperoxidase (MPO) activity, and persistent inflammation have been associated with the development of fibrosis, as a long-term effect post radiation. MPO activity was quantified by using intravenously administered luminol and chemiluminescence IVIS imaging at several time points post radiation. We also compared the antitumor efficacy of proton Flash radiation with Proton Standard radiation using syngeneic mice bearing orthotopic fibrosarcoma tumors on the hind leg. Tumors were established by intramuscularly injecting radiation-induced fibrosarcoma (RIF) cells in C57BL/6J mice. Tumor growth was monitored by tumor volume measurements, using a caliper. Results: Acute effects of radiation on skin began with erythema, edema, pigment changes and depilation and was followed by complete loss of the epidermis, constant fibrinous exudates and edema. Skin reaction of C3H/HeJ mice exposed to proton FLASH radiation blindly received a 60% lower score than the CONV-irradiated mice at day 24 post IR, whereas C57BL/6J mice received 60% lower score at day 39, as they appear to be more radiosensitive. Leg contracture was clearly observed by day 68 in the C3H/HeJ whereas in C57BL/6J appeared much earlier, at 10 days post-radiation. Proton FLASH-irradiated group of C57BL/6J mice presented 50% less leg contracture as compared to the CONV-irradiated group, by day 46. MPO activity peaked for both strains at day 18 post radiation and FLASH-irradiated mice showed significantly higher levels of MPO activity compared to the CONV-irradiated mice. The MPO activity was still significantly different between proton FLASH and CONV groups up to day 27 in C57BL/6J mice. Moreover, proton FLASH and CONV radiation inhibited fibrosarcoma tumor growth to the same extent, and there was no significant difference in tumor growth delay or overall survival between the two groups. Conclusions: Proton FLASH radiation causes significant less skin and soft tissue injury as well as lower levels of inflammation post-radiation compared to proton CONV radiation whereas tumor growth inhibition is indistinguishable between the two modalities. Citation Format: Anastasia Velalopoulou, Ilias V. Karagounis, Ioannis I. Verginadis, Denisa Goia, Michele Kim, Khayrullo Shoniyozov, Keith Cengel, Eric Diffenderfer, Lei Dong, James Metz, Theresa Busch, Amit Maity, Costas Koumenis. Proton FLASH radiation spares normal skin and soft tissues of the murine leg from radiation-induced damage while being equipotent with standard proton radiation in controlling sarcoma growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5349.

Small animal x-ray irradiation platforms are expanding the capabilities and future pathways for radiobiology research. Meanwhile, proton radiotherapy is transitioning to a standard treatment modality in the clinician’s precision radiotherapy toolbox, highlighting a gap between state-of-the-art clinical radiotherapy and small animal radiobiology research. Comparative research of the biological differences between proton and x-ray beams could benefit from an integrated small animal irradiation system for in vivo experiments and corresponding quality assurance (QA) protocols to ensure rigor and reproducibility. The objective of this study is to incorporate a proton beam into a small animal radiotherapy platform while implementing QA modelled after clinical protocols. A 225 kV x-ray small animal radiation research platform (SARRP) was installed on rails to align with a modified proton experimental beamline from a 230 MeV cyclotron-based clinical system. Collimated spread out Bragg peaks (SOBP) were produced with beam parameters compatible with small animal irradiation. Proton beam characteristics were measured and alignment reproducibility with the x-ray system isocenter was evaluated. A QA protocol was designed to ensure consistent proton beam quality and alignment. As a preliminary study, cellular damage via γ-H2AX immunofluorescence staining in an irradiated mouse tumor model was used to verify the beam range in vivo. The beam line was commissioned to deliver Bragg peaks with range 4–30 mm in water at 2 Gy min−1. SOBPs were delivered with width up to 25 mm. Proton beam alignment with the x-ray system agreed within 0.5 mm. A QA phantom was created to ensure reproducible alignment of the platform and verify beam delivery. γ-H2AX staining verified expected proton range in vivo. An image-guided small animal proton/x-ray research system was developed to enable in vivo investigations of radiobiological effects of proton beams, comparative studies between proton and x-ray beams, and investigations into novel proton treatment methods.

