Proton minibeam (pMB) radiotherapy, delivers highly heterogeneous dose distributions alternating high-dose peaks and low-dose valleys. This aims to widen the therapeutic window by improving normal tissue sparing while maintaining the same or even better tumour control. The performance of pMB strongly depends on the collimator design and physical parameters. To better understand the physical and radiobiological drivers of this enhanced therapeutic window, we perform a detailed microdosimetric characterization of proton minibeams and assess their impact. We characterize radiation quality with microdosimetry through Monte Carlo simulations. Then we extend the Generalized Stochastic Microdosimetric Model to predict the normal tissue complication probability (NTCP) at different depths in water, 1cm, 2cm, and 4cm, for 100MeV proton minibeams realized with varying configurations of collimator. Results are compared with conventional homogeneous field (HF) irradiation after dose normalization to the tumor. The developed model is applied by considering tissues as divided into several functional subunits, connected by a seriality parameter. Microdosimetric characterization of proton minibeam irradiation shows differences between peak and valley regions in shaping lineal energy spectra, especially at low depth, while radiation quality uniforms progressively getting closer to the tumor. NTCP calculations results suggest an increased sparing effect for pMB over conventional HF. A strong dependence is found on the peak-to-valley dose ratio (PVDR), and on the seriality parameter. Predictions indicate substantial sparing from pMB, especially for PVDR > 15, including relatively serial organs with seriality around 0.7. This integrated dose-microdosimetric-biological framework elucidates how spatial fractionation, radiation quality, and organ architecture collectively shape tissue sparing in pMB.

Accurate knowledge of nuclear fragmentation cross-sections is essential for optimizing charged particle therapy. In this study, conducted within the framework of the FOOT (FragmentatiOn Of Target) experiment, we present the first measurements with a large angular acceptance of total charge-changing cross-section and the cross-section for the production of fragments (production cross-section) for ^16O ions interacting with Carbon (C) and Polyethylene (C_2H_4) targets in the kinetic energy range of 80 to 200 MeV/nucleon. Measurements were performed using the Emulsion Cloud Chamber (ECC) technique, which combines high spatial resolution and angular acceptance, up to 45^∘. The results are compared with Monte Carlo model predictions. Moreover, the total charge-changing and fragment production cross-sections for ^16O on Hydrogen in the same energy range are derived.

IntroductionWe used the non-tumorigenic MCF10A and triple-negative MDA-MB-231 breast cell lines to compare cell response to CONV and FLASH dose-rates under normoxic conditions. Beyond evaluating cell survival and DNA/microtubule damage, we assessed transcriptomic and immunological profiles to describe putative molecular changes. Oxidative stress induced by two different irradiation modalities was also investigated.MethodsBreast cell lines were irradiated with electron beams at increasing doses of 2, 4, 6, 9, 11, and 15 Gy delivered at either FLASH (230 Gy/s) or CONV (6 Gy/min) dose rates. Survival fractions were determined by clonogenic assay and dose-response curves. DNA damage was quantified by γ-H2AX and 53BP1 foci counting at 0.5, 1, and 24 hours after treatment with 2 and 5 Gy, while microtubule damage was evaluated by confocal microscopy. Transcriptomic profiling was performed by RNA sequencing 24 hours after RT with doses of 9 and 15 Gy. Immunological profiles were analyzed by using Luminex technique at 24, 48, and 72 hours post-RT. The GSH/GSSG ratio was also measured by mass spectrometry at 24 hours post-treatments to assess differences in cellular oxidative status.ResultsCell survival was comparable between FLASH and CONV regimens in both cell lines. However, a dose-rate effect was observed at the level of early DNA damage, with increased γ-H2AX foci 1 hour after FLASH-RT in both cell lines, and greater persistence of 53BP1 foci at 24 hours in MDA-MB-231 cells, suggesting a dose-dependent response. Immunological profiling showed no qualitative differences between dose rates; nevertheless, MDA-MB-231 cells produced higher levels of several factors after FLASH-RT, whereas MCF10A cells displayed minimal variation. Transcriptomic profiling revealed a broader gene modulation following FLASH-RT, with mitochondrial gene upregulation in MCF10A cells and induction of structural genes in MDA-MB-231 cells. No significant differences in glutathione balance were detected between FLASH and CONV irradiation in either breast cell line.DiscussionOur findings provide new insights into the early biological responses to ultra-high dose rates in vitro under normoxic conditions, suggesting that the dose-rate effect may influence early cellular processes at different levels, without directly affecting cell survival.

