ABSTRACT. Research in the field of biological effects of heavy charged particles is needed for both heavy-ion therapy (hadrontherapy) and protection from the exposure to galactic cosmic radiation in long-term manned space missions. Although the exposure conditions (e.g. high- vs. low-dose rate) are different in therapy and space, it is clear that a substantial overlap exists in several research topics, such as individual radiosensitivity, mixed radiation fields, and tissue degenerative effects. Late effects of heavy ions are arguably the main health risk for human space exploration, and with the increasing number of cancer patients treated by heavy-ion therapy, including young adults and children, this issue is now becoming the main source of uncertainty for the success of hadrontherapy as well. Reducing uncertainty in both cancer and noncancer late risk estimates is therefore the first priority in heavy-ion radiobiology. In addition, researchers involved either in experimental studies on space radiation protection or heavy-ion therapy often use the same accelerator facilities. Several heavy-ion therapy facilities are now under construction or planned in Europe, USA, and Japan. Beamtime will be available at these facilities for clinical radiobiology and basic heavy-ion effects experimental research, as already happens since several years at the HIMAC in Japan. The NASA Space Radiation Laboratory (NSRL) in Brookhaven (Long Island, NY) provides beams of very heavy ions at energies around 1 GeV/n which are of specific interest for space radiobiology. In Europe, these very high energy beams are available at GSI in Germany, where the new Facility for Antiprotons and Ion Research (FAIR) is currently under construction. It is foreseeable that the availability of beamtime and the presence of many dedicated research programs will lead to great improvements in our knowledge of biological effects of heavy ions in the coming few years.

ABSTRACT. Exploration mission safety assurance requires the understanding of radiation risks and limiting risks to acceptable levels, however the large uncertainties in risk predictions are an impediment to mission design. Predicting the health risks from galactic cosmic ray (GCR) exposures carries large uncertainties due to the qualitatively distinct microscopic energy deposition and early biochemistry of heavy ions compared to low linear energy transfer (LET) radiation, such as X-ray or gamma-rays. Radiobiology research with heavy ions has identified important qualitative differences that suggest conventional approaches based on scaling to epidemiology data for gamma-rays using quality factors have inherent limitations [1,2]. We describe an alternate approach using the direct application of data from animal studies with heavy ions and fission neutrons [2]. GCR risk estimates suggest fatal cancer risks for long-term space missions could approach 20% fatality [3]. This high efficiency of heavy ions in causing solid cancers has several components. High LET radiation causes non-targeted effects (NTE), which dramatically increases risk at low dose. The quality of heavy ion tumors is also distinct with more aggressive tumors, shorter latency, and decrease immune cell infiltration into tumor volumes. This later aspect suggests a possible role for synergistic risks from space radiation and immune changes due to microgravity. Space radiation risks for non-cancer effects are diverse in nature. The risk of acute radiation syndromes from solar particle events is readily avoided using passive shielding and alert dosimetry. There is a significant probability of vision impairing cataracts with short latency (<3 years), and cognitive and memory detriments during a mission. We review results from heavy ion accelerator-based animal experiments with heavy ions on risks to cognition that suggest a potentially significant risk for a Mars mission [4,5], and discuss modeling approaches to make quantitative estimates of cognitive risks.

1. Cucinotta FA, et al. Life Sci Space Res 31, 59-70, 2021. 2. Cucinotta FA. Int J Molecular Sci 23(8), 4324, 2022. 3. Cucinotta FA, et al. Acta Astronautica 166, 529-536, 2020. 4. Cucinotta FA, Cacao E. Int J Radiat Biol 95, 985-998, 2019. 5. Cucinotta FA, Cacao E. Life Sci Space Res 25, 129-135, 2019.

