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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 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.

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.

Recent studies suggest that ultrahigh-dose-rate, “FLASH,” radiation therapy (RT) decreases normal tissue damage while maintaining tumor response compared with conventional dose rate RT. Our group has designed and tested the first system to accurately deliver dosimetrically identical FLASH Proton RT (F-PRT; 60-110 Gy/sec) or Standard Proton RT (S-PRT; 0.5-1 Gy/sec) using double-scattered protons. Our purpose is to identify if F-PRT is superior to S-PRT in protecting normal tissues, while equipotent in controlling tumor growth. We previously demonstrated that compared to S-PRT, F-PRT significantly decreases overall mortality from late epithelial gastrointestinal (GI) fibrosis (p Citation Format: Ioannis I. Verginadis, Clara Morral Martinez, Tristan Lim, Priyanka Chowdhury, Giorgos Skoufos, Michele M. Kim, Anastasia Velalopoulou, Denisa Goia, Leo Luo, Caitlin J. Foley, Zachary J. Reitman, Andrea R. Daniel, Eric Diffenderfer, Lei Dong, James Metz, David G. Kirsch, Andy Minn, Constantinos Koumenis. Single-cell RNA sequencing analysis and transgenic mouse models reveal differential effects of flash vs. standard proton radiotherapy on gastrointestinal tissues and tumors [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 3320.

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.

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.