Radiation-induced DNA double-strand breaks in cortisol exposed fibroblasts as quantified with the novel foci-integrated damage complexity score (FIDCS).

Without the protective shielding of Earth's atmosphere, astronauts face higher doses of ionizing radiation in space, causing serious health concerns. Highly charged and high energy (HZE) particles are particularly effective in causing complex and difficult-to-repair DNA double-strand breaks compared to low linear energy transfer. Additionally, chronic cortisol exposure during spaceflight raises further concerns, although its specific impact on DNA damage and repair remains unknown. This study explorers the effect of different radiation qualities (photons, protons, carbon, and iron ions) on the DNA damage and repair of cortisol-conditioned primary human dermal fibroblasts. Besides, we introduce a new measure, the Foci-Integrated Damage Complexity Score (FIDCS), to assess DNA damage complexity by analyzing focus area and fluorescent intensity. Our results show that the FIDCS captured the DNA damage induced by different radiation qualities better than counting the number of foci, as traditionally done. Besides, using this measure, we were able to identify differences in DNA damage between cortisol-exposed cells and controls. This suggests that, besides measuring the total number of foci, considering the complexity of the DNA damage by means of the FIDCS can provide additional and, in our case, improved information when comparing different radiation qualities.

Lost in Space? Unmasking the T Cell Reaction to Simulated Space Stressors.

The space environment will expose astronauts to stressors like ionizing radiation, altered gravity fields and elevated cortisol levels, which pose a health risk. Understanding how the interplay between these stressors changes T cells' response is important to better characterize space-related immune dysfunction. We have exposed stimulated Jurkat cells to simulated space stressors (1 Gy, carbon ions/1 Gy photons, 1 µM hydrocortisone (HC), Mars, moon, and microgravity) in a single or combined manner. Pro-inflammatory cytokine IL-2 was measured in the supernatant of Jurkat cells and at the mRNA level. Results show that alone, HC, Mars gravity and microgravity significantly decrease IL-2 presence in the supernatant. 1 Gy carbon ion irradiation showed a smaller impact on IL-2 levels than photon irradiation. Combining exposure to different simulated space stressors seems to have less immunosuppressive effects. Gene expression was less impacted at the time-point collected. These findings showcase a complex T cell response to different conditions and suggest the importance of elevated cortisol levels in the context of space flight, also highlighting the need to use simulated partial gravity technologies to better understand the immune system's response to the space environment.

Gravitational effects on fibroblasts' function in relation to wound healing.

The spaceflight environment imposes risks for maintaining a healthy skin function as the observed delayed wound healing can contribute to increased risks of infection. To counteract delayed wound healing in space, a better understanding of the fibroblasts' reaction to altered gravity levels is needed. In this paper, we describe experiments that were carried out at the Large Diameter Centrifuge located in ESA-ESTEC as part of the ESA Academy 2021 Spin Your Thesis! Campaign. We exposed dermal fibroblasts to a set of altered gravity levels, including transitions between simulated microgravity and hypergravity. The addition of the stress hormone cortisol to the cell culture medium was done to account for possible interaction effects of gravity and cortisol exposure. Results show a main impact of cortisol on the secretion of pro-inflammatory cytokines as well as extracellular matrix proteins. Altered gravity mostly induced a delay in cellular migration and changes in mechanosensitive cell structures. Furthermore, 20 × g hypergravity transitions induced changes in nuclear morphology. These findings provide insights into the effect of gravity transitions on the fibroblasts' function related to wound healing, which may be useful for the development of countermeasures.

Comparison of in vitro scratch wound assay experimental procedures.

Fibroblast migration is an important aspect of wound healing. Different factors can influence migration and as such proper wound healing. In vitro scratch wound assays are used to examine cellular migration. However, the wide array of techniques available reduces reproducibility of findings. In this paper, we compare two techniques for wound creation; i.e. the exclusion method or scratching of cell monolayers. Furthermore, we investigate if analysis software influences experimental outcome by comparing both commercially and freely available analysis software. Besides, we examine the effect of cortisol on migration behavior of fibroblasts and identify possible caveats in experimental design. Results show a significantly reduced migration of fibroblasts when wounds are created using a cell exclusion method. Furthermore, addition of cortisol to the cell culture media only reduced migration of fibroblast monolayers that had been scratched but not in those where wounds were created using the exclusion method. A possible explanation related to cytokine expression is discussed.

The Effects of Combined Exposure to Simulated Microgravity, Ionizing Radiation, and Cortisol on the In Vitro Wound Healing Process.

