High-LET charged particles: radiobiology and application for new approaches in radiotherapy.

The number of patients treated with charged-particle radiotherapy as well as the number of treatment centers is increasing worldwide, particularly regarding protons. However, high-linear energy transfer (LET) particles, mainly carbon ions, are of special interest for application in radiotherapy, as their special physical features result in high precision and hence lower toxicity, and at the same time in increased efficiency in cell inactivation in the target region, i.e., the tumor. The radiobiology of high-LET particles differs with respect to DNA damage repair, cytogenetic damage, and cell death type, and their increased LET can tackle cells' resistance to hypoxia. Recent developments and perspectives, e.g., the return of high-LET particle therapy to the US with a center planned at Mayo clinics, the application of carbon ion radiotherapy using cost-reducing cyclotrons and the application of helium is foreseen to increase the interest in this type of radiotherapy. However, further preclinical research is needed to better understand the differential radiobiological mechanisms as opposed to photon radiotherapy, which will help to guide future clinical studies for optimal exploitation of high-LET particle therapy, in particular related to new concepts and innovative approaches. Herein, we summarize the basics and recent progress in high-LET particle radiobiology with a focus on carbon ions and discuss the implications of current knowledge for charged-particle radiotherapy. We emphasize the potential of high-LET particles with respect to immunogenicity and especially their combination with immunotherapy.

Immune checkpoint inhibitors and Carbon iON radiotherapy In solid Cancers with stable disease (ICONIC).

ICONIC is a multicenter, open-label, nonrandomized phase II clinical trial aiming to assess the feasibility and clinical activity of the addition of carbon ion radiotherapy to immune checkpoint inhibitors in cancer patients who have obtained disease stability with pembrolizumab administered as per standard-of-care. The primary end point is objective response rate, and the secondary end points are safety, survival and disease control rate. Translational research is an exploratory aim. The planned sample size is 27 patients. The study combination will be considered worth investigating if at least four objective responses are observed. If the null hypothesis is rejected, ICONIC will be the first proof of concept of the feasibility and clinical activity of the addition of carbon ion radiotherapy to immune checkpoint inhibitors in oncology.

ICONIC is a multicenter, open-label, nonrandomized, phase II clinical trial aiming to evaluate the feasibility and clinical activity of the addition of carbon ion radiotherapy to immune checkpoint inhibitors in cancer patients who have obtained disease stability with pembrolizumab administered as per standard-of-care. Considering that no clinical trials have been conducted thus far to assess the safety of the association between immune checkpoint inhibitors and carbon ion radiotherapy, the current clinical study will provide controlled data about the safety of this unprecedented therapeutic combination. Clinical Trial Registration: NCT05229614 (ClinicalTrials.gov).

Particle radiotherapy and molecular therapies: mechanisms and strategies towards clinical applications.

Immunotherapy and targeted therapy are now commonly used in clinical trials in combination with radiotherapy for several cancers. While results are promising and encouraging, the molecular mechanisms of the interaction between the drugs and radiation remain largely unknown. This is especially important when switching from conventional photon therapy to particle therapy using protons or heavier ions. Different dose deposition patterns and molecular radiobiology can in fact modify the interaction with drugs and their effectiveness. We will show here that whilst the main molecular players are the same after low and high linear energy transfer radiation exposure, significant differences are observed in post-exposure signalling pathways that may lead to different effects of the drugs. We will also emphasise that the problem of the timing between drug administration and radiation and the fractionation regime are critical issues that need to be addressed urgently to achieve optimal results in combined treatments with particle therapy.

Hibernation and Radioprotection: Gene Expression in the Liver and Testicle of Rats Irradiated under Synthetic Torpor.

Hibernation has been proposed as a tool for human space travel. In recent years, a procedure to induce a metabolic state known as "synthetic torpor" in non-hibernating mammals was successfully developed. Synthetic torpor may not only be an efficient method to spare resources and reduce psychological problems in long-term exploratory-class missions, but may also represent a countermeasure against cosmic rays. Here we show the preliminary results from an experiment in rats exposed to ionizing radiation in normothermic conditions or synthetic torpor. Animals were irradiated with 3 Gy X-rays and organs were collected 4 h after exposure. Histological analysis of liver and testicle showed a reduced toxicity in animals irradiated in torpor compared to controls irradiated at normal temperature and metabolic activity. The expression of ataxia telangiectasia mutated (ATM) in the liver was significantly downregulated in the group of animal in synthetic torpor. In the testicle, more genes involved in the DNA damage signaling were downregulated during synthetic torpor. These data show for the first time that synthetic torpor is a radioprotector in non-hibernators, similarly to natural torpor in hibernating animals. Synthetic torpor can be an effective strategy to protect humans during long term space exploration of the solar system.