ABSTRACT. Space radiation can be the single limiting factor of human and robotic space exploration. The space radiation environment is complex, with different qualities and quantities of radiation. It can have detrimental effects to human health, as well as hardware on board of spacecrafts. As the European Space Agency (ESA) enters a new era of space missions beyond Low Earth Orbit, to the Moon and beyond, new measures need to be taken to safeguard astronauts and protect spacecrafts on deep space missions. ESA’s “Science in Space Environment” (SciSpacE) programme is dedicated to facilitating state of the art science in space and on Earth, to enable human and robotic space exploration, as well as to deliver solutions to problems back on Earth. SciSpacE offers an extensive programme for radiation research, with the use of European irradiation facilities, as well as current (the International Space Station) and future (Gateway — the Moon orbiting station, the Moon’s surface) platforms. In this presentation, we will elaborate on the complex space radiation environment and operational constrains of current and future space missions. Current astronaut radiation protection strategies and future challenges will be described. We will present ESA-supported current and future opportunities for radiation research and discuss the ESA's objectives of science in and for space.

ABSTRACT. Human space exploration is one of the most effective drivers for scientific research and technological innovation. In view of the steadily growing international relevance of human space exploration beyond Low Earth Orbit, the Italian Space Agency (ASI) is continuously reinforcing the network of the national scientific community and industrial stakeholders for the success of future Moon and Mars exploration missions. This requires tackling unsolved issues related to the effects of long-duration space flights and developing the required, enabling technologies.

ASI has activated four working groups with the participation of national experts from the scientific community on the macro-areas of space life sciences: integrated physiology, microbiology, biological systems for life support, and radiations. The groups have analyzed the current scenario of space life science research and have identified the most relevant objectives and key issues to enhance the national contribution and competitiveness towards enabling human deep space exploration in collaboration with international partners and agencies.

In this contribution, we will present and discuss the results produced by the working groups, with emphasis on radiation risk mitigation and development of countermeasures for human space exploration.

ABSTRACT. Cosmic radiation represents an important threat to the health of astronauts during long space flights [1]. So far, the only strategy to counteract such a threat is the use of passive or active shielding, but radiation shields are expensive and cannot provide a full protection from the damage [2]. Radiations are also used in current radiotherapy. While radiation is very effective in killing cancer cells, the therapeutic dose must be weighed against the possible damage to healthy tissue, that limits the efficacy of radiotherapy. A peculiar biological condition that enhances radioprotection is hibernation/torpor [3]. Torpor is an hypometabolic state used by many mammals such as bears, squirrels, hamsters, mice and many others to save energy in harsh conditions [4]. Although already known in the past [5], the idea to exploit hibernation-induced radioprotection for interplanetary travels [6, 7] or for medical purposes [8] was renewed by recent studies showing new ways to effectively induce a safe and reversible state similar to hibernation in non-hibernators [9-12]. Such a state can be referred to as synthetic torpor [8, 13]. As a first step on the path to the unravelling of the molecular mechanism mediating hibernation-induced radioprotection, we conducted an experiment funded by INFN: project HIBRAD. In this experiments, non-hibernating animals (rats) were exposed to 3Gy of X-rays in synthetic torpor or in euthermic conditions. To observe the early molecular response, organs were sampled 4 hours after the irradiation. Tissue damage was evaluated by immunohistochemistry and gene expression by RNAseq. Here we report preliminary results from HIBRAD, showing that animals irradiated in synthetic torpor don’t show sign of liver damage, compared with control animals. Moreover, we have identified a small set of genes that are expressed only in the animals irradiated in synthetic torpor, among which ETNK1 and CISH, possibly responsible for the resistance to the radiation damage.

ABSTRACT. In this work, the BIANCA (BIophysical ANalysis of Cell death and chromosome Aberrations) biophysical model was extended to heavy ions up to Fe, with the goal of evaluating the biological damage induced in astronauts exposed to space radiation. Specifically, two radiobiological databases were generated, the first one describing Human Skin Fibroblast (HSF) cell survival, and the second one describing the induction of lymphocyte dicentric chromosomes, as a function of ion type (1≤Z≤26) and LET, as well as dose. Using an interface between BIANCA and the FLUKA Monte Carlo transport code, astronaut doses and the corresponding relative biological effectiveness (RBE) values for Galactic Cosmic Rays (GCR) in deep space were calculated, under different shielding conditions. To compare the results with cancer and non-cancer dose limits for astronauts, the RBE calculated for HSF cell survival was used to estimate the equivalent dose for deterministic effects (Gy-Eq), whereas the RBE for lymphocyte dicentrics was used to estimate the equivalent and the effective dose for stochastic effects (Sv). Concerning the deterministic limits, for 650 days in free space at solar minimum (as representative of a Mars mission) the RBE-weighted doses for HSF cell survival were lower than the career limit recommended by NCRP report no. 132 for skin. For 365 days, the simulation values were lower than the 1-year limits, both for skin and for blood forming organs (BFO). Concerning stochastic effects, the equivalent doses calculated using lymphocyte dicentrics were similar to those calculated using the Q values recommended by ICRP. Furthermore, for a 650-day mission the results were always lower than the career limit recommended by ICRP (1 Sv), which has been adopted by ESA and RSA; the comparison with the NCRP limits adopted by NASA was more complex, since they are age- and sex-dependent. Following this work BIANCA, when interfaced to a MC transport code like FLUKA, can now predict RBE values for cell death and lymphocyte dicentrics following GCR exposure, taking into account the ion type (up to Fe-ions), LET and dose.

