FLASH Proton Radiation Therapy Mitigates Inflammatory and Fibrotic Pathways and Preserves Cardiac Function in a Preclinical Mouse Model of Radiation-Induced Heart Disease.

PURPOSE: Studies during the past 9 years suggest that delivering radiation at dose rates exceeding 40 Gy/s, known as "FLASH" radiation therapy, enhances the therapeutic index of radiation therapy (RT) by decreasing normal tissue damage while maintaining tumor response compared with conventional (or standard) RT. This study demonstrates the cardioprotective benefits of FLASH proton RT (F-PRT) compared with standard (conventional) proton RT (S-PRT), as evidenced by reduced acute and chronic cardiac toxicities. METHODS AND MATERIALS: Mice were imaged using cone beam computed tomography to precisely determine the heart's apex as the beam isocenter. Irradiation was conducted using a shoot-through technique with a 5-mm diameter circular collimator. Bulk RNA-sequencing was performed on nonirradiated samples, as well as apexes treated with F-PRT or S-PRT, at 2 weeks after a single 40 Gy dose. Inflammatory responses were assessed through multiplex cytokine/chemokine microbead assay and immunofluorescence analyses. Levels of perivascular fibrosis were quantified using Masson's Trichrome and Picrosirius red staining. Additionally, cardiac tissue functionality was evaluated by 2-dimensional echocardiograms at 8- and 30-weeks post-PRT. RESULTS: Radiation damage was specifically localized to the heart's apex. RNA profiling of cardiac tissues treated with PRT revealed that S-PRT uniquely upregulated pathways associated with DNA damage response, induction of tumor necrosis factor superfamily, and inflammatory response, and F-PRT primarily affected cytoplasmic translation, mitochondrion organization, and adenosine triphosphate synthesis. Notably, F-PRT led to a milder inflammatory response, accompanied by significantly attenuated changes in transforming growth factor ß1 and α smooth muscle actin levels. Critically, F-PRT decreased collagen deposition and better preserved cardiac functionality compared with S-PRT. CONCLUSIONS: This study demonstrated that F-PRT reduces the induction of an inflammatory environment with lower expression of inflammatory cytokines and profibrotic factors. Importantly, the results indicate that F-PRT better preserves cardiac functionality, as confirmed by echocardiography analysis, while also mitigating the development of long-term fibrosis.

Proton FLASH Radiotherapy Ameliorates Radiation-induced Salivary Gland Dysfunction and Oral Mucositis and Increases Survival in a Mouse Model of Head and Neck Cancer.

Head and neck cancer radiotherapy often damages salivary glands and oral mucosa, severely negatively impacting patients' quality of life. The ability of FLASH proton radiotherapy (F-PRT) to decrease normal tissue toxicity while maintaining tumor control compared with standard proton radiotherapy (S-PRT) has been previously demonstrated for several tissues. However, its potential in ameliorating radiation-induced salivary gland dysfunction and oral mucositis and controlling orthotopic head and neck tumor growth has not been reported. The head and neck area of C57BL/6 mice was irradiated with a single dose of radiotherapy (ranging from 14-18 Gy) or a fractionated dose of 8 Gy × 3 of F-PRT (128 Gy/second) or S-PRT (0.95 Gy/second). Following irradiation, the mice were studied for radiation-induced xerostomia by measuring their salivary flow. Oral mucositis was analyzed by histopathologic examination. To determine the ability of F-PRT to control orthotopic head and neck tumors, tongue tumors were generated in the mice and then irradiated with either F-PRT or S-PRT. Mice treated with either a single dose or fractionated dose of F-PRT showed significantly improved survival than those irradiated with S-PRT. F-PRT-treated mice showed improvement in their salivary flow. S-PRT-irradiated mice demonstrated increased fibrosis in their tongue epithelium. F-PRT significantly increased the overall survival of the mice with orthotopic tumors compared with the S-PRT-treated mice. The demonstration that F-PRT decreases radiation-induced normal tissue toxicity without compromising tumor control, suggests that this modality could be useful for the clinical management of patients with head and neck cancer.