Carbon Ion and Photon Radiation Therapy Show Enhanced Antitumoral Therapeutic Efficacy With Neoantigen RNA-LPX Vaccines in Preclinical Colon Carcinoma Models.

PURPOSE: Personalized liposome-formulated mRNA vaccines (RNA-LPX) are a powerful new tool in cancer immunotherapy. In preclinical tumor models, RNA-LPX vaccines are known to achieve potent results when combined with conventional X-ray radiation therapy (XRT). Densely ionizing radiation used in carbon ion radiation therapy (CIRT) may induce distinct effects in combination with immunotherapy compared with sparsely ionizing X-rays. METHODS AND MATERIALS: Within this study, we investigate the potential of CIRT and isoeffective doses of XRT to mediate tumor growth inhibition and survival in murine colon adenocarcinoma models in conjunction with neoantigen (neoAg)-specific RNA-LPX vaccines encoding both major histocompatibility complex (MHC) class I- and class II-restricted tumor-specific neoantigens. We characterize tumor immune infiltrates and antigen-specific T cell responses by flow cytometry and interferon-γ enzyme-linked immunosorbent spot (ELISpot) analyses, respectively. RESULTS: NeoAg RNA-LPX vaccines significantly potentiate radiation therapy-mediated tumor growth inhibition. CIRT and XRT alone marginally prime neoAg-specific T cell responses detected in the tumors but not in the blood or spleens of mice. Infiltration and cytotoxicity of neoAg-specific T cells is strongly driven by RNA-LPX vaccines and is accompanied by reduced expression of the inhibitory markers PD-1 and Tim-3 on these cells. The neoAg RNA-LPX vaccine shows similar overall therapeutic efficacy in combination with both CIRT and XRT, even if the physical radiation dose is lower for carbon ions than for X-rays. CONCLUSIONS: We hence conclude that the combination of CIRT and neoAg RNA-LPX vaccines is a promising strategy for the treatment of radioresistant tumors.

Automated synthesis of [68Ga]Ga-FAPI-46 without pre-purification of the generator eluate on three common synthesis modules and two generator types.

BACKGROUND: The recent development of quinoline-based radiotracers, which act as fibroblast activation protein inhibitors (FAPIs), has shown promising preclinical and clinical advantages. [68Ga]Ga-FAPI-46 is a new radiotracer for in vivo detection of the fibroblast activation protein by positron emission tomography (PET). Recently, the automated synthesis of [68Ga]Ga-FAPI-46 was reported based on pre-concentration and purification of the generator eluate by using a cation exchange-cartridge. Our aim was to simplify the synthesis and shorten the automated synthesis of [68Ga]Ga-FAPI-46 to make it accessible and thus even more attractive to a broader clinical and scientific community. RESULTS: We developed and evaluated the GMP compliant automatic synthesis of [68Ga]Ga-FAPI-46 using two different 68Ge/68Ga generators (an Eckert & Ziegler, GalliaPharm generator, 1.85 GBq/50 mCi and an iThemba generator, 1.85 GBq/50 mCi) Somerset West, South Africa) and three different commercial and customized systems: the EasyOne module from Trasis; the GaSy module from Synthra with a customized synthesis template and a customized single use cassette. Additionally, the automatic synthesis of [68Ga]Ga-FAPI-46 was established on a GallElut synthesis module from Scintomics with fixed tubing. CONCLUSIONS: Independent of the synthesis modules or the generators employed we were able to complete the synthesis of [68Ga]Ga-FAPI-46 in 12 min including the process of purification and formulation. In all cases, the final products showed more than 99.5% chemical purity and the radiochemical yield reached around 92.5% (decay corrected). All quality control parameters (e.g. sterility, stability and radiochemical purity) were conform to the European Pharmacopoeia.

FLASH with carbon ions: Tumor control, normal tissue sparing, and distal metastasis in a mouse osteosarcoma model.