ABSTRACT. The challenging part of radiation therapy is needed to successfully eradicate cancer and, at the same time, not harm the healthy tissue or cells. Radiation is also still the primary concern for space travelers, such as astronauts who are at risk of being exposed to heavy-ion radiation, affecting their health. Therefore, it is essential to find a reasonable way to protect the healthy tissue from radiation. We treated rats with adenosine agonist 5’-monophosphate monohydrate (5’-AMP) i.p., immediately after whole-body irradiation with 8 Gy or 2 Gy of Carbon (C-) ions and then housed them in a cold room (16oC) for 6 hours. The body temperature of the 5’-AMP-treated rats decreased compared to those treated with saline injection. The animals treated with 5’-AMP showed higher survival following 8 Gy of C-ions than when treated with saline. Furthermore, the histology results showed that one week after 2 Gy of C-ion irradiation, activated microglia in the brain and apoptotic cells in the liver of 5’-AMP treated rats showed fewer numbers than in saline-treated animals after irradiation. Additionally, in vitro experiments using rats' retinal pigmentosum cells (RPE-J) showed that 5’-AMP treatment in combination with hypoxia or lower temperature immediately after 2 Gy of C-ions leads to delayed DNA repair suppresses the radiation-induced mitotic catastrophe. Thus, results suggested that using 5’-AMP together with hypoxia or low temperatures may increase cell resistance to radiation damage.

ABSTRACT. Since the first human journeyed into outer space in 1961, boundaries were pushed and it became possible to visit space for longer periods of time. However, sending humans further into space and extending mission durations challenges the current capabilities of space medicine. When leaving the Earth’s surface, the human body experiences big environmental challenges caused by an extreme environment. These challenges, either physical (cosmic radiation and altered gravity levels) or psychological (stress) in nature, disrupt the body’s homeostasis resulting in adverse health effects.

One of the important spaceflight associated health problems which is considered particularly important for the success of long-term, exploratory-type human space missions is immune dysfunction. Physical and psychological space stressors have been shown to weaken the immune defense capacity, e.g. cell skeleton alterations and lowered immune cell numbers. This weakening in combination with an increased microbial virulence, growth and resistance in space increases the health risks to astronauts. At the moment, however, the exact effects of the induced immune dysfunction are unknown and more research is needed to evaluate how different spaceflight stressors can affect the immune system.

To this aim we are using both in-flight experiments on board the international space station as well as space-analogue models (e.g. Antarctic expeditions, bed rest), as a platform to analyze T cell behavior. Furthermore, in vitro and in vivo ground-based experiments using space simulating models are also currently being performed at SCK CEN with a specific focus on further elucidating the signaling pathways that control immune cell alterations induced by space flight stressors.

Acknowledgements: This research is financially supported by the ESA/BELSPO/Prodex PRODEX IMPULSE contract (CO-90-11-2801-03) and via the ELGRA Research Prize 2020-21.

ABSTRACT. The flux of Galactic Cosmic Rays near Earth is not representative of the Local Interstellar Spectrum at energies below ~30 GeV due to a variety of physical processes arising in their propagation through the heliosphere. The changes in the GCR intensities and energy spectra are related to the solar activity, and are referred to as CR solar modulation. A numerical modulation model to study the transport of galactic protons in the heliosphere is presented. The model was applied to the 27-day averaged galactic proton flux recently released by the PAMELA and AMS02 experiments, covering overall an extended time period from mid-2006 to mid-2017. The time evolution of the model parameters and their relationship with solar activity proxies is shown. As we will discuss, our data-driven approach, based on the availability of new precision measurements, leads to new insights on the solar modulation phenomena.