Improving the efficiency of small animal 3D-printed compensator IMRT with beamlet intensity total variation regularization.

PURPOSE: There is growing interest in the use of modern 3D printing technology to implement intensity-modulated radiation therapy (IMRT) on the preclinical scale that 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 less accurate compensator fabrication. In this work, we have developed a total variation regularization (TVR) approach to address these issues. METHODS: TVR-IMRT was used to optimize the beamlet intensity map, which was then converted to a thickness of the corresponding compensator attenuation region in copper-doped polylactic acid (PLA) filament. IMRT and TVR-IMRT heart and lung plans were generated for two different mice using three, five, or seven gantry angles. The total compensator thickness, total variation of compensator beamlet thicknesses, total variation of beamlet intensities, and exposure time were compared. The individual field doses and composite dose were delivered to film for one plan and gamma analysis was performed. RESULTS: In total, 12 mice heart and lung plans were generated for both IMRT and TVR-IMRT cases. Across all cases, it was found that TVR-IMRT reduced the total variation of compensator beamlet thicknesses and beamlet intensities by 54 ± 4 % $54\pm 4\%$ and 50 ± 3 % $50\pm 3\%$ on average when compared to standard 3D-printed compensator IMRT. On average, the total mass of compensator material consumed and radiation beam-on time were reduced by 45 ± 6 % $45\pm 6\%$ and 24 ± 4 % $24\pm 4\%$ , respectively, whereas dose metrics remained comparable. Heart plan compensators were printed and delivered to film and subsequent gamma analysis performed for each of the single fields as well as the composite dose. For the composite delivery, a passing rate of 89.1% for IMRT and 95.4% for TVR-IMRT was achieved for a 3 % / 0.3 $3\%/0.3$ mm criterion. CONCLUSIONS: TVR can be applied to small animal IMRT beamlet intensities to produce fluence maps and subsequent 3D-printed compensator patterns with significantly less complexity while still maintaining similar dose conformity to traditional IMRT. This can simplify/accelerate the 3D printing process, reduce the amount of filament required, and reduce overall beam-on time to deliver a plan.

Oxygen Monitoring in Model Solutions and In Vivo in Mice During Proton Irradiation at Conventional and FLASH Dose Rates.

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. pO2 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 pO2 virtually unchanged at Conv dose rates. However, during FLASH irradiation there was a stepwise decrease in pO2 (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 pO2 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.

Ultrafast Tracking of Oxygen Dynamics During Proton FLASH.

PURPOSE: Radiation therapy delivered at ultrafast dose rates, known as FLASH RT, has been shown to provide a therapeutic advantage compared with conventional radiation therapy by selectively protecting normal tissues. Radiochemical depletion of oxygen has been proposed to underpin the FLASH effect; however, experimental validation of this hypothesis has been lacking, in part owing 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: In vitro in solutions containing bovine serum albumin the O2 depletion g values (moles/L of O2 depleted per radiation dose, eg, µM/Gy) are higher for conventional irradiation (by ∼13% at 75 µM [O2]) than for FLASH, and in the low-oxygen region (<25 µM [O2]) they decrease with oxygen concentration. In vivo, depletion of oxygen by a single FLASH is insufficient to achieve severe hypoxia in initially well-oxygenated tissue, and the g values measured appear to correlate with baseline oxygen levels. CONCLUSIONS: The developed method should be instrumental in radiobiological studies, such as studies aimed at unraveling the mechanism of the FLASH effect. The FLASH effect could in part originate from the difference in the oxygen dependencies of the oxygen consumption g values for conventional versus FLASH RT.

A Novel Mouse Model of Radiation-Induced Cardiac Injury Reveals Biological and Radiological Biomarkers of Cardiac Dysfunction with Potential Clinical Relevance.

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.

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.

FLASH Proton Radiotherapy Spares Normal Epithelial and Mesenchymal Tissues While Preserving Sarcoma Response.

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 TGFß1 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.

Design, Implementation, and in Vivo Validation of a Novel Proton FLASH Radiation Therapy System.

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.

Design and commissioning of an image-guided small animal radiation platform and quality assurance protocol for integrated proton and x-ray radiobiology research.

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.