Long-term neurocognitive benefits of FLASH radiotherapy driven by reduced reactive oxygen species.

Here, we highlight the potential translational benefits of delivering FLASH radiotherapy using ultra-high dose rates (>100 Gy⋅s-1). Compared with conventional dose-rate (CONV; 0.07-0.1 Gy⋅s-1) modalities, we showed that FLASH did not cause radiation-induced deficits in learning and memory in mice. Moreover, 6 months after exposure, CONV caused permanent alterations in neurocognitive end points, whereas FLASH did not induce behaviors characteristic of anxiety and depression and did not impair extinction memory. Mechanistic investigations showed that increasing the oxygen tension in the brain through carbogen breathing reversed the neuroprotective effects of FLASH, while radiochemical studies confirmed that FLASH produced lower levels of the toxic reactive oxygen species hydrogen peroxide. In addition, FLASH did not induce neuroinflammation, a process described as oxidative stress-dependent, and was also associated with a marked preservation of neuronal morphology and dendritic spine density. The remarkable normal tissue sparing afforded by FLASH may someday provide heretofore unrealized opportunities for dose escalation to the tumor bed, capabilities that promise to hasten the translation of this groundbreaking irradiation modality into clinical practice.

Implementation and validation of a beam-current transformer on a medical pulsed electron beam LINAC for FLASH-RT beam monitoring.

PURPOSE: To implement and validate a beam current transformer as a passive monitoring device on a pulsed electron beam medical linear accelerator (LINAC) for ultra-high dose rate (UHDR) irradiations in the operational range of at least 3 Gy to improve dosimetric procedures currently in use for FLASH radiotherapy (FLASH-RT) studies. METHODS: Two beam current transformers (BCTs) were placed at the exit of a medical LINAC capable of UHDR irradiations. The BCTs were validated as monitoring devices by verifying beam parameters consistency between nominal values and measured values, determining the relationship between the charge measured and the absorbed dose, and checking the short- and long-term stability of the charge-absorbed dose ratio. RESULTS: The beam parameters measured by the BCTs coincide with the nominal values. The charge-dose relationship was found to be linear and independent of pulse width and frequency. Short- and long-term stabilities were measured to be within acceptable limits. CONCLUSIONS: The BCTs were implemented and validated on a pulsed electron beam medical LINAC, thus improving current dosimetric procedures and allowing for a more complete analysis of beam characteristics. BCTs were shown to be a valid method for beam monitoring for UHDR (and therefore FLASH) experiments.

Experimental Drainage Device to Reduce Lymphoedema in a Rat Model.

OBJECTIVE: Despite recent advances in pharmacological research and microsurgery, lymphoedema remains an incurable disease that deeply affects quality of life. There is an urgent need for innovative approaches to restore continuous lymph flow in affected tissues. To this end, the efficacy of a subcutaneously implanted draining device in reducing lymphoedema volume in a rat hindlimb lymphoedema model was tested. METHODS: A rat model of chronic lymphoedema was developed by surgical removal of popliteal and inguinal lymph nodes, followed by irradiation. The model was characterised by monitoring limb volume via tape measure, skin water content via dielectric constant measurement, and lymphatic drainage via lymphofluoroscopy. After lymphoedema establishment in 16 Wistar rats, a device made of fenestrated tubing equipped with a miniaturised pumping system, was implanted subcutaneously in the affected limb to restore continuous recirculation of interstitial fluid. RESULTS: Lymphofluoroscopy imaging showed impaired lymphatic drainage following lymphadenectomy and irradiation. Affected limb volume and skin water content increased significantly compared with the untreated limb, with a median (interquartile range) of 3.85 (0.38) cm3 versus 3.03 (0.43) cm3 for volume (n = 16, p = .001) and 26.6 (9.1) versus 16.6 (3.7) cm3 for skin dielectric constant (n = 16, p = .001). Treatment of lymphoedema with the implanted drainage device showed that 5 weeks post-implant excess volume was significantly reduced by 51 ± 18% compared with the pre-implant situation (n = 9 sham group, n = 7 pump group). CONCLUSION: Lymphoedema volume in the rat model was significantly reduced by restoring continuous drainage of excess fluid using a novel subcutaneously implanted device, opening the way to the development of an artificial lymphatic vessel.

