The role of hypoxia and radiation in developing a CTCs-like phenotype in murine osteosarcoma cells.

Introduction: Cancer treatment has evolved significantly, yet concerns about tumor recurrence and metastasis persist. Within the dynamic tumor microenvironment, a subpopulation of mesenchymal tumor cells, known as Circulating Cancer Stem Cells (CCSCs), express markers like CD133, TrkB, and CD47, making them radioresistant and pivotal to metastasis. Hypoxia intensifies their stemness, complicating their identification in the bloodstream. This study investigates the interplay of acute and chronic hypoxia and radiation exposure in selecting and characterizing cells with a CCSC-like phenotype. Methods: LM8 murine osteosarcoma cells were cultured and subjected to normoxic (21% O2) and hypoxic (1% O2) conditions. We employed Sphere Formation and Migration Assays, Western Blot analysis, CD133 Cell Sorting, and CD133+ Fluorescent Activated Cell Sorting (FACS) analysis with a focus on TrkB antibody to assess the effects of acute and chronic hypoxia, along with radiation exposure. Results: Our findings demonstrate that the combination of radiation and acute hypoxia enhances stemness, while chronic hypoxia imparts a cancer stem-like phenotype in murine osteosarcoma cells, marked by increased migration and upregulation of CCSC markers, particularly TrkB and CD47. These insights offer a comprehensive understanding of the interactions between radiation, hypoxia, and cellular responses in the context of cancer treatment. Discussion: This study elucidates the complex interplay among radiation, hypoxia, and cellular responses, offering valuable insights into the intricacies and potential advancements in cancer treatment.

Establishment of Primary Adult Skin Fibroblast Cell Lines from African Savanna Elephants (Loxodonta africana).

Following population declines of the African savanna elephant (Loxodonta africana) across the African continent, the establishment of primary cell lines of endangered wildlife species is paramount for the preservation of their genetic resources. In addition, it allows molecular and functional studies on the cancer suppression mechanisms of elephants, which have previously been linked to a redundancy of tumor suppressor gene TP53. This methodology describes the establishment of primary elephant dermal fibroblast (EDF) cell lines from skin punch biopsy samples (diameter: ±4 mm) of African savanna elephants (n = 4, 14-35 years). The applied tissue collection technique is minimally invasive and paves the way for future remote biopsy darting. On average, the first explant outgrowth was observed after 15.75 ± 6.30 days. The average doubling time (Td) was 93.02 ± 16.94 h and 52.39 ± 0.46 h at passage 1 and 4, respectively. Metaphase spreads confirmed the diploid number of 56 chromosomes. The successful establishment of EDF cell lines allows for future elephant cell characterization studies and for research on the cancer resistance mechanisms of elephants, which can be harnessed for human cancer prevention and treatment and contributes to the conservation of their genetic material.

Towards sustainable human space exploration-priorities for radiation research to quantify and mitigate radiation risks.

Human spaceflight is entering a new era of sustainable human space exploration. By 2030 humans will regularly fly to the Moon's orbit, return to the Moon's surface and preparations for crewed Mars missions will intensify. In planning these undertakings, several challenges will need to be addressed in order to ensure the safety of astronauts during their space travels. One of the important challenges to overcome, that could be a major showstopper of the space endeavor, is the exposure to the space radiation environment. There is an urgent need for quantifying, managing and limiting the detrimental health risks and electronics damage induced by space radiation exposure. Such risks raise key priority topics for space research programs. Risk limitation involves obtaining a better understanding of space weather phenomena and the complex radiation environment in spaceflight, as well as developing and applying accurate dosimetric instruments, understanding related short- and long-term health risks, and strategies for effective countermeasures to minimize both exposure to space radiation and the remaining effects post exposure. The ESA/SciSpacE Space Radiation White Paper identifies those topics and underlines priorities for future research and development, to enable safe human and robotic exploration of space beyond Low Earth Orbit.

Synthetic torpor protects rats from exposure to accelerated heavy ions.