BACKGROUND:The clinical translation of Minibeam RT (MBRT) has recently started thanks to the first human treatments recently performed. However, despite experimental evidence, the impact of the dose distribution parameters involved on the magnitude of the effect itself and the underlying radiobiological mechanisms are still only partially understood. To address this issue, systematic investigations are needed through the implementation of advanced quantitative experiments with a multidisciplinary approach, which is the one proposed in the framework of the INFN funded MIRO (MInibeam RadiOtherapy) project. PURPOSE:The aim of this work is to report on the on-going main activities recently carried out in the framework of the project, showing some of the main results achieved during the first 2 years of the project, in terms of: (i) facilities development and characterization; (ii) new dosimetric approaches; (iii) biological investigation of the effect; and (iv) development of the first tools for dose planning. The multidisciplinary approach adopted by this national Collaboration to tackle the main challenges of minibeam radiotherapy for a reliable and solid clinical translation is discussed. METHODS:The facilities involved in the project are: (i) two facilities dedicated to low (up to 9 MeV) and medium (up to 18 MeV) energy electron minibeam studies, one of them also equipped with an electron FLASH LINAC to study possible synergistic effects with UHDR beams; (ii) one facility dedicated to proton minibeam studies, with energies from 70 to 140 MeV. Novel dosimetric approaches, are presented, mainly based on scintillators, silicon and silicon carbide detectors. Novel techniques for the analysis of biological samples are described, leveraging both integrated averaged data and spatially resolved analysis. Furthermore, a framework for biological modeling using a multiscale approach is presented. A dedicated dose-planning tool is currently under development to compare "virtual" minibeam plans with conventional ones and, consequently, to quantitatively investigate the potential for clinical translation. RESULTS:All the facilities were dosimetrically characterized, demonstrating the capability of producing controlled and reproducible electron and proton minibeams. These beams exhibited diverse physical parameters, including peak-to-valley ratios between 3 and 30 at the entrance and center-to-center distances between 2 and 3 mm. The developed detectors, specifically scintillators and silicon detectors, successfully measured the minibeam patterns with sub-millimeter resolution, while large-area silicon carbide detectors were used for average dose measurements. First in-vitro biological investigations performed with low energy minibeams clearly showed an enhanced survival fraction in the 16HBE healthy lung cells, while maintaining iso-effective cell killing in A549 cancer cells. Furthermore, a synergy was observed when combining UHDR electron beams with minibeams. CONCLUSIONS:The multidisciplinary approach was consolidated during these first 2 years of the MIRO project, as demonstrated by the results obtained both in terms of dosimetric characterization and biological investigations. The dedicated framework for modelling is being optimized to support the biological findings, allowing for a better interpretation of the results. The tool for dose planning, still under development, will allow the investigation of peculiar configurations to explore new frontiers in the perspective of future human trials.

In conventional radiotherapy, the probability of controlling tumor growth is quantified using Tumor Control Probability (TCP) models. Instead, the probability of experiencing a side effect after the irradiation of healthy tissues and organs is typically assessed using the concept of Normal Tissue Complication Probability (NTCP), an additional crucial metric for evaluating and comparing treatment plans. This work is dedicated to the development, implementation, and application of a general mechanistic model to describe the effects of particle therapy (PT) on different tissue organizations beyond Poissonian assumptions, extending the Generalized Stochastic Microdosimetric Model (GSM2), i.e., a stochastic radiobiological model that describes the time evolution of DNA lesions in a cell nucleus according to microdosimetric principles, to the study of macroscopic biological systems. Specifically, we extend the biological stage of radiation damage of the GSM2 model to larger spatial and temporal scales, involving cell populations with a specific geometric and functional architecture. The model's single-cell resolution allows it to account for energy deposition and tissue heterogeneity, considering different organ volume effects, cell type distributions, and oxygen gradients for different radiation qualities of the beam, that is, type, energy, and LET of radiation, and various fractionation schemes. We show the interplay between physical and environmental parameters on the induction of side effects on healthy tissues, for different radiation qualities and fractionation schemes, and we highlight the impact of biochemical heterogeneities in the target environment, for tumor response.