BACKGROUND AND PURPOSE: The FLASH effect is a potential breakthrough in radiotherapy because ultra-high dose-rate irradiation can substantially widen the therapeutic window. While the normal tissue sparing at high doses and short irradiation times has been demonstrated with electrons, photons, and protons, so far evidence with heavy ions is limited to in vitro cell experiments. Here we present the first in vivo results with high-energy 12C-ions delivered at an ultra-high dose rate. MATERIALS AND METHODS: LM8 osteosarcoma cells were subcutaneously injected in the posterior limb of female C3H/He mice 7 days before radiation exposure. Both hind limbs of the animals were irradiated with 240 MeV/n 12C-ions at ultra-high (18 Gy in 150 ms) or conventional dose rate (∼18 Gy/min). Tumor size was measured until 28 days post-exposure, when animals were sacrificed and lungs, limb muscles, and tumors were collected for further histological analysis. RESULTS: Irradiation with carbon ions was able to control the tumour both at conventional and ultra-high dose rate. FLASH decreases normal tissue toxicity as demonstrated by the reduced structural changes in muscle compared to conventional dose-rate irradiation. Carbon ion irradiation in FLASH conditions significantly reduced lung metastasis compared to conventional dose-rate irradiation and sham-irradiated animals. CONCLUSIONS: We demonstrated the FLASH effect in vivo with high-energy carbon ions. In addition to normal tissue sparing, we observed tumor control and a substantial reduction of lung metastasis in an osteosarcoma mouse model.

A simple, reliable and accurate approach for assessing [131I]-capsule activity leading to significant reduction of radiation exposure of medical staff during radioiodine therapy.

PURPOSE: According to German law, the [131I]-capsule activity has to be checked in the context of radioiodine therapy (RIT) immediately before application. The measurement leads to significant radiation exposure of the medical personnel, especially of their hands. We aimed to establish a method for estimating [131I]-capsule activity by measuring the dose rate (DR) at contact of the delivered lead closed container carrying the [131I]-capsules and to evaluate radiation exposure in comparison to conventional [131I]-capsule measurement using a dose calibrator. METHODS: DR on the surface of the closed lead container was measured at two locations and correlated linearly with the [131I]-capsule activity measured in a dose calibrator to create calibrating curves. The hand and whole body (effective) doses were determined with official dose meters during validation of our method in clinical practice. RESULTS: The determination coefficients (R2) of linear calibration curves were greater than 0.9974. The total relative uncertainty for estimating [131I]-capsule activity with our method was <±7.5% which is lower than the uncertainty of the nominal activity and quite close to the threshold limit for the maximum allowed uncertainty of ± 5% for measuring activity in radioactive drugs. The reduction of the hand dose caused by our method was 97% compared with the conventional measurements of the [131I]-capsules in a dose calibrator. CONCLUSION: Measuring DR on the surface of the closed lead containers enables the [131I]-capsules activity to be estimated simply, reliably and with sufficient accuracy leading to significant reduction of the radiation exposure for the medical staff.

A Combination of Cabozantinib and Radiation Does Not Lead to an Improved Growth Control of Tumors in a Preclinical 4T1 Breast Cancer Model.

The tyrosine kinase inhibitor Cabozantinib has been applied in clinical studies in combination with radiotherapy. We investigated the effect of such combination on triple-negative 4T1 cells as a metastatic breast cancer model in vitro and in vivo upon inoculation in BALB/c mice. In vitro assays indicated a potential for improved effects using the combination. Both Cabozantinib (2.5 µM) and 10 Gy of 250 kV x-rays were able to cease the growth of 4T1 cells as revealed by growth curves. In a clonogenic survival assay, the effect of Cabozantinib added on the effects of irradiation and the effectiveness of inhibiting the clonogenic survival was found to be 2 (RBE10). Additionally, cell death measurements of apoptosis plus necrosis revealed a synergistic effect when combining irradiation with Cabozantinib. Surprisingly, however, in vivo tumor growth kinetics showed no additional effect in growth control when irradiation was used together with Cabozantinib. Since both ionizing radiation and Cabozantinib are acknowledged to feature immunogenic effects, we additionally investigated the effect of the treatments on lung metastases. No difference to the control groups was found here, neither for irradiation nor Cabozantinib alone nor in combination. Yet, upon analysis of the mice' livers, CD11b-positive cells, indicating immune suppressive myeloid derived suppressor cells were found diminished following treatment with Cabozantinib. In conclusion, despite promising in vitro controls of the combination of Cabozantinib and irradiation, tumor growth control was not increased by the combination, which was true also for the occurrence of lung metastases.

Reduction of Lung Metastases in a Mouse Osteosarcoma Model Treated With Carbon Ions and Immune Checkpoint Inhibitors.