Hibernation or torpor is considered a possible tool to protect astronauts from the deleterious effects of space radiation that contains high-energy heavy ions. We induced synthetic torpor in rats by injecting adenosine 5'-monophosphate monohydrate (5'-AMP) i.p. and maintaining in low ambient temperature room (+ 16 °C) for 6 h immediately after total body irradiation (TBI) with accelerated carbon ions (C-ions). The 5'-AMP treatment in combination with low ambient temperature reduced skin temperature and increased survival following 8 Gy C-ion irradiation compared to saline-injected animals. Analysis of the histology of the brain, liver and lungs showed that 5'-AMP treatment following 2 Gy TBI reduced activated microglia, Iba1 positive cells in the brain, apoptotic cells in the liver, and damage to the lungs, suggesting that synthetic torpor spares tissues from energetic ion radiation. The application of 5'-AMP in combination with either hypoxia or low temperature environment for six hours following irradiation of rat retinal pigment epithelial cells delays DNA repair and suppresses the radiation-induced mitotic catastrophe compared to control cells. We conclude that synthetic torpor protects animals from cosmic ray-simulated radiation and the mechanism involves both hypothermia and hypoxia.

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.

Ultra-High Dose Rate (FLASH) Carbon Ion Irradiation: Dosimetry and First Cell Experiments.

PURPOSE: To establish a beam monitoring and dosimetry system to enable the FLASH dose rate carbon ion irradiation and investigate, at different oxygen concentrations, the in vitro biological response in comparison to the conventional dose rate. METHODS AND MATERIALS: CHO-K1 cell response to irradiation at different dose rates and at different levels of oxygenation was studied using clonogenic assay. The Heidelberg Ion-Beam Therapy Center (HIT) synchrotron, after technical improvements, was adjusted to extract ≥5 × 108 12C ions within approximately 150 milliseconds. The beam monitors were filled with helium. RESULTS: The FLASH irradiation with beam scanning yields a dose of 7.5 Gy (homogeneity of ±5%) for a 280 MeV/u beam in a volume of at least 8 mm in diameter and a corresponding dose rate of 70 Gy/s (±20%). The dose repetition accuracy is better than 2%, the systematic uncertainty is better than 2%. Clonogenic assay demonstrates a significant FLASH sparing effect which is strongly oxygenation-dependent and mostly pronounced at 0.5% O2 but absent at 0% and 21% O2. CONCLUSION: The FLASH dose rates >40 Gy/s were achieved with carbon beams. Cell survival analysis revealed FLASH dose rate sparing in hypoxia (0.5%-4% O2).

Radioactive Beams for Image-Guided Particle Therapy: The BARB Experiment at GSI.

Several techniques are under development for image-guidance in particle therapy. Positron (ß+) emission tomography (PET) is in use since many years, because accelerated ions generate positron-emitting isotopes by nuclear fragmentation in the human body. In heavy ion therapy, a major part of the PET signals is produced by ß+-emitters generated via projectile fragmentation. A much higher intensity for the PET signal can be obtained using ß+-radioactive beams directly for treatment. This idea has always been hampered by the low intensity of the secondary beams, produced by fragmentation of the primary, stable beams. With the intensity upgrade of the SIS-18 synchrotron and the isotopic separation with the fragment separator FRS in the FAIR-phase-0 in Darmstadt, it is now possible to reach radioactive ion beams with sufficient intensity to treat a tumor in small animals. This was the motivation of the BARB (Biomedical Applications of Radioactive ion Beams) experiment that is ongoing at GSI in Darmstadt. This paper will present the plans and instruments developed by the BARB collaboration for testing the use of radioactive beams in cancer therapy.

What can space radiation protection learn from radiation oncology?

Protection from cosmic radiation of crews of long-term space missions is now becoming an urgent requirement to allow a safe colonization of the moon and Mars. Epidemiology provides little help to quantify the risk, because the astronaut group is small and as yet mostly involved in low-Earth orbit mission, whilst the usual cohorts used for radiation protection on Earth (e.g. atomic bomb survivors) were exposed to a radiation quality substantially different from the energetic charged particle field found in space. However, there are over 260,000 patients treated with accelerated protons or heavier ions for different types of cancer, and this cohort may be useful for quantifying the effects of space-like radiation in humans. Space radiation protection and particle therapy research also share the same tools and devices, such as accelerators and detectors, as well as several research topics, from nuclear fragmentation cross sections to the radiobiology of densely ionizing radiation. The transfer of the information from the cancer radiotherapy field to space is manifestly complicated, yet the two field should strengthen their relationship and exchange methods and data.

Hypoxia Transcriptomic Modifications Induced by Proton Irradiation in U87 Glioblastoma Multiforme Cell Line.