ABSTRACT. Human long-term, deep space exploration missions are becoming a reality in the 21st century [1]: the NASA’s Artemis program and the launch of the Lunar Gateway will lead humanity forward to the Moon and prepare us for the next giant leap, the exploration of Mars. Nevertheless, space radiation may place astronauts at significant risk for radiation sickness, increased lifetime risk of tumors, degenerative diseases, thus representing the main potential showstopper for safe human exploration of the Solar system [2]. The scientific competences required to properly tackle these issues are broad and interdisciplinary. The tools and strategies needed to further consolidate the radiation research efforts are often still not well known to the wide scientific community and the relevant knowledge is often scattered and difficult to reach especially to the non-expert user. The European Radiation Facility Network - Data Hub (ERFNet-DH) project, funded and coordinated by ESA and implemented by ALTEC (Turin, Italy), aims at providing a solution to the above challenges. The ERFNet-DH focuses on processing and managing data of space radiation research missions. It also provides a service that promotes data sharing and cooperating among the different experts of the international scientific community, with the aim to support space radiation research. In particular, the ERFNet-DH will offer the possibility to: • collect, process, store and distribute radiation data from present and past space missions • provide a data hub for physics, biology and medical research groups interested in working with space and space-related radiation data • provide operational support to ESA’s radiation payloads on the Lunar Gateway • support the coordination of ESA’s radiation research and applications activities • perform data analysis and run numerical simulations • be the boost for the development of European Radiation Risk Model.

All the details about the ERFNet-DH design and development will be presented.

References [1] ISECG, 2013. The Global Exploration Roadmap. NASA, Washington, DC. [2] Chancellor, J., Scott, G., Sutton, J., 2014. Space radiation: the number one risk to astronaut health beyond low earth orbit. Life 4, 491–510. https://doi.org/10.3390/ life4030491

ABSTRACT. Data obtained in our laboratory suggest that telomere damaging agents are also capable of sensitizing tumor cells to ionizing radiation (IR) exposure. In recent years, the nucleoside analogue 6-thio-2'-deoxyguanosine (6-thio-dG) has been shown to inhibit the proliferation of telomerase-positive cancer cells through its incorporation into telomeres and subsequent induction of DNA damage, cell cycle arrest and cell death. These effects were investigated in several tumor models, such as lung cancer, glioma and melanoma both in vitro and in vivo, but to date no data are available in breast cancer cell lines. In this work we characterized the response to 6-thio-dG in breast cancer lines and tested the efficacy of the compound in combination with IR in MCF7 cells. In particular, the anti-proliferative capacity of the nucleoside analogue was studied in the three telomerase-positive breast cancer lines and in primary human fibroblasts (HFFF2, telomerase negative). Furthermore, effect on cell cycle modulation and induction of genomic and telomeric DNA damage (in the first seven days from treatment), were assessed. These experiments let us to identify 6-thio-dG concentrations and treatment duration capable of inducing telomere dysfunction without drastically affect cell proliferation and plating efficiency in order to set up conditions for 6-thio-dG and IR combined treatments. Surviving fraction experiments indicate that 6-thio-dG is able to synergistically increase the sensitivity to IR in breast adenocarcinoma cells. In addition, molecular cytogenetic analysis and experiments on three-dimensional breast cancer models are in progress to further validate the results obtained. Data so far collected confirm telomere targeting as a promising radiosensitizing strategy.