PURPOSE: The combination of radiation therapy and immunotherapy is recognized as a very promising strategy for metastatic cancer treatment. The purpose of this work is to compare the effectiveness of x-ray and high-energy carbon ion therapy in combination with checkpoint inhibitors in a murine model. METHODS AND MATERIALS: We used an osteosarcoma mouse model irradiated with either carbon ions or x-rays in combination with 2 immune checkpoint inhibitors (anti-PD-1 and anti-CTLA-4). LM8 osteosarcoma cells were injected in both hind limbs of female C3H/He mice 7 days before exposure to carbon ions or x-rays. In experimental groups receiving irradiation, only the tumor on the left limb was exposed, whereas the tumor on the right limb served as an abscopal mimic. Checkpoint inhibitors were injected intraperitoneally 1 day before exposure as well as concomitant to and after exposure. Tumor growth was measured regularly up to day 21 after exposure, when mice were sacrificed. Both tumors as well as lungs were extracted. RESULTS: A reduced growth of the abscopal tumor was most pronounced after the combined protocol of carbon ions and the immune checkpoint inhibitors administered sequentially. Radiation or checkpoint inhibitors alone were not sufficient to reduce the growth of the abscopal tumors. Carbon ions alone reduced the number of lung metastases more efficiently than x-rays, and in combination with immunotherapy both radiation types essentially suppressed the metastasis, with carbon ions being again more efficient. Investigation of the infiltration of immune cells in the abscopal tumors of animals treated with combination revealed an increase in CD8+ cells. CONCLUSIONS: Combination of checkpoint inhibitors with high-energy carbon ion radiation therapy can be an effective strategy for the treatment of advanced tumors.

Heart in space: effect of the extraterrestrial environment on the cardiovascular system.

National space agencies and private corporations aim at an extended presence of humans in space in the medium to long term. Together with currently suboptimal technology, microgravity and cosmic rays raise health concerns about deep-space exploration missions. Both of these physical factors affect the cardiovascular system, whose gravity-dependence is pronounced. Heart and vascular function are, therefore, susceptible to substantial changes in weightlessness. The altered cardiovascular function in space causes physiological problems in the postflight period. A compromised cardiovascular system can be excessively vulnerable to space radiation, synergistically resulting in increased damage. The space radiation dose is significantly lower than in patients undergoing radiotherapy, in whom cardiac damage is well-documented following cancer therapy in the thoracic region. Nevertheless, epidemiological findings suggest an increased risk of late cardiovascular disease even with low doses of radiation. Moreover, the peculiar biological effectiveness of heavy ions in cosmic rays might increase this risk substantially. However, whether radiation-induced cardiovascular effects have a threshold at low doses is still unclear. The main countermeasures to mitigate the effect of the space environment on cardiac function are physical exercise, antioxidants, nutraceuticals, and radiation shielding.

Combining Heavy-Ion Therapy with Immunotherapy: An Update on Recent Developments.

Clinical trials and case reports of cancer therapies combining radiation therapy with immunotherapy have at times demonstrated total reduction or elimination of metastatic disease. While virtually all trials focus on the use of immunotherapy combined with conventional photon irradiation, the dose-distributive benefits of particles, in particular the distinct biological effects of heavy ions, have unknown potential vis-a-vis systemic disease response. Here, we review recent developments and evidence with a focus on the potential for heavy-ion combination therapy.

Advances in Radiation Biology of Particle Irradiation.

The increasing number of centers providing proton or carbon beam therapy underlines the growing importance of charged particle therapy within the spectrum of cancer radiotherapy. Whereas protons are more widely used around the world, carbon ions, which are known to bear a higher efficacy as compared to protons, are still neglected to some extent, especially due to a lack of clinical data on adverse side effects. Yet, an increasing amount of clinical data indicates the distinguished efficacy of carbon ion therapy. Notwithstanding, the radiobiological mechanisms of particle radiation are not completely understood and lag behind advances in technology, which potentially enable new therapy regimens. However, an increased knowledge is required for their application with maximal benefit and sufficient risk estimation. Differential gene expression, distinct molecular mechanisms and signal pathways in the radiation response, and systemic effects, such as increased immunogenicity, and the possibilities of combined treatments arising from them, are important fields of particle radiobiology in which new discoveries and advances have occurred. These aspects are contemplated with respect to an individualization of radiotherapy; radiation type and treatment regimen might be chosen on the basis of the radiosensitivity of the individual and the cancer type. Here, we provide an update on a few recent findings and advances in particle radiobiology. A comprehensive essay on the basics of particle radiobiology is beyond the scope of this article. The focus is directed on a few subjects currently undergoing intense study and which are of current interest with respect to advances in therapy.

The Immunoregulatory Potential of Particle Radiation in Cancer Therapy.

