There has been increased interest in the immune stimulatory properties of ionizing radiation based on several preclinical models and recently completed clinical studies performed in combination with checkpoint inhibitors. This is a paradigm shift in that it considers the role of radiation beyond its direct cytotoxic effects, however, the factors that promote or limit radiation-induced immunogenicity are still unclear. Here we review the role of radiation in modulating the various aspects of the tumor immune microenvironment and discuss in particular the direct effects of radiation on the DNA damage response and its immediate consequences to neighboring cells. The latter “danger response” in particular can enhance recruitment of dendritic and macrophage cells to the tumor microenvironment, which in turn can activate or diminish subsequent T-cell priming. Identification of the critical factors that modulate the interaction between radiation-induced cell damage and the immune system will allow for rational combinational therapy design and the development of biomarkers that predict effective immune responses.

Customized open-source software is used to characterize, exemplify, compare and critically evaluate mathematical/computational synergy analysis methods currently used in biology, and used or potentially applicable in radiobiology. As examples, we reanalyze some published results on murine Harderian gland tumors and on in vitro chromosome aberrations induced by exposure to single-ion radiations that simulate components of the galactic cosmic ray field. Baseline no-synergy/no-antagonism-mixture dose-effect relationships are calculated for corresponding mixed fields. No new experimental results are presented. Synergy analysis of effects due to a mixed radiation field whose components' individual dose-effect relationships are highly curvilinear should not consist of simply comparing to the sum of the components' effects. Such curvilinearity must often be allowed for in current radiobiology, especially when studying possible non-targeted (“bystander”) effects. Consequently, many different synergy analysis theories are currently used in biology to replace simple effect additivity. We give evidence that for most synergy experiments and observations, incremental effect additivity is the most appropriate replacement. It has a large domain of applicability, being useful even when pronounced individual dose-effect relationship curvilinearity is a confounding factor. It allows calculation of 95% confidence intervals for baseline mixture dose-effect relationships taking into account parameter correlations; if non-targeted effects are important this gives much tighter intervals than neglecting the correlations. It always obeys two consistency conditions that simple effect additivity usually fails to obey: a “mixture of mixtures principle” and the standard “sham mixture principle”. The mixture of mixtures principle is important in radiobiology because even nominally single-ion radiations are usually mixtures when they strike the biological target, due to intervening material. It is not yet clear whether mixing galactic cosmic ray components sometimes leads to statistically significant synergy for animal tumorigenesis. The substantial limitations of synergy theories are sometimes overlooked, and they warrant further study.

In this work, we examined the DNA sequence preference of gamma-radiation-induced DNA damage in purified DNA sequences after heat treatment. DNA was fluorescently end-labeled and gamma-radiation-induced DNA cleavage was examined using capillary electrophoresis with laser-induced fluorescence detection. Our findings provide evidence that gamma-radiation-induced DNA damage to end-labeled DNA is nonrandom and has a sequence preference. The degree of cleavage was quantified at each nucleotide, and we observed that preferential cleavage occurred at C nucleotides with lesser cleavage at G nucleotides, while being very low at T nucleotides. The differences in percentage cleavage at individual nucleotides ranged up to sixfold. The DNA sequences surrounding the most intense radiation-induced DNA cleavage sites were examined and a consensus sequence 5′-AGGC*C (where C* is the cleavage site) was found. The highest intensity gamma-radiation-induced DNA cleavage sites were found at the dinucleotides, 5′-GG*, 5′-GC*, 5′-C*C and 5′-G*G and at the trinucleotides, 5′-GG*C, 5′-TC*A, 5′-GG*G and 5′-GC*C. These findings have implications for our understanding of ionizing radiation-induced DNA damage.

Long noncoding RNAs (lncRNAs) are emerging as key molecules in regulating many biological processes and have been implicated in development and disease pathogenesis. Biomarkers of cancer and normal tissue response to treatment are of great interest in precision medicine, as well as in public health and medical management, such as for assessment of radiation injury after an accidental or intentional exposure. Circulating and functional RNAs, including microRNAs (miRNAs) and lncRNAs, in whole blood and other body fluids are potential valuable candidates as biomarkers. Early prediction of possible acute, intermediate and delayed effects of radiation exposure enables timely therapeutic interventions. To address whether long noncoding RNAs (lncRNAs) could serve as biomarkers for radiation biodosimetry we performed whole genome transcriptome analysis in a mouse model after whole-body irradiation. Differential lncRNA expression patterns were evaluated at 16, 24 and 48 h postirradiation in total RNA isolated from whole blood of mice exposed to 1, 2, 4, 8 and 12 Gy of X rays. Sham-irradiated animals served as controls. Significant alterations in the expression patterns of lncRNAs were observed after different radiation doses at the various time points. We identified several radiation-induced lncRNAs known for DNA damage response as well as immune response. Long noncoding RNA targets of tumor protein 53 (P53), Trp53cor1, Dino, Pvt1 and Tug1 and an upstream regulator of p53, Meg3, were altered in response to radiation. Gm14005 (Morrbid) and Tmevpg1 were regulated by radiation across all time points and doses. These two lncRNAs have important potential as blood-based radiation biomarkers; Gm14005 (Morrbid) has recently been shown to play a key role in inflammatory response, while Tmevpg1 has been implicated in the regulation of interferon gamma. Precise molecular biomarkers, likely involving a diverse group of inducible molecules, will not only enable the development and effective use of medical countermeasures but may also be used to detect and circumvent or mitigate normal tissue injury in cancer radiotherapy.