[Management of refractory ventricular tachycardia using stereotactic body radiotherapy]. / Prise en charge des tachycardies ventriculaires réfractaires par radiothérapie stéréotaxique.

Stereotactic body radiotherapy (SBRT) is routinely used in oncology to treat non-invasively solid tumors with high precision and efficacy. Recently, this technology has been evaluated in the treatment of ventricular tachycardia (VT). This article presents the basic underlying principles, proofs of concept and main results of clinical studies that used SBRT for the treatment of VT.

La radiothérapie stéréotaxique (SBRT) est une technologie couramment utilisée en oncologie pour traiter de façon non invasive les tumeurs solides avec précision et efficacité. Récemment, cette technologie a été évaluée dans le traitement des tachycardies ventriculaires (TV). Cet article présente les principes de base sous-jacents, le concept ainsi que les résultats des premières études cliniques ayant traité avec succès des patients souffrant de TV avec la SBRT.

CSF1R inhibition prevents radiation pulmonary fibrosis by depletion of interstitial macrophages.

Radiation-induced lung fibrosis (RIF) is a delayed side-effect of chest radiotherapy, frequently associated with macrophage infiltration.We aimed to characterise the role of pulmonary macrophages in RIF using human lung biopsies from patients receiving radiotherapy for thorax malignancies and a RIF model developed in C57BL/6 mice after 16-Gy thorax irradiation.High numbers of macrophages (both interstitial and alveolar) were detected in clinical and preclinical RIF. In the preclinical model, upregulation of T-helper (Th)2 cytokines was measured, whereas Th1 cytokines were downregulated in RIF tissue lysate. Bronchoalveolar lavage demonstrated upregulation of both types of cytokines. At steady state, tissue-infiltrating macrophages (IMs) expressed 10-fold more arginase (Arg)-1 than alveolar macrophages (AMs), and a 40-fold upregulation of Arg-1 was found in IMs isolated from RIF. IMs, but not AMs, were able to induce myofibroblast activation in vitro In addition, whereas depletion of AMs using Clodrosome didn't affect RIF score, depletion of IMs using a clinically available colony-stimulating factor receptor-1 (CSF1R) neutralising antibody was antifibrotic.These findings suggest differential contributions of alveolar versus interstitial macrophages in RIF, highlighting the fibrogenic role of IMs. The CSF1/CSF1R pathway was identified as a new therapeutic target to inhibit RIF.

Acute Hypoxia Does Not Alter Tumor Sensitivity to FLASH Radiation Therapy.

PURPOSE: Tumor hypoxia is a major cause of treatment resistance, especially to radiation therapy at conventional dose rate (CONV), and we wanted to assess whether hypoxia does alter tumor sensitivity to FLASH. METHODS AND MATERIALS: We engrafted several tumor types (glioblastoma [GBM], head and neck cancer, and lung adenocarcinoma) subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20-Gy fraction at either CONV or FLASH, measured oxygen tension, monitored tumor growth, and sampled tumors for bulk RNAseq and pimonidazole analysis. Next, we inhibited glycolysis with trametinib in GBM tumors to enhance FLASH efficacy. RESULTS: Using various subcutaneous tumor models, and in contrast to CONV, FLASH retained antitumor efficacy under acute hypoxia. These findings show that in addition to normal tissue sparing, FLASH could overcome hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered a FLASH-specific profile in human GBM that involved cell-cycle arrest, decreased ribosomal biogenesis, and a switch from oxidative phosphorylation to glycolysis. Glycolysis inhibition by trametinib enhanced FLASH efficacy in both normal and clamped conditions. CONCLUSIONS: These data provide new and specific insights showing the efficacy of FLASH in a radiation-resistant context, proving an additional benefit of FLASH over CONV.