Human spaceflight is associated with several health-related issues as a result of long-term exposure to microgravity, ionizing radiation, and higher levels of psychological stress. Frequent reported skin problems in space include rashes, itches, and a delayed wound healing. Access to space is restricted by financial and logistical issues; as a consequence, experimental sample sizes are often small, which limits the generalization of the results. Earth-based simulation models can be used to investigate cellular responses as a result of exposure to certain spaceflight stressors. Here, we describe the development of an in vitro model of the simulated spaceflight environment, which we used to investigate the combined effect of simulated microgravity using the random positioning machine (RPM), ionizing radiation, and stress hormones on the wound-healing capacity of human dermal fibroblasts. Fibroblasts were exposed to cortisol, after which they were irradiated with different radiation qualities (including X-rays, protons, carbon ions, and iron ions) followed by exposure to simulated microgravity using a random positioning machine (RPM). Data related to the inflammatory, proliferation, and remodeling phase of wound healing has been collected. Results show that spaceflight stressors can interfere with the wound healing process at any phase. Moreover, several interactions between the different spaceflight stressors were found. This highlights the complexity that needs to be taken into account when studying the effect of spaceflight stressors on certain biological processes and for the aim of countermeasures development.

Monitoring functional immune responses with a cytokine release assay: ISS flight hardware design and experimental protocol for whole blood cultures executed under microgravity conditions.

Introduction: Long-term space missions trigger a prolonged neuroendocrine stress response leading to immune system dysregulation evidenced by susceptibility to infections, viral reactivation, and skin irritations. However, due to existing technical constraints, real-time functional immune assessments are not currently available to crew inflight. The in vitro cytokine release assay (CRA) has been effectively employed to study the stimulated cytokine response of immune cells in whole blood albeit limited to pre- and post-flight sessions. A novel two-valve reaction tube (RT) has been developed to enable the execution of the CRA on the International Space Station (ISS). Methods: In a comprehensive test campaign, we assessed the suitability of three materials (silicone, C-Flex, and PVC) for the RT design in terms of biochemical compatibility, chemical stability, and final data quality analysis. Furthermore, we thoroughly examined additional quality criteria such as safety, handling, and the frozen storage of antigens within the RTs. The validation of the proposed crew procedure was conducted during a parabolic flight campaign. Results: The selected material and procedure proved to be both feasible and secure yielding consistent and dependable data outcomes. This new hardware allows for the stimulation of blood samples on board the ISS, with subsequent analysis still conducted on the ground. Discussion: The resultant data promises to offer a more accurate understanding of the stress-induced neuroendocrine modulation of immunity during space travel providing valuable insights for the scientific community. Furthermore, the versatile nature of the RT suggests its potential utility as a testing platform for various other assays or sample types.

B-Cell Homeostasis Is Maintained During Two Months of Head-Down Tilt Bed Rest With or Without Antioxidant Supplementation.

Alterations of the immune system could seriously impair the ability to combat infections during future long-duration space missions. However, little is known about the effects of spaceflight on the B-cell compartment. Given the limited access to astronaut samples, we addressed this question using blood samples collected from 20 healthy male volunteers subjected to long-duration bed rest, an Earth-based analog of spaceflight. Hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, four B-cell subsets, immunoglobulin isotypes, six cytokines involved in inflammation, cortisone and cortisol were quantified at five time points. Tibia microarchitecture was also studied. Moreover, we investigated the efficiency of antioxidant supplementation with a cocktail including polyphenols, omega 3, vitamin E and selenium. Our results show that circulating hematopoietic progenitors, white blood cells, total lymphocytes and B-cells, and B-cell subsets were not affected by bed rest. Cytokine quantification suggested a lower systemic inflammatory status, supported by an increase in serum cortisone, during bed rest. These data confirm the in vivo hormonal dysregulation of immunity observed in astronauts and show that bed rest does not alter B-cell homeostasis. This lack of an impact of long-term bed rest on B-cell homeostasis can, at least partially, be explained by limited bone remodeling. None of the evaluated parameters were affected by the administration of the antioxidant supplement. The non-effectiveness of the supplement may be because the diet provided to the non-supplemented and supplemented volunteers already contained sufficient antioxidants. Given the limitations of this model, further studies will be required to determine whether B-cell homeostasis is affected, especially during future deep-space exploration missions that will be of unprecedented durations.

Three-Dimensional Growth of Prostate Cancer Cells Exposed to Simulated Microgravity.