Objective . Radiation biophysical modelling of the spatio-temporal events following energy deposition in a tissue-like medium is a useful tool for investigating mechanistic features of radiobiological processes. The present study focuses on the description of complex milieux and long time domains. Approach . Monte Carlo (MC) chemical track structure algorithms allow the formation, transport, and recombination of radical species under various irradiation conditions to be followed. This feature has been proposed to have outermost relevance, e.g. in the comprehension of the FLASH effect. Nevertheless, to extend the simulations predictability range in both temporal scales and realistic environments, while avoiding prohibitive running times, computationally lighter approaches have to be used in combination with the accurate step-by-step descriptions provided by MC. To this end, TRAX-CHEMxt has been implemented. Main results . We propose here an upgraded version of the code, capable now to investigate the chemical effects of radiation up to 1 s and in a more complex environment, featured not only by oxygenated water, but also by a representative biomolecule, RH, and an antioxidant component, XSH. The robustness of the code in this new configuration has been proven. Its predictions are compared with both full MC counterparts at the overlapping time scale, (1–10) µ s, and available experimental data at longer temporal points, showing in all cases good agreements. The change in the chemical yields due to the presence of RH and XSH is then investigated, as a function of primary particle type, energy, LET, and target oxygenation. Significance . TRAX-CHEMxt can thus be effectively applied to study the impact of radiation-induced radicals at larger time scales on more complex systems, allowing for specific biological targets simulations.

This paper presents the measurements of the angular differential cross sections for the forward production of He, Li, Be, B, C, and N nuclei in the fragmentation process of a 400 MeV/nucleon O-16 beam interacting with a graphite target. Due to the limited data available in this energy regime, these measurements of nuclear fragmentation cross sections are relevant to improve nuclear interaction models for particle therapy and space radioprotection applications. The data analyzed in this paper were collected during a measurement campaign carried out at the GSI Helmholtz Center for Heavy Ion Research facility in Darmstadt (Germany) by the FOOT Collaboration. The results are compared with similar results found in the literature and with a previous FOOT measurement of the same process, using the same setup, from a previous pilot run performed at GSI. The pilot run data, however, had limited statistics and only allowed for the measurement of elemental fragmentation cross sections integrated in the setup acceptance. This data set, with statistics more than 100 times larger compared to the data collected in the previous run, enabled the measurement of angular differential cross sections, fully exploiting the granularity of the FOOT Delta E-TOF (time-of-flight) system. Furthermore, a better comprehension of the FOOT apparatus allowed to improve the analysis techniques, leading to a reduction in the final systematic uncertainties. The cross section results have been compared with some of the prominent Monte Carlo models of FLUKA and Geant4 dedicated to the energy range of interest for light ion fragmentation physics.