Structural plasticity of pyramidal cell neurons measured after FLASH and conventional dose-rate irradiation.

Evidence shows that ultra-high dose-rate FLASH-radiotherapy (FLASH-RT) protects against normal tissue complications and functional decrements in the irradiated brain. Past work has shown that radiation-induced cognitive impairment, neuroinflammation and reduced structural complexity of granule cell neurons were not observed to the same extent after FLASH-RT (> MGy/s) compared to conventional dose-rate (CONV, 0.1 Gy/s) delivery. To explore the sensitivity of different neuronal populations to cranial irradiation and dose-rate modulation, hippocampal CA1 and medial prefrontal cortex (PFC) pyramidal neurons were analyzed by electron and confocal microscopy. Neuron ultrastructural analyses by electron microscopy after 10 Gy FLASH- or CONV-RT exposures indicated that irradiation had little impact on dendritic complexity and synapse density in the CA1, but did increase length and head diameter of smaller non-perforated synapses. Similarly, irradiation caused no change in PFC prelimbic/infralimbic axospinous synapse density, but reductions in non-perforated synapse diameters. While irradiation resulted in thinner myelin sheaths compared to controls, none of these metrics were dose-rate sensitive. Analysis of fluorescently labeled CA1 neurons revealed no radiation-induced or dose-rate-dependent changes in overall dendritic complexity or spine density, in contrast to our past analysis of granule cell neurons. Super-resolution confocal microscopy following a clinical dosing paradigm (3×10Gy) showed significant reductions in excitatory vesicular glutamate transporter 1 and inhibitory vesicular GABA transporter puncta density within the CA1 that were largely dose-rate independent. Collectively, these data reveal that, compared to granule cell neurons, CA1 and mPFC neurons are more radioresistant irrespective of radiation dose-rate.

Infrared microspectroscopy to elucidate the underlying biomolecular mechanisms of FLASH radiotherapy.

BACKGROUND: FLASH-radiotherapy (FLASH-RT) is an emerging modality that uses ultra-high dose rates of radiation to enable curative doses to the tumor while preserving normal tissue. The biological studies showed the potential of FLASH-RT to revolutionize radiotherapy cancer treatments. However, the complex biological basis of FLASH-RT is not fully known yet. AIM: Within this context, our aim is to get deeper insights into the biomolecular mechanisms underlying FLASH-RT through Fourier Transform Infrared Microspectroscopy (FTIRM). METHODS: C57Bl/6J female mice were whole brain irradiated at 10 Gy with the eRT6-Oriatron system. 10 Gy FLASH-RT was delivered in 1 pulse of 1.8µs and conventional irradiations at 0.1 Gy/s. Brains were sampled and prepared for analysis 24 h post-RT. FTIRM was performed at the MIRAS beamline of ALBA Synchrotron. Infrared raster scanning maps of the whole mice brain sections were collected for each sample condition. Hyperspectral imaging and Principal Component Analysis (PCA) were performed in several regions of the brain. RESULTS: PCA results evidenced a clear separation between conventional and FLASH irradiations in the 1800-950 cm-1 region, with a significant overlap between FLASH and Control groups. An analysis of the loading plots revealed that most of the variance accounting for the separation between groups was associated to modifications in the protein backbone (Amide I). This protein degradation and/or conformational rearrangement was concomitant with nucleic acid fragmentation/condensation. Cluster separation between FLASH and conventional groups was also present in the 3000-2800 cm-1 region, being correlated with changes in the methylene and methyl group concentrations and in the lipid chain length. Specific vibrational features were detected as a function of the brain region. CONCLUSION: This work provided new insights into the biomolecular effects involved in FLASH-RT through FTIRM. Our results showed that beyond nucleic acid investigations, one should take into account other dose-rate responsive molecules such as proteins, as they might be key to understand FLASH effect.