ABSTRACT. Ionizing radiation (IR) has been shown to modulate a variety of immune response processes both in vitro and in vivo. IR-mediated immune system modulation is a complex phenomenon in which several players (such as stress sensors and cytokines) take part regulating immune and inflammatory response. Among them, the cyclic -GMP-AMP synthase (cGAS), a free cytosolic DNA sensor, is capable of recognizing DNA fragments that, after treatment with IR, could be observed as micronuclei (MNi)1. Activation of cGAS causes GAMP dependent-STING activation and promotes phosphorylation and translocation into the nucleus of transcription factors (such as IRF3 and IRF7) that induce innate immune responses and type I interferon (IFN)2. In the present work the induction of cGAS-positive MNi was evaluated in immortalized human keratinocytes (HaCaT) exposed to X-rays (250 Kev; 0.5, 1 and 2Gy) and fixed 24-120h after treatment, to determine the dose-response and activation kinetic. Data obtained showed that the highest induction of cGAS-positive MNi was observed after 48h from X-rays irradiation and despite a dose-dependent increase in the number of total MN, cGAS-positive MNi increase after 0.5 Gy reaching a plateau in the dose range of 1-2 Gy. Pathway activation kinetic was obtained by analyzing the cGAS-positive MN frequency at 3, 6, 9, 24, 48, 72, 96 and 120 hours after exposure to 1 Gy of X-rays. In addition, type I IFN gene expression was verified by RT-qPCR and ISG15 induction, a ubiquitin-like protein transcriptionally induce by type I IFN, was analyzed by western blot. In addition, literature data suggested instability of nuclear lamina in MNi leading to membrane rupture and allowing DNA fragment recognition by cGAS. It would be important to characterize MNi lamina integrity, to study the relationship between DNA damage and innate immune system activation and to provide useful information for the role of radiation exposure in immune response.

ABSTRACT. In vivo experiments using positron emission tomography (PET) to evaluate the efficacy of the dose on the biodistribution of new radiolabeled chelators are a necessary step of translational research. In this paper we present an innovative method based on radiomics to process and analyze data extracted from images obtained from a microPET/CT study conducted on nude Balb/c mice treated with 7MBq of a novel [64Cu] chelator. The scans of the mice were acquired at three different time points, specifically 1 hour, 4 hours and 24 hours after inoculation of the 64Cu-labeled compound (EC/β+ 61.5%; t1/2 = 12.701 h). In particular, an image segmentation was conducted on PET studies as follows: all images were registered using a 3D whole-body Digimouse atlas; features extraction was performed from seven organs to compare time-course [64Cu]chelator uptake values. Statistical analysis showed a different in vivo biodistribution of the 64Cu-labeled chelator over time, with a significant variation of many features between groups observed in bladder and liver (greater than 60% and 50%, respectively). Due to the importance of the biodistribution of radioactive substances in preclinical studies, a method transferable to human PET studies such as the one proposed based on radiomics, never used in the preclinical setting to date, it may be useful to improve future results in the context of in vivo studies in which radionuclides for biomedical purposes will be used.

ABSTRACT. NECTAR (NEutron Capture Enhanced Treatment of neurotoxic Amyloid aggRegates) project, funded by the European Commission, aims to study the efficacy of neutron capture reaction on B-10 and Gd-157 in the degradation of β-amyloid (Aβ) protein aggregates involved in Alzheimer's disease (AD).

AD is a neurodegenerative disorder that affects a wide range of the world population. Although this pathology was discovered in the early 1900s, the triggers factors are still unclear. A key role is attributed to the Aβ protein which accumulates in the extra-cellular matrix of the brain compromising the proper functioning of the nervous system cells. Nowaday the only drug capable of interfering in the AD accumulation process is aducanumab, a very expensive monoclonal antibody that still requires years of use in patients to prove its effectiveness even in the long term. It is therefore necessary to conduct further research on performing AD treatments. A study has shown the effectiveness of X radiation in damaging the Aβ aggregates: both in vitro and in vivo experiments were conducted, the first case showed not relevant changing in the protein structure, instead of the latter characterized by a relevant Aβ burden reduction. Finally, a pilot study on a small cohort of patients showed how different CT scans caused an improvement in terms of their cognitive and behavioural sphere.

The NECTAR project proposes an innovative idea: to exploit the selectivity of neutron capture reactions on B-10 and Gd-157 in order to degrade protein structures. The range of secondary particles coupled well with the aggregates size, allowing them to deposit their energy locally, saving healthy tissue. This process will be combined with the action of gamma rays, produced by the same neutron capture reactions, which act over a long distance causing a neuroinflammatory response of the glia cells which should promote a further clearance of Aβ aggregates. The talk will present the preliminary results achieved. In particular, the first in vitro experiments carried out at the nuclear reactor of the University of Pavia and the first Monte Carlo simulations conducted at a microdosimetric scale. In addition, studies will be focused on simulations concerning the irradiation effects on AD transgenic mice in view of the NECTAR developments which involves in vivo activity.