This review explores the effects of ionizing radiation on blood and its components, focusing on its applications, biological impacts, and implications for medical and occupational settings. Ionizing radiation is a cornerstone of modern medicine, playing a critical role in diagnostic imaging, cancer treatment, and preventive measures, such as the irradiation of blood units to prevent transfusion-associated graft-versus-host disease. However, it also induces significant alterations in blood cells, including genetic damage, immune suppression, and changes in hematological, biochemical, and hemorheological parameters, depending on the dose, dose rate, and type of radiation. Conventional radiotherapy, hadron therapy, and the emerging FLASH modality exhibit distinct effects on blood. Hadron therapy and FLASH radiotherapy could reduce oxidative stress preserving red blood cell deformability more effectively than conventional methods, thereby minimizing systemic toxicity. However, the underlying mechanisms remain a topic of ongoing investigation. Additionally, studies reveal how different types of radiation, including gamma rays, X-rays, electron beams, and hadrons, uniquely influence blood cells, underscoring the complexity of radiobiological interactions. Challenges and controversies, such as the long-term hematological impact of radiation exposure, individual variability in response, and the potential of radioprotective strategies and immune system stimulation are also addressed. Insights into hemorheological changes and the development of personalized approaches are critical for optimizing therapeutic outcomes and safety protocols. By synthesizing current knowledge, this review emphasizes the need for further research on the effects of ionizing radiation on blood to bridge gaps in understanding and enhance clinical and practical applications.

Objectives: To investigate how the FLASH effect modulates radiation response on multiple developmental endpoints of zebrafish embryos under normoxic and hypoxic conditions, after irradiation with proton beams at a conventional and an ultra-high dose rate (UHDR). Methods: Embryos were obtained from adult zebrafish and irradiated with a 228 MeV proton beam 24 h post-fertilization (hpf) at a dose rate of 0.6 and 317 Gy/s. For the hypoxic group, samples were kept inside a hypoxic chamber prior to irradiation, while standard incubation was adopted for the normoxic group. After irradiation, images of single embryos were acquired, and radiation effects on larval length, yolk absorption, pericardial edema, head size, eye size, and spinal curvature were assessed at specific time points. Results: Data indicate a general trend of significantly reduced toxicity after exposure to a UHDR compared to conventional regimes, which is maintained under both normoxic and hypoxic conditions. Differences are significant for the levels of pericardial edema induced by a UHDR versus conventional irradiation in normoxic conditions, and for eye and head size in hypoxic conditions. The toxicity scoring analysis shows a tendency toward a protective effect of the UHDR, which appears to be associated with a lower percentage of embryos in the high score categories. Conclusions: A radioprotective effect at a UHDR is observed both for normoxic (pericardial edema) and hypoxic (head and eye size) conditions. These results suggest that while the UHDR may preserve a potential to reduce radiation-induced damage, its protective effects are endpoint-dependent; the role of oxygenation might also be dependent on the tissue involved.

BACKGROUND AND PURPOSE:While the advantages of ultra-high dose-rate (UHDR) irradiation have been well highlighted experimentally, the biological mechanism underlying the FLASH effect is still unclear and highly debated. The aim of this work is to reproduce the main in-vitro UHDR experiments and to try to explain the different in-vivo response between healthy tissues and tumors, developing a fully consistent radiation biophysical model for UHDR regime. MATERIALS AND METHODS:We developed the MultiScale Generalized Stochastic Microdosimetric Model (MS-GSM2), a multi-stage extension of the GSM2, which is a probabilistic model describing the time evolution of the lesions in an irradiated cell nucleus. We coupled the slow DNA damage evolution with the fast chemical reaction kinetics, including the impact of the redox environment. RESULTS:The MS-GSM2 can investigate the combined effects of chemical species, DNA damage formation and time evolution. We demonstrate that the MS-GSM2 predictions are coherent with the in-vitro UHDR experimental results across various oxygenation levels, and radiation qualities. We analyze the role of the chemical environmental conditions of the irradiated medium, i.e. oxygenation, and scavengers concentration, discussing possible factors that can attenuate or level out the dose rate dependence of the cell survival, to understand the differential effect that occurs in-vivo between normal tissue and tumor. CONCLUSION:The MS-GSM2 can accurately describe multiple aspects of the FLASH effect and be consistent with the main evidence from the in-vitro experiments with different types of radiation and oxygenations. Our model proposes a consistent explanation for the differential outcomes observed in normal tissues and tumors, in-vivo and in-vitro.