OBJECTIVES/SPECIFIC AIMS: The overall objective of this study is to develop a novel, clinically-relevant, image-guided mouse model for radiation-induced cardiotoxicity, which can be used to gain insight into clinically-targetable, pathophysiologic mechanisms of cardiac injury in thoracic radiotherapy patients. METHODS/STUDY POPULATION: Photon or sham radiation will be administered at differential doses to a defined portion of the heart and/or lungs of C57BL/6 female mice using micro-CT visualization of the heart with Xstrahl’s MuriSlice Software applied to the Small Animal Radiation Research Platform (SARRP). Cardiac and lung segments from a subset of mice will be harvested at specific time points for confirmation of radiation targeting, local apoptosis assessment, and evaluation of fibrosis and vascular tissue morphology. Quantitative echocardiography, myocardial 18F-fluorodeoxyglucose positron emission tomography computed tomography (18F-FDG PET/CT), and myocardial perfusion imaging (MPI) with Technicium-99 (Tc-99) sestamibi will be implemented to identify sensitive imaging measures of cardiac injury and asses myocardial mechanics, inflammation, and perfusion deficits, respectively. Concurrently, a multiparametric analysis will be conducted to identify novel, circulating biomarkers of cardiotoxicity. RESULTS/ANTICIPATED RESULTS: We hypothesize that a clinically-relevant mouse model can be generated by the in situ, focal irradiation of a portion of heart and/or lung tissue segments, and can be used to elucidate molecular mechanisms of radiation-induced cardiac damage. We anticipate time-dependent and dose-dependent, focal histopathologic changes in the mouse heart, with cardiac fibrosis development, vascular damage, and cellular apoptosis in irradiated mice. Additionally, we anticipate that our mouse model of focal heart irradiation will reveal radiologic and biochemical changes that can be used to characterize and predict radiation-induced cardiac injury. Specifically, we expect our quantitative echocardiography, FDG-PET, and MPI parameters to identify and characterize cardiac damage that topographically matches histopathological analysis, and expect levels of select biochemical markers to differentially vary with time. DISCUSSION/SIGNIFICANCE OF IMPACT: Our mouse model of radiation-induced cardiotoxicity has the potential to shift current preclinical research paradigms to more closely mimic the radiation plans most commonly administered in clinical practice. The primary technologic innovation to be developed here is the use of the SARRP to deliver image-guided, in situ, focal radiation to a defined portion of the mouse heart. From a conceptual perspective, we propose a novel approach for phenotyping radiation-induced cardiac damage in patients undergoing chest radiation therapy, integrating sensitive radiomic and biochemical markers into a predictive model of cardiotoxicity.

The response of fast plastic scintillators to protons is unknown for energies below approximately 300 keV. The response of BC-418 plastic scintillator to protons from 100 keV to 1 MeV was measured using elastic scattering of neutrons at the University of Kentucky. Protons of precise energies from a Van de Graaff accelerator impinged on a thin LiF target to produce neutrons in narrow energy bands. A coincidence was detected between the recoiling proton in the plastic scintillator and the elastically scattered neutron in a liquid scintillator. The energy of the recoil proton was determined by the elastic scattering kinematics, with the scattered neutron energy precisely determined by time of flight. The results are compared with previous measurements of the response of similar plastic scintillators in the energy region where they overlap.

There have been very few measurements of the total cross section for np scattering below 500 keV. In order to differentiate among NN potential models, improved cross section data between 20 and 600 keV are required. We measured the np and nC total cross sections in this energy region by transmission; a collimated neutron beam was passed through CH2 and C samples and transmitted neutrons were detected by a BC-501A liquid scintillator. Cross sections were obtained with a precision of 1.1-2.0% between 150 and 800 keV using ratios of normalized neutron yields measured with and without the scattering samples in the beam. In energy regions where they overlap, the present results are consistent with existing precision measurements, and fill in a significant gap in the data between En = 150 and 500 keV., 7 pages, 7 figures, published in Phys. Rev. C

We report on recent efforts to design a solid plastic scintillation hodoscope to measure neutron production cross sections at low energies. Our program includes not only the development of the detector itself, but also a set of auxiliary measurements which will help characterize its low-energy response. A novel scintillation counter has been developed to detect sub-MeV neutrons while rejecting gamma-ray backgrounds with good efficiency. The detector uses multiple layers of thin solid scintillator, with optical isolation between the adjacent layers. Incident low-energy neutrons produce ionizing recoil particles which remain within just one of the scintillator layers, while background gamma rays create electrons which most often cross the boundary between layers. By observing the trigger pattern within the layers, most gamma-ray backgrounds can be distinguished from the low-energy neutrons of interest. We report on the results of our Monte Carlo studies of this design, as well as on the operation of a prototype detector unit. We also have undertaken a new measurement of the neutron-proton total cross section below 1 MeV. Calculations of the efficiency for detecting low energy neutrons in plastic scintillator rely on accurate low energy n-p cross sections, yet surprisingly few such data currently exist. New measurements which span the region from 150 to 800 keV neutron (lab) energy are reported and discussed. Additionally, we have measured the light response of BC 418 scintillator for recoil proton energies as low as 100 keV. Recoil protons are produced at a known energy in the scintillator by placing it in a neutron beam and detecting in coincidence the elastically scattered neutrons at fixed angle. Our new results extend the energy range of previous measurements of the light response of solid organic scintillators, and may indicate a significantly modified response at the lowest observed energies., United States. Dept. of Energy (Grant No. DE-FG52-10NA29651)