Cancer treatment, today, consists of surgery, chemotherapy, radiation, and most recently immunotherapy. Combination immunotherapy-radiotherapy (CIR) has experienced a surge in public attention due to numerous clinical publications outlining the reduction or elimination of metastatic disease, following treatment with specifically ipilimumab and radiotherapy. The mechanism behind CIR, however, remains unclear, though it is hypothesized that radiation transforms the tumor into an in situ vaccine which immunotherapy modulates into a larger immune response. To date, the majority of attention has focused on rotating out immunotherapeutics with conventional radiation; however, the unique biological and physical benefits of particle irradiation may prove superior in generation of systemic effect. Here, we review recent advances in CIR, with a particular focus on the usage of charged particles to induce or enhance response to cancerous disease.

The Influence of C-Ions and X-rays on Human Umbilical Vein Endothelial Cells.

Damage to the endothelium of blood vessels, which may occur during radiotherapy, is discussed as a potential precursor to the development of cardiovascular disease. We thus chose human umbilical vein endothelial cells as a model system to examine the effect of low- and high-linear energy transfer (LET) radiation. Cells were exposed to 250 kV X-rays or carbon ions (C-ions) with the energies of either 9.8 MeV/u (LET = 170 keV/µm) or 91 MeV/u (LET = 28 keV/µm). Subculture of cells was performed regularly up to 46 days (~22 population doublings) post-irradiation. Immediately after exposure, cells were seeded for the colony forming assay. Additionally, at regular intervals, mitochondrial membrane potential (MMP) (JC-1 staining) and cellular senescence (senescence-associated ß-galactosidase staining) were assessed. Cytogenetic damage was investigated by the micronucleus assay and the high-resolution multiplex fluorescence in situ hybridization (mFISH) technique. Analysis of radiation-induced damage shortly after exposure showed that C-ions are more effective than X-rays with respect to cell inactivation or the induction of cytogenetic damage (micronucleus assay) as observed in other cell systems. For 9.8 and 91 MeV/u C-ions, relative biological effectiveness values of 2.4 and 1.5 were obtained for cell inactivation. At the subsequent time points, the number of micronucleated cells decreased to the control level. Analysis of chromosomal damage by mFISH technique revealed aberrations frequently involving chromosome 13 irrespective of dose or radiation quality. Disruption of the MMP was seen only a few days after exposure to X-rays or C-ions. Cellular senescence was not altered by radiation at any time point investigated. Altogether, our data indicate that shortly after exposure C-ions were more effective in damaging endothelial cells than X-rays. However, late damage to endothelial cells was not found for the applied conditions and endpoints.

Ionizing Radiation Impacts on Cardiac Differentiation of Mouse Embryonic Stem Cells.

Little is known about the effects of ionizing radiation on the earliest stages of embryonic development although it is well recognized that ionizing radiation is a natural part of our environment and further exposure may occur due to medical applications. The current study addresses this issue using D3 mouse embryonic stem cells as a model system. Cells were irradiated with either X-rays or carbon ions representing sparsely and densely ionizing radiation and their effect on the differentiation of D3 cells into spontaneously contracting cardiomyocytes through embryoid body (EB) formation was measured. This study is the first to demonstrate that ionizing radiation impairs the formation of beating cardiomyocytes with carbon ions being more detrimental than X-rays. However, after prolonged culture time, the number of beating EBs derived from carbon ion irradiated cells almost reached control levels indicating that the surviving cells are still capable of developing along the cardiac lineage although with considerable delay. Reduced EB size, failure to downregulate pluripotency markers, and impaired expression of cardiac markers were identified as the cause of compromised cardiomyocyte formation. Dysregulation of cardiac differentiation was accompanied by alterations in the expression of endodermal and ectodermal markers that were more severe after carbon ion irradiation than after exposure to X-rays. In conclusion, our data show that carbon ion irradiation profoundly affects differentiation and thus may pose a higher risk to the early embryo than X-rays.

Hibernation for space travel: Impact on radioprotection.

Hibernation is a state of reduced metabolic activity used by some animals to survive in harsh environmental conditions. The idea of exploiting hibernation for space exploration has been proposed many years ago, but in recent years it is becoming more realistic, thanks to the introduction of specific methods to induce hibernation-like conditions (synthetic torpor) in non-hibernating animals. In addition to the expected advantages in long-term exploratory-class missions in terms of resource consumptions, aging, and psychology, hibernation may provide protection from cosmic radiation damage to the crew. Data from over half century ago in animal models suggest indeed that radiation effects are reduced during hibernation. We will review the mechanisms of increased radioprotection in hibernation, and discuss possible impact on human space exploration.