FOOT (FragmentatiOn Of Target) is a nuclear physics experiment currently under construction that will measure differential cross sections for the production of secondary fragments induced by the interactions of proton and ion beams, up to 400 MeV/u, with human tissues. By extending the energy range to about 800 MeV/u, FOOT will also provide data useful to radio-protection in space, as understanding fragmentation processes that take place in spacecraft shieldings is crucial to their optimisation. The FOOT collaboration is building a detector designed for the identification of heavy fragments in an inverse-kinematics configuration, through the measurement of their momentum, energy and time-of-flight with very high resolution. The kinetic energy of the fragments will be measured with a BGO calorimeter that must cover a dynamic range from tens of MeVs to about 10 GeV and achieve an energy resolution smaller than 2%. In this work we report about the R&D steps that led to the design choices and we assess the performance of the calorimeter prototype. Several beam tests have been performed at CNAO (Pavia, Italy) to choose the best photodetector, crystal wrapping, front-end electronics and readout, in order to achieve the required performance in terms of linearity and energy resolution. Measurements on the first assembled module, made of 3 × 3 BGO crystals with truncated pyramid shape, coupled to SiPM photodetectors, show that, up to at least 5 GeV of deposited energy, there is no saturation effect related to optical photon pileup in the SiPM microcells and the energy resolution ranges from about 2% standard deviation for 70 MeV protons to less than 0.5% for 400 MeV/u carbon ions. This level of performance has been achieved on data collected within a temperature range of about 10 ∘ C. Deviations from linearity were studied by calibrating the crystals with monochromatic beams impinging both on the front face and at different positions along its side. Correction methods to compensate for the signal loss as a function of the range (i.e., the energy) and for temperature fluctuations, were developed and validated on experimental data. Presently, the full calorimeter construction (320 BGO crystals) is complete.

BACKGROUND:Hypoxia significantly affects radiotherapy by increasing tumor radioresistance. High linear energy transfer radiation, such as carbon ion therapy, can help mitigate this issue. Carbon ion arc therapy offers the potential to increase LET in hypoxic tumor regions, offering a promising approach to improve treatment outcomes, but lacks robustness. PURPOSE:We introduced and quantitatively evaluated a new heavy ion therapy treatment strategy named LET bOost by heavy Particle Arc RaDiation (LEOPARD), which combines intensity-modulated particle therapy (IMPT) with spot-scanning hadron arc (SHArc) strategy. LEOPARD aims to increase dose and the dose-averaged linear energy transfer ( LET d ${\rm LET}_{\mathrm{d}}$ ) within the hypoxic target volume (HTV) while maintaining plan quality and robustness. IMPT and SHArcBoost are used as reference strategies for comparison. METHODS:A proof of concept for LEOPARD was realized on 15 head & neck cancer patients with an artificially contoured HTV generated from isotropic shrinking of the clinical target volume (CTV). The LEOPARD plans integrate a full arc field (0 ∘ $^\circ$ to 358 ∘ $^\circ$ , step = 2 ∘ $^\circ$ , 180 fields) delivering a boost dose to the HTV with additional IMPT fields(10 ∘ $^\circ$ and 170 ∘ $^\circ$ ) that simultaneously provide the prescribed dose to the CTV, combining both approaches in a unified treatment plan. To evaluate the potential of LEOPARD, we compared it to intensity modulated particle therapy dose boost plans (IMPTBoost) which included 2 IMPT fields (10 ∘ $^\circ$ and 170 ∘ $^\circ$ ) for CTV, with 2 identical fields added to boost dose in HTV, and SHArc-only boost plans (SHArcBoost) which used same arc fields as LEOPARD aimed at both CTV and HTV but without IMPT fields. Plans were evaluated for dosimetric accuracy, robustness, LET d ${\rm LET}_{\mathrm{d}}$ , and cell survival fraction considering the oxygen-enhancement ratio. Treatment delivery times were calculated using an in-house dynamic carbon ion arc therapy delivery simulator. RESULTS:LEOPARD combined treatment demonstrated a capacity to balance plan quality by mitigating the robustness issues inherent in SHArc plans while improving the LET distribution compared to IMPTBoost plans. Compared with SHArcBoost, LEOPARD showed higher mean D95 in the HTV (96.1% vs. 95.4%; p < 0.001 $p < 0.001$ ), lower mean D2 (113.2% vs. 117.0%; p < 0.001 $p < 0.001$ ) and HI (18.3% vs. 22.6%; p < 0.001 $p < 0.001$ ). Compared with IMPTBoost, mean LET d ${\rm LET}_{\mathrm{d}}$ 50 in the HTV increased by 16.9% ( p < 0.001 $p < 0.001$ ), and mean SF50 in the HTV reduced by 3.8% ( p < 0.001 $p < 0.001$ ). CONCLUSION:We developed a new treatment strategy-LEOPARD, which can generate higher LET d ${\rm LET}_{\mathrm{d}}$ in the HTV than IMPTBoost, while achieving better dose distribution and plan robustness compared to SHArcBoost. This work therefore indicates promising potential of LEOPARD for the treatment of hypoxic tumors.