ABSTRACT. The ability of the Proton-Boron Capture Therapy (PBCT)1 approach to enhance proton biological effectiveness, has been recently demonstrated using the 11B carrier sodium mercaptododecaborate (BSH)2-4. The rationale resides in the highly DNA damaging α-particles generated by the nuclear p+11B→3α (pB) reaction, whose cross-section peaks as protons slow down across the tumour-conformed Spread-Out Bragg Peak (SOBP). Here, a novel data are presented using another binary strategy based on the reaction p+19F→α+16O (pF), which presents a resonance around 2000 keV5. Compared to the pB process, the peak of the energy spectrum of the generated α-particles is shifted to higher values (up to 13 MeV, versus 4 MeV for pB) thus generating secondary tracks able to traverse multiple cells. Moreover, a much more densely ionizing ion is produced (16O). 19F-labelled p-boronophenylalanine (F BPA), whose in vitro internalization in human cancer pancreatic cell line (PANC 1) was recently measured6, was used as 11B-19F carrier. Three different cell lines (DU-145 prostate cancer, PANC-1 pancreatic cancer and MCF-10A normal breast epithelial cells) were irradiated at a 3-MV tandem accelerator (CIRCE laboratory, Caserta, Italy) with proton energies close to either the pB or pF reaction cross-section maximum (~700 keV and ~2000 keV, respectively). To investigate the clinical usefulness of the combined pB-pF approach, DU-145 and PANC-1 were also irradiated along a clinical proton SOBP (energy range: 131.5-164.8 MeV) at CNAO (Pavia, Italy). Clonogenic survival and micronucleus induction were measured in cells irradiated in presence of F-BPA (120 ppm of 11B) suggesting 11B-19F synergic mediated radiosensitization.

1Yoon, D.K. et al. Appl. Phys. Lett. 2014, 105, 223507/1; 2Cirrone, G.A.P. et al. Sci. Rep; 2018. 8(1):1141; 3Bláha, P. et al. Front. Oncol; 2021; 11:682647; 4Ricciardi, V. et al. Appl. Sci. 2021, 11:11986; 5Torrisi, L. et al. NIM in Phys. Res. B 2021, 486: 28-36; 6Ciardiello, A. et al. Physica Medica 2022, 94: 75-84.

ABSTRACT. The need to improve radiobiological effectiveness of charged particles have increased the interest to exploit protons and boron nuclear thermal reaction to generate three alpha particles, a phenomenon biologically known as Proton Boron Capture Therapy (PBCT) [1][2][3]. This strategy may hold important therapeutic benefits to treat radioresistant and inoperable brain tumors, including the high-grade glioma, defined glioblastoma (GBM). This work shows a first PBCT preclinical evaluation in a heterotopic GBM mouse model aimed at analyzing pathophysiological and molecular effects in response to this innovative treatment modality. We first validated the reliability of our GBM mouse model by the ultrasound and photoacoustic multimodal imaging technique, allowing the assessment of the tumor oxygenation over the tumor growth. Blood oxygenation and hemoglobin were found significantly reduced over time in tumor masses. Reduced levels of 18F-2-deoxy-2-fluoroglucose (FDG) uptake after PBCT treatment were detected by micro positron emission tomography-assisted scanning. Further pathological analyses were performed to examine biological effects such as apoptosis, mitotic index, proliferation and autophagy. Interestingly, mitophagy and caspases levels were observed significantly higher in PBCT compared to proton irradiated GBM. Finally, RNA-seq revealed several deregulated gene and molecular pathways involved in major types of radiation-induced cell death and survival effects after PBCT treatment. Further studies will be fundamental to validate specific biological mechanisms emerged from omics analysis. Moreover, gene and molecular signatures in combination with biomarkers and radiomics data from preclinical imaging may reveal fundamental mechanisms underlying PBCT response leading to a more personalized targeted radiotherapy.