Prostate cancer metastasis has an enormous impact on the mortality of cancer patients. Factors involved in cancer progression and metastasis are known to be key players in microgravity (µg)-driven three-dimensional (3D) cancer spheroid formation. We investigated PC-3 prostate cancer cells for 30 min, 2, 4 and 24 h on the random positioning machine (RPM), a device simulating µg on Earth. After a 24 h RPM-exposure, the cells could be divided into two groups: one grew as 3D multicellular spheroids (MCS), the other one as adherent monolayer (AD). No signs of apoptosis were visible. Among others, we focused on cytokines involved in the events of metastasis and MCS formation. After 24 h of exposure, in the MCS group we measured an increase in ACTB, MSN, COL1A1, LAMA3, FN1, TIMP1, FLT1, EGFR1, IL1A, IL6, CXCL8, and HIF1A mRNA expression, and in the AD group an elevation of LAMA3, COL1A1, FN1, MMP9, VEGFA, IL6, and CXCL8 mRNAs compared to samples subjected to 1 g conditions. Significant downregulations in AD cells were detected in the mRNA levels of TUBB, KRT8, IL1B, IL7, PIK3CB, AKT1 and MTOR after 24 h. The release of collagen-1α1 and fibronectin protein in the supernatant was decreased, whereas the secretion of IL-6 was elevated in 24 h RPM samples. The secretion of IL-1α, IL-1ß, IL-7, IL-2, IL-8, IL-17, TNF-α, laminin, MMP-2, TIMP-1, osteopontin and EGF was not significantly altered after 24 h compared to 1 g conditions. The release of soluble factors was significantly reduced after 2 h (IL-1α, IL-2, IL-7, IL-8, IL-17, TNF-α, collagen-1α1, MMP-2, osteopontin) and elevated after 4 h (IL-1ß, IL-2, IL-6, IL-7, IL-8, TNF-α, laminin) in RPM samples. Taken together, simulated µg induced 3D growth of PC-3 cancer cells combined with a differential expression of the cytokines IL-1α, IL-1ß, IL-6 and IL-8, supporting their involvement in growth and progression of prostate cancer cells.

The CellBox-2 Mission to the International Space Station: Thyroid Cancer Cells in Space.

A spaceflight to the International Space Station (ISS) is a dream of many researchers. We had the chance to investigate the effect of real microgravity (CellBox-2 Space mission) on the transcriptome and proteome of FTC-133 human follicular thyroid cancer cells (TCC). The cells had been sent to the ISS by a Falcon 9 rocket of SpaceX CRS-13 from Cape Canaveral (United States) and cultured in six automated hardware units on the ISS before they were fixed and returned to Earth. Multicellular spheroids (MCS) were detectable in all spaceflight hardware units. The VCL, PXN, ITGB1, RELA, ERK1 and ERK2 mRNA levels were significantly downregulated after 5 days in space in adherently growing cells (AD) and MCS compared with ground controls (1g), whereas the MIK67 and SRC mRNA levels were both suppressed in MCS. By contrast, the ICAM1, COL1A1 and IL6 mRNA levels were significantly upregulated in AD cells compared with 1g and MCS. The protein secretion measured by multianalyte profiling technology and enzyme-linked immunosorbent assay (AngiogenesisMAP®, extracellular matrix proteins) was not significantly altered, with the exception of elevated angiopoietin 2. TCC in space formed MCS, and the response to microgravity was mainly anti-proliferative. We identified ERK/RELA as a major microgravity regulatory pathway.

In vitro Assessment of the DNA Damage Response in Dental Mesenchymal Stromal Cells Following Low Dose X-ray Exposure.

Stem cells contained within the dental mesenchymal stromal cell (MSC) population are crucial for tissue homeostasis. Assuring their genomic stability is therefore essential. Exposure of stem cells to ionizing radiation (IR) is potentially detrimental for normal tissue homeostasis. Although it has been established that exposure to high doses of ionizing radiation (IR) has severe adverse effects on MSCs, knowledge about the impact of low doses of IR is lacking. Here we investigated the effect of low doses of X-irradiation with medical imaging beam settings (<0.1 Gray; 900 mGray per hour), in vitro, on pediatric dental mesenchymal stromal cells containing dental pulp stem cells from deciduous teeth, dental follicle progenitor cells and stem cells from the apical papilla. DNA double strand break (DSB) formation and repair kinetics were monitored by immunocytochemistry of γH2AX and 53BP1 as well as cell cycle progression by flow cytometry and cellular senescence by senescence-associated ß-galactosidase assay and ELISA. Increased DNA DSB repair foci, after exposure to low doses of X-rays, were measured as early as 30 min post-irradiation. The number of DSBs returned to baseline levels 24 h after irradiation. Cell cycle analysis revealed marginal effects of IR on cell cycle progression, although a slight G2/M phase arrest was seen in dental pulp stromal cells from deciduous teeth 72 h after irradiation. Despite this cell cycle arrest, no radiation-induced senescence was observed. In conclusion, low X-ray IR doses (< 0.1 Gray; 900 mGray per hour), were able to induce significant increases in the number of DNA DSBs repair foci, but cell cycle progression seems to be minimally affected. This highlights the need for more detailed and extensive studies on the effects of exposure to low IR doses on different mesenchymal stromal cells.

