Alexander Helm

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.

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 β-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.

Fate of D3 mouse embryonic stem cells exposed to X-rays or carbon ions

The risk of radiation exposure during embryonic development is still a major problem in radiotoxicology. In this study we investigated the response of the murine embryonic stem cell (mESC) line D3 to two radiation qualities: sparsely ionizing X-rays and densely ionizing carbon ions. We analyzed clonogenic cell survival, proliferation, induction of chromosome aberrations as well as the capability of cells to differentiate to beating cardiomyocytes up to 3 days after exposure. Our results show that, for all endpoints investigated, carbon ions are more effective than X-rays at the same radiation dose. Additionally, in long term studies (≥8 days post-irradiation) chromosomal damage and the pluripotency state were investigated. These studies reveal that pluripotency markers are present in the progeny of cells surviving the exposure to both radiation types. However, only in the progeny of X-ray exposed cells the aberration frequency was comparable to that of the control population, while the progeny of carbon ion irradiated cells harbored significantly more aberrations than the control, generally translocations. We conclude that cells surviving the radiation exposure maintain pluripotency but may carry stable chromosomal rearrangements after densely ionizing radiation.

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.

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.

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.

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.

Generating and grading the abscopal effect: proposal for comprehensive evaluation of combination immunoradiotherapy in mouse models

Numerous published case reports and forthcoming clinical trials combine immunotherapy and radiotherapy with the goal of demonstrating an abscopal effect reaction. However, reports and results are varied, and a comprehensive animal study combining radiotherapy modalities and immunotherapy agents has yet to be performed. Further, clinical reports are mixed, with inconsistent generation of the effect as well as abscopal effects seen ranging from widespread disease elimination to growth stagnation of local lymph node metastasis. We propose a grading system for use in differentiating abscopal effects seen in animal and clinical trials. Further, we will conduct a comprehensive study in mice, evaluating three radiotherapeutic modalities (photon, proton, and carbon-ion) combined with five immunotherapeutic agents with differing actions. Comprehensive cell analysis will be conducted with the aim of improving upon draft models of abscopal effect generation, as well as differentiating between locoregional and systemic methods of action. Further, evaluation of radiation fractionation, as well as combination with ex vivo activated dendritic cell (DC) inoculation, will be performed. Though the abscopal effect has been reported for nearly 70 years, only recently has delineation of its mechanism seemed possible. Comprehensive evaluation of available modalities may shed light on the precise requirements for generating the effect, potentially enabling its regular usage in the treatment of disease.

Clinical Evidence and Radiobiological Background of Particle Radiation Therapy

Charged particle therapy compared to the conventional radiotherapy offers many advantages. The particles’ peculiar inversed dose-depth profile characteristics provide the possibility, contrarily to photon irradiation, to deposit the energy more precisely to the tumour leading to a higher tumour local control, a lower probability to damage the surrounding healthy tissue and a lower risk of complications.

The effect of X-rays and C-ions on pluripotent embryonic stem cells

Embryonic stem cells (ESC) are characterized by both the capacity of infinite self-renewal and the ability to give rise to all the three germ layers emphasizing the need to strictly control the genetic integrity. To date, ESC are a powerful tool in disease modeling, tissue engineering and drug testing. However, in the field of radiation research, their potential has not been exploited. We used the mouse ESC line D3 as a model to examine the effects of X-rays or C-ions (spread out Bragg peak, energy 106–147 MeV/u, average LET = 75 keV/µm) [ 1]. Doses of 0.5–5 Gy were applied and endpoints such as cell cycle progression (measured by flow cytometry), apoptosis (microscopic analysis of cell nucleus morphology), induction of chromosome aberrations (mFISH analysis), presence of pluripotency markers Oct3/4 and SOX2 (western blotting) and differentiation capacity by means of an embryoid body formation assay were analyzed up to 17 days post-irradiation. The experiments show that cells undergo a transient G2 arrest following exposure. After G2 checkpoint release, an increase in the apoptotic index is observed for both radiation types (3.7-fold increase for 2 Gy X-ray and 2.4-fold increase for 2 Gy C-ions). C-ions induce more structural chromosomal aberrations in first cycle cells than X-rays. During subsequent cell divisions, the frequency of chromosome aberrations declines: After >7 population doublings (8 days after exposure), the aberration frequency in the progeny of X-ray exposed cells returns to the control level (7% aberrant cells), while the progeny of C-ion exposed cells still harbor significantly more aberrations than control cells, which is mainly due to transmissible translocations. The expression of pluripotency markers is maintained in cells surviving X-ray or C-ion exposure. This finding is supported by examining the differentiation capacity of ESC through the formation of embryoid bodies. Our experiments show that after X-ray or C-ion exposure, cells are able to develop spontaneous beating activity, indicating the differentiation ability into mesodermal cell lineages, i.e. beating cardiomyocytes. However, following C-ion exposure, the formation of beating clusters was delayed compared with control cells. Moreover, our chromosome studies revealed that unexposed cells carry a high frequency of numerical aberrations. These comprise trisomies of chromosome 8 and 11 with a frequency of 29 ± 8% and 26 ± 6% respectively, as well as nullisomy of chromosome Y with a frequency of 35 ± 3%. Aneuploidy is a typical feature of mouse ESC and has been related to cell culture methods [ 2] and passage number. Because aneuploidy may affect gene expression and influence the properties of a cell population, the relevance of experiments based on mouse ESC is limited. To overcome this problem, we recently extended our studies to human ESC. Human ESC are known to be cytogenetically more stable than mouse ESC, and represent a model that is closer to human embryonic development. Indeed, first investigations revealed a lower faction of cells with numerical and structural aberrations in the human ESC line H9 [ 3] compared with the mouse ESC line D3 (2% vs. 73% and 3% vs. 7%, respectively).