In Glioblastoma Multiforme (GBM), hypoxia is associated with radioresistance and poor prognosis. Since standard GBM treatments are not always effective, new strategies are needed to overcome resistance to therapeutic treatments, including radiotherapy (RT). Our study aims to shed light on the biomarker network involved in a hypoxic (0.2% oxygen) GBM cell line that is radioresistant after proton therapy (PT). For cultivating cells in acute hypoxia, GSI's hypoxic chambers were used. Cells were irradiated in the middle of a spread-out Bragg peak with increasing PT doses to verify the greater radioresistance in hypoxic conditions. Whole-genome cDNA microarray gene expression analyses were performed for samples treated with 2 and 10 Gy to highlight biological processes activated in GBM following PT in the hypoxic condition. We describe cell survival response and significant deregulated pathways responsible for the cell death/survival balance and gene signatures linked to the PT/hypoxia configurations assayed. Highlighting the molecular pathways involved in GBM resistance following hypoxia and ionizing radiation (IR), this work could suggest new molecular targets, allowing the development of targeted drugs to be suggested in association with PT.

Hibernation as a Tool for Radiation Protection in Space Exploration.

With new and advanced technology, human exploration has reached outside of the Earth's boundaries. There are plans for reaching Mars and the satellites of Jupiter and Saturn, and even to build a permanent base on the Moon. However, human beings have evolved on Earth with levels of gravity and radiation that are very different from those that we have to face in space. These issues seem to pose a significant limitation on exploration. Although there are plausible solutions for problems related to the lack of gravity, it is still unclear how to address the radiation problem. Several solutions have been proposed, such as passive or active shielding or the use of specific drugs that could reduce the effects of radiation. Recently, a method that reproduces a mechanism similar to hibernation or torpor, known as synthetic torpor, has started to become possible. Several studies show that hibernators are resistant to acute high-dose-rate radiation exposure. However, the underlying mechanism of how this occurs remains unclear, and further investigation is needed. Whether synthetic hibernation will also protect from the deleterious effects of chronic low-dose-rate radiation exposure is currently unknown. Hibernators can modulate their neuronal firing, adjust their cardiovascular function, regulate their body temperature, preserve their muscles during prolonged inactivity, regulate their immune system, and most importantly, increase their radioresistance during the inactive period. According to recent studies, synthetic hibernation, just like natural hibernation, could mitigate radiation-induced toxicity. In this review, we see what artificial hibernation is and how it could help the next generation of astronauts in future interplanetary missions.

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.

Tumor Hypoxia and Circulating Tumor Cells.

Circulating tumor cells (CTCs) are a rare tumor cell subpopulation induced and selected by the tumor microenvironment's extreme conditions. Under hypoxia and starvation, these aggressive and invasive cells are able to invade the lymphatic and circulatory systems. Escaping from the primary tumor, CTCs enter into the bloodstream to form metastatic deposits or re-establish themselves in cancer's primary site. Although radiotherapy is widely used to cure solid malignancies, it can promote metastasis. Radiation can disrupt the primary tumor vasculature, increasing the dissemination of CTCs. Radiation also induces epithelial-mesenchymal transition (EMT) and eliminates suppressive signaling, causing the proliferation of existent, but previously dormant, disseminated tumor cells (DTCs). In this review, we collect the results and evidence underlying the molecular mechanisms of CTCs and DTCs and the effects of radiation and hypoxia in developing these cells.

Carbon Ion Radiobiology.

Radiotherapy using accelerated charged particles is rapidly growing worldwide. About 85% of the cancer patients receiving particle therapy are irradiated with protons, which have physical advantages compared to X-rays but a similar biological response. In addition to the ballistic advantages, heavy ions present specific radiobiological features that can make them attractive for treating radioresistant, hypoxic tumors. An ideal heavy ion should have lower toxicity in the entrance channel (normal tissue) and be exquisitely effective in the target region (tumor). Carbon ions have been chosen because they represent the best combination in this direction. Normal tissue toxicities and second cancer risk are similar to those observed in conventional radiotherapy. In the target region, they have increased relative biological effectiveness and a reduced oxygen enhancement ratio compared to X-rays. Some radiobiological properties of densely ionizing carbon ions are so distinct from X-rays and protons that they can be considered as a different "drug" in oncology, and may elicit favorable responses such as an increased immune response and reduced angiogenesis and metastatic potential. The radiobiological properties of carbon ions should guide patient selection and treatment protocols to achieve optimal clinical results.

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.

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.

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.

Helium ions for radiotherapy? Physical and biological verifications of a novel treatment modality.