Quantification of DNA Double Strand Breaks and Oxidation Response in Children and Adults Undergoing Dental CBCT Scan.

Assessing the possible biological effects of exposure to low doses of ionizing radiation (IR) is one of the prime challenges in radiation protection, especially in medical imaging. Today, radiobiological data on cone beam CT (CBCT) related biological effects are scarce. In children and adults, the induction of DNA double strand breaks (DSBs) in buccal mucosa cells and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and antioxidant capacity in saliva samples after CBCT examination were examined. No DNA DSBs induction was observed in children nor adults. In children only, an increase in 8-oxo-dG levels was observed 30 minutes after CBCT. At the same time an increase in antioxidant capacity was observed in children, whereas a decrease was observed in adults. Our data indicate that children and adults react differently to IR doses associated with CBCT. Fully understanding these differences could lead to an optimal use of CBCT in different age categories as well as improved radiation protection guidelines.

Targeting the Hedgehog pathway in combination with X­ray or carbon ion radiation decreases migration of MCF­7 breast cancer cells.

The use of carbon ion therapy for cancer treatment is becoming more widespread due to the advantages of carbon ions compared with X­rays. Breast cancer patients may benefit from these advantages, as the surrounding healthy tissues receive a lower dose, and the increased biological effectiveness of carbon ions can better control radioresistant cancer cells. Accumulating evidence indicates that the Hedgehog (Hh) pathway is linked to the development and progression of breast cancer, as well as to resistance to X­irradiation and the migratory capacity of cancer cells. Hence, there is an increasing interest in targeting the Hh pathway in combination with radiotherapy. Several studies have already investigated this treatment strategy with conventional radiotherapy. However, to the best of our knowledge, the combination of Hh inhibitors with particle therapy has not yet been explored. The aim of the present study was to investigate the potential of the Hh inhibitor GANT61 as an effective modulator of radiosensitivity and migration potential in MCF­7 breast cancer cells, and compare potential differences between carbon ion irradiation and X­ray exposure. Although Hh targeting was not able to radiosensitise cells to any radiation type used, the combination of GANT61 with X­rays or carbon ions (energy: 95 MeV/n; linear energy transfer: 73 keV/µm) was more effective in decreasing MCF­7 cell migration compared with either radiation type alone. Gene expression of the Hh pathway was affected to different degrees in response to X­ray and carbon ion irradiation, as well as in response to the combination of GANT61 with irradiation. In conclusion, combining Hh inhibition with radiation (X­rays or carbon ions) more effectively decreased breast cancer cell migration compared with radiation treatment alone.

The Combination of Particle Irradiation With the Hedgehog Inhibitor GANT61 Differently Modulates the Radiosensitivity and Migration of Cancer Cells Compared to X-Ray Irradiation.

Due to the advantages of charged particles compared to conventional radiotherapy, a vast increase is noted in the use of particle therapy in the clinic. These advantages include an improved dose deposition and increased biological effectiveness. Metastasis is still an important cause of mortality in cancer patients and evidence has shown that conventional radiotherapy can increase the formation of metastasizing cells. An important pathway involved in the process of metastasis is the Hedgehog (Hh) signaling pathway. Recent studies have demonstrated that activation of the Hh pathway, in response to X-rays, can lead to radioresistance and increased migratory, and invasive capabilities of cancer cells. Here, we investigated the effect of X-rays, protons, and carbon ions on cell survival, migration, and Hh pathway gene expression in prostate cancer (PC3) and medulloblastoma (DAOY) cell lines. In addition, the potential modulation of cell survival and migration by the Hh pathway inhibitor GANT61 was investigated. We found that in both cell lines, carbon ions were more effective in decreasing cell survival and migration as well as inducing more significant alterations in the Hh pathway genes compared to X-rays or protons. In addition, we show here for the first time that the Hh inhibitor GANT61 is able to sensitize DAOY medulloblastoma cells to particle radiation (proton and carbon ion) but not to conventional X-rays. This important finding demonstrates that the results of combination treatment strategies with X-ray radiotherapy cannot be automatically extrapolated to particle therapy and should be investigated separately. In conclusion, combining GANT61 with particle radiation could offer a benefit for specific cancer types with regard to cancer cell survival.