Silicon carbide (SiC) detectors have been widely demonstrated to be suitable alternative detectors for dosimetry in FLASH radiotherapy, showing radiation hardness and dose-rate independence at the FLASH radiotherapy instantaneous dose rates (IDRs). However, the practical use of such devices in the preclinical/clinical environment still requires the development of special handy housing enabling the quality assurance (QA) measurements under the reference dosimetric conditions. A 10μm thick, 4.5 mm2area SiC detector produced by the STLab company was recently embedded at the INFN-Catania Division inside a plastic waterproof 15 mm diameter cylindrical housing. This encapsulated version of SiC (eSiC) allows the measurement of the dose in reference conditions and of the dose profiles in liquid/solid water phantoms for assuring high accuracy dosimetry QA procedures. Dosimetric characterizations were performed with both electron and proton beams at conventional and ultra-high dose rates (UHDR). A first experiment was carried out at the Centro Pisano for Flash Radiotherapy using UHDR 9 MeV electron beams to confirm the linearity of the charge response as a function of the dose per pulse after the encapsulation procedure. A linearity from 1.8 Gy/pulse up to about 12 Gy/pulse, corresponding to an IDR of 3 MGy s-1, was found. The percentage depth dose (PDD) distribution in water of 9 MeV electron beams was also measured and compared with the PDD measured with a Freiburg Physikalisch-Technische Werkstätten Dr. Pychlau GmbH (PTW) flash diamond detector, used as reference dosimeter. The eSiC detector was also tested with proton beams accelerated by the IBA Proteus 235 cyclotron at the Trento Proton Therapy facility. A response independence on the total delivered dose (1-30 Gy) and average dose rate (50-530 Gy s-1) was found using the UHDR 228 MeV proton beam available along the experimental beamline. The depth dose distribution measured with the eSiC within a liquid water phantom was successfully compared with the one simultaneously measured by the IBA PPC05 reference chamber, using 180 MeV clinical proton beams. The excellent results demonstrated that this first realized eSiC prototype can be used to accurately perform reference and relative dosimetry with UHDR electron and proton beams, contributing to support the clinical translation of FLASH radiotherapy.

The FOOT (FragmentatiOn Of Target) experiment aims to measure double differential fragmentation cross-sections for applications in Particle Therapy and space radiation protection. A critical component of the apparatus is its magnetic spectrometer, composed of two Halbach-configured dipole magnets (M1 and M2) using NdFeB permanent magnets, designed for high field uniformity and stability. A full 3D magnetic model was developed using the OPERA solver, incorporating detailed BH curves and a refined meshing strategy to ensure precision along the beam axis. The resulting field map, essential for Monte Carlo simulations in FLUKA, was validated through high-resolution magnetic measurements along the longitudinal and radial axes using a Hall probe. The comparison revealed agreement within 1.4% over most of the field region. Radial scans confirmed uniformity within 1% up to 10 mm from the axis, with minor misalignments attributed to mechanical tolerances. These results confirm the accuracy of the magnetic model and its suitability for precise momentum reconstruction in the FOOT spectrometer.