In vivo measurements of change in tissue oxygen level during irradiation reveal novel dose rate dependence.

BACKGROUND AND PURPOSE: This study aimed to investigate the radiochemical oxygen depletion (ROD) in vivo by directly measuring oxygen levels in various mouse tissues during ultra-high dose rate (UHDR) irradiation at clinically relevant doses and dose rates. MATERIALS AND METHODS: Mice bearing subcutaneous human glioblastoma (U-87 MG) tumors were used for tumor and normal tissue (skin, muscle, brain) measurements. An oxygen-sensitive phosphorescent probe (Oxyphor PtG4) was injected into the tissues, and oxygen levels were monitored using a fiberoptic phosphorometer during UHDR irradiation with a 6 MeV electron linear accelerator (LINAC). Dose escalation experiments (10-40 Gy) were performed at a dose rate of 1300 Gy/s, and dose rate escalation experiments were conducted at a fixed dose of 40 Gy with dose rates ranging from 2 to 101 Gy/s. RESULTS: Radiation-induced change in tissue oxygenation (ΔpO2) increased linearly with dose and correlated with baseline tissue oxygenation levels in the range of 0 - 30 mmHg. At higher baseline tissue oxygenation levels, such as those observed in muscle and brain, there was no corresponding increase in ΔpO2. When we modulated dose rate, ΔpO2 increased steeply up to âˆ¼ 20 Gy/s and plateaued thereafter. The relationship between ΔpO2 and dose rate showcases the interplay between ROD and reoxygenation. CONCLUSION: While UHDR irradiation induces measurable oxygen depletion in tissues, the observed changes in oxygenation levels do not support the hypothesis that ROD-induced radioresistance is responsible for the FLASH tissue-sparing effect at clinically relevant doses and dose rates. These findings highlight the need for further investigation into alternative mechanisms underlying the FLASH effect.

Dosimetric and biologic intercomparison between electron and proton FLASH beams.

BACKGROUND AND PURPOSE: The FLASH effect has been validated in different preclinical experiments with electrons (eFLASH) and protons (pFLASH) operating at an average dose rate above 40 Gy/s. However, no systematic intercomparison of the FLASH effect produced by eFLASHvs. pFLASH has yet been performed and constitutes the aim of the present study. MATERIALS AND METHODS: The electron eRT6/Oriatron/CHUV/5.5 MeV and proton Gantry1/PSI/170 MeV were used to deliver conventional (0.1 Gy/s eCONV and pCONV) and FLASH (≥110 Gy/s eFLASH and pFLASH) dose rates. Protons were delivered in transmission. Dosimetric and biologic intercomparisons were performed using previously validated dosimetric approaches and experimental murine models. RESULTS: The difference between the average absorbed dose measured at Gantry 1 with PSI reference dosimeters and with CHUV/IRA dosimeters was -1.9 % (0.1 Gy/s) and + 2.5 % (110 Gy/s). The neurocognitive capacity of eFLASH and pFLASH irradiated mice was indistinguishable from the control, while both eCONV and pCONV irradiated cohorts showed cognitive decrements. Complete tumor response was obtained after an ablative dose of 20 Gy delivered with the two beams at CONV and FLASH dose rates. Tumor rejection upon rechallenge indicates that anti-tumor immunity was activated independently of the beam-type and the dose-rate. CONCLUSION: Despite major differences in the temporal microstructure of proton and electron beams, this study shows that dosimetric standards can be established. Normal brain protection and tumor control were produced by the two beams. More specifically, normal brain protection was achieved when a single dose of 10 Gy was delivered in 90 ms or less, suggesting that the most important physical parameter driving the FLASH sparing effect might be the mean dose rate. In addition, a systemic anti-tumor immunological memory response was observed in mice exposed to high ablative dose of electron and proton delivered at CONV and FLASH dose rate.