PURPOSE: Modern facilities for actively scanned ion beam radiotherapy allow in principle the use of helium beams, which could present specific advantages, especially for pediatric tumors. In order to assess the potential use of these beams for radiotherapy, i.e., to create realistic treatment plans, the authors set up a dedicated (4)He beam model, providing base data for their treatment planning system TRiP98, and they have reported that in this work together with its physical and biological validations. METHODS: A semiempirical beam model for the physical depth dose deposition and the production of nuclear fragments was developed and introduced in TRiP98. For the biological effect calculations the last version of the local effect model was used. The model predictions were experimentally verified at the HIT facility. The primary beam attenuation and the characteristics of secondary charged particles at various depth in water were investigated using (4)He ion beams of 200 MeV/u. The nuclear charge of secondary fragments was identified using a ΔE/E telescope. 3D absorbed dose distributions were measured with pin point ionization chambers and the biological dosimetry experiments were realized irradiating a Chinese hamster ovary cells stack arranged in an extended target. RESULTS: The few experimental data available on basic physical processes are reproduced by their beam model. The experimental verification of absorbed dose distributions in extended target volumes yields an overall agreement, with a slight underestimation of the lateral spread. Cell survival along a 4 cm extended target is reproduced with remarkable accuracy. CONCLUSIONS: The authors presented a simple simulation model for therapeutical (4)He beams which they introduced in TRiP98, and which is validated experimentally by means of physical and biological dosimetries. Thus, it is now possible to perform detailed treatment planning studies with (4)He beams, either exclusively or in combination with other ion modalities.

DNA Damage Response Proteins and Oxygen Modulate Prostaglandin E2 Growth Factor Release in Response to Low and High LET Ionizing Radiation.

Common cancer therapies employ chemicals or radiation that damage DNA. Cancer and normal cells respond to DNA damage by activating complex networks of DNA damage sensor, signal transducer, and effector proteins that arrest cell cycle progression, and repair damaged DNA. If damage is severe enough, the DNA damage response (DDR) triggers programed cell death by apoptosis or other pathways. Caspase 3 is a protease that is activated upon damage and triggers apoptosis, and production of prostaglandin E2 (PGE2), a potent growth factor that can enhance growth of surviving cancer cells leading to accelerated tumor repopulation. Thus, dying tumor cells can promote growth of surviving tumor cells, a pathway aptly named Phoenix Rising. In the present study, we surveyed Phoenix Rising responses in a variety of normal and established cancer cell lines, and in cancer cell lines freshly derived from patients. We demonstrate that IR induces a Phoenix Rising response in many, but not all cell lines, and that PGE2 production generally correlates with enhanced growth of cells that survive irradiation, and of unirradiated cells co-cultured with irradiated cells. We show that PGE2 production is stimulated by low and high LET ionizing radiation, and can be enhanced or suppressed by inhibitors of key DDR proteins. PGE2 is produced downstream of caspase 3 and the cyclooxygenases COX1 and COX2, and we show that the pan COX1-2 inhibitor indomethacin blocks IR-induced PGE2 production in the presence or absence of DDR inhibitors. COX1-2 require oxygen for catalytic activity, and we further show that PGE2 production is markedly suppressed in cells cultured under low (1%) oxygen concentration. Thus, Phoenix Rising is most likely to cause repopulation of tumors with relatively high oxygen, but not in hypoxic tumors. This survey lays a foundation for future studies to further define tumor responses to radiation and inhibitors of the DDR and Phoenix Rising to enhance the efficacy of radiotherapy with the ultimate goal of precision medicine informed by deep understanding of specific tumor responses to radiation and adjunct chemotherapy targeting key factors in the DDR and Phoenix Rising pathways.

Kill-painting of hypoxic tumours in charged particle therapy.

Solid tumours often present regions with severe oxygen deprivation (hypoxia), which are resistant to both chemotherapy and radiotherapy. Increased radiosensitivity as a function of the oxygen concentration is well described for X-rays. It has also been demonstrated that radioresistance in anoxia is reduced using high-LET radiation rather than conventional X-rays. However, the dependence of the oxygen enhancement ratio (OER) on radiation quality in the regions of intermediate oxygen concentrations, those normally found in tumours, had never been measured and biophysical models were based on extrapolations. Here we present a complete survival dataset of mammalian cells exposed to different ions in oxygen concentration ranging from normoxia (21%) to anoxia (0%). The data were used to generate a model of the dependence of the OER on oxygen concentration and particle energy. The model was implemented in the ion beam treatment planning system to prescribe uniform cell killing across volumes with heterogeneous radiosensitivity. The adaptive treatment plans have been validated in two different accelerator facilities, using a biological phantom where cells can be irradiated simultaneously at three different oxygen concentrations. We thus realized a hypoxia-adapted treatment plan, which will be used for painting by voxel of hypoxic tumours visualized by functional imaging.