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.

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).

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.

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.

X-irradiation of developing hippocampal neurons causes changes in neuron population phenotypes, dendritic morphology and synaptic protein expression in surviving neurons at maturity.

The effects of X-irradiation on developing neurons and their functions are unclear. We used primary cultures of mouse hippocampal neurons to investigate the effects of X-irradiation on cell death in developing neurons by analyzing caspase-3, MAP2 and DAPI-labeled cells, and the phenotypes and function of surviving neurons, by examining GAD67-positive cells as a GABAergic marker, and the synaptic markers synapsin 1, drebrin and PSD-95 through its maturation. One-day in vitro (DIV 1) cells were exposed to 0.5 Gy or 1 Gy of X-rays. A significant increase in the percentage of activated caspase-3, a decrease in the number of MAP2/DAPI-positive cells and change in the percentage of GAD67 positive neurons, compared with sham controls, were found 6 days after 1 Gy and 13 days after 0.5 Gy of X-rays. The expression of PSD-95 and drebrin, as well as drebrin clusters, in the remaining neurons was decreased at DIV 21, in both 0.5 Gy and on 1 Gy-irradiation there was a reduced number of dendritic intersection as well. Together, our findings show that 0.5 Gy and 1 Gy of X-irradiation at DIV 1 not only causes neuronal cell death but elicits an increase in the percentage of inhibitory neurons, changes in the dendrites and decrease in expression of important synaptic proteins in the surviving neurons at maturity 3 weeks after exposure.

Development and performance evaluation of a three-dimensional clinostat synchronized heavy-ion irradiation system.

Outer space is an environment characterized by microgravity and space radiation, including high-energy charged particles. Astronauts are constantly exposed to both microgravity and radiation during long-term stays in space. However, many aspects of the biological effects of combined microgravity and space radiation remain unclear. We developed a new three-dimensional (3D) clinostat synchronized heavy-ion irradiation system for use in ground-based studies of the combined exposures. Our new system uses a particle accelerator and a respiratory gating system from heavy-ion radiotherapy to irradiate samples being rotated in the 3D clinostat with carbon-ion beams only when the samples are in the horizontal position. A Peltier module and special sample holder were loaded on a static stage (standing condition) and the 3D clinostat (rotation condition) to maintain a suitable temperature under atmospheric conditions. The performance of the new device was investigated with normal human fibroblasts 1BR-hTERT in a disposable closed cell culture chamber. Live imaging revealed that cellular adhesion and growth were almost the same for the standing control sample and rotation sample over 48h. Dose flatness and symmetry were judged according to the relative density of Gafchromic films along the X-axis and Y-axis of the positions of the irradiated sample to confirm irradiation accuracy. Doses calculated using the carbon-ion calibration curve were almost the same for standing and rotation conditions, with the difference being less than 5% at 1Gy carbon-ion irradiation. Our new device can accurately synchronize carbon-ion irradiation and simulated microgravity while maintaining the temperature under atmospheric conditions at ground level.

X Irradiation Induces Acute Cognitive Decline via Transient Synaptic Dysfunction.

Cranial X irradiation can severely impair higher brain function, resulting in neurocognitive deficits. Radiation-induced brain injury is characterized by acute, early and late delayed changes, and morbidity is evident more than 6 months after irradiation. While the acute effects of radiation exposure on the brain are known, the underlying mechanisms remain unclear. In this study, we examined the acute effect of X radiation on synaptic function using behavioral analysis and immunohistochemistry. We found that 10 Gy whole-brain irradiation immediately after conditioning (within 30 min) impaired the formation of fear memory, whereas irradiation 24 h prior to conditioning did not. To investigate the mechanisms underlying these behavioral changes, we irradiated one hemisphere of the brain and analyzed synaptic function and adult neurogenesis immunohistochemically. We focused on drebrin, whose loss from dendritic spines is a surrogate marker of synaptopathy. The intensity of drebrin immunoreactivity started to decrease in the irradiated hemisphere 2 h after exposure. The immunostaining intensity recovered to preirradiation levels by 24 h, indicating that X radiation induced transient synaptic dysfunction. Interestingly, the number of newly generated neurons was not changed at 2 h postirradiation, whereas it was significantly decreased at 8 and 24 h postirradiation. Because irradiation 24 h prior to conditioning had no effect on fear memory, our findings suggest that radiation-induced death of newly-generated neurons does not substantially impact fear memory formation. The radiation-induced synaptic dysfunction likely caused a transient memory deficit during the critical period for fear memory formation (approximately 1-3 h after conditioning), which coincides with a change in drebrin immunostaining in the hippocampus, a structure critical for fear memory formation.