To experimentally investigate the role of hydration in the initial process of the decomposition of 2-deoxy-d-ribose (dR), which is a major component of the DNA backbone, we used mass spectrometry to monitor the ions desorbing from hydrated dR films exposed to monochromatic soft X rays (560 eV). The X-ray photons preferentially ionize the K-shell electrons of the oxygen atoms in DNA. Hydrated dR samples were prepared under vacuum by exposing a cooled (∼150 K) dR film deposited on a Si substrate to water vapor. Using a quadrupole mass spectrometer, we observed the desorption of ions such as H , CH_x , C₂H_x , CH_xO , C₃H_x and C₂H_xO (x = 1, 2, 3 and 4). In addition, the desorption of H₂O or H₃O was observed in the mass spectra of hydrated dR films. Except for H , the yields of these ions decreased when one layer of water molecules was deposited onto the film. These ions are produced by C–C or C–O bond scission. This result suggests that the water molecules act as a quencher, suppressing Coulomb repulsion and thus the extensive molecular decomposition of dR. Ab initio molecular dynamics simulations were performed to rationalize the fragments observed in the experiments. The results of the dynamical process of a hydrated dR molecule after oxygen K-ionization revealed elongation of a C–O bond of dR and the O–H bonds of both dR and water molecules prior to the Auger process, resulting in the ejection of H ions. These results strongly suggest that the very early process contributes to reducing the dR fragmentation, producing the H₃O and H detected from the hydrated dR films. These desorbed ions may be involved in the induction of other types of damage, such as oxidatively generated base lesions, concomitantly produced with a strand break when produced in DNA.

Astronauts on deep space missions will be required to work more autonomously than on previous missions, and thus their ability to perform executive functions could be critical to mission success. One of the most common measures of executive function in humans is the ability to perform attentional set shifting, which requires contributions from working memory, discrimination, reversal learning, attentional set shifting and attention. Rodent attentional set shifting assays require rats to form an association between the presence of the food reward and an associative cue, which is either the digging media or the scent that is placed in the bowl; by altering the combination of scent and digging media, progressively more complex cognitive processes can be tested. In this study, we have determined the effect that exposure to 5–20 cGy of 600 MeV/n ²⁸Si particles has on the ability of male retired breeder Wistar rats to perform attentional set shifting at three months postirradiation. All doses of Si resulted in a significant impairment in the ability of the rats to perform the first and most simple step of the ATSET assay, the simple discrimination (SD) task. If astronauts were to experience HZE-induced SD impairments, they would be unable to identify key factors to successfully resolve a situation. Performance in at least one other component of the ATSET test was impaired at all doses studied, however, these varied according to the dose. Compared with our previous studies using 1 GeV/n ⁵⁶Fe and ⁴⁸Ti particles, 600 MeV/n ²⁸Si ions impaired attentional set-shifting performance at lower doses than the heavier ions. However, when the effect of isofluences of the three HZE ions were compared, there were no significant differences in the severity of the impaired performance; there were, however, ion-specific decrements in the ability of rats to perform within the various stages of the test. This study further supports the notion that “mission-relevant” doses of HZE particles (<20 cGy) can impair certain aspects of attentional set-shifting performance in retired breeder rats, but there may be some ion-specific changes in the specific cognitive domains impaired.

AZD9291 is a novel, irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), which is administered orally. It has been proven effective in non-small cell lung cancer (NSCLC) patients, with both EGFR-sensitizing and EGFR T790M mutations in preclinical models. However, the potential therapeutic effects of AZD9291 combined with other modalities, including ionizing radiation, are not well understood. The presence of AZD9291 significantly increases the cell-killing effects of radiation in PC-9-IR cells with a secondary EGFR mutation (T790M), which was developed from NSCLC PC-9 cells (human lung adenocarcinoma cell with EGFR 19 exon 15 bp deletion) after chronic exposure to increasing doses of gefitinib, and in H1975 cells (human lung adenocarcinoma cell with EGFR exon 20 T790M mutation de novo), but not in PC-9 cells or in H460 cells (human lung adenocarcinoma cell with wild-type EGFR). In PC-9-IR cells, AZD9291 remarkably decreases phosphorylation levels of EGFR, extracellular regulated protein kinase (ERK), and protein kinase B (AKT). AZD9291 increases sensitivity to radiation in PC-9-IR cells by delaying deoxyribonucleic acid (DNA) damage repair after irradiation and inducing apoptosis, and enhances tumor growth inhibition when combined with radiation in PC-9-IR xenografts. Our findings suggest a potential therapeutic effect of AZD9291 as a radiation sensitizer in lung cancer cells with an acquired EGFR T790M mutation, providing a rationale for a clinical trial using the combination of AZD9291 and radiation in NSCLCs harboring acquired T790M mutation.

Future long-duration space missions will involve travel outside of the Earth's magnetosphere, which will result in increased radiation exposure for astronauts. Exposure could permanently damage multiple tissues, including the central nervous system (CNS), and result in deleterious effects on cognition and behavior during and beyond the mission. Here, we assessed the effects of whole-body oxygen ion (¹⁶O; 1,000 MeV/n) exposure (5 or 25 cGy) on social odor recognition memory in male Long-Evans rats at one and six months after exposure. At one month postirradiation, all rats displayed a preference for a novel 1 (N1) social odor experienced during the habituation phase. When assessed for recognition memory 24 h later, only sham-irradiated rats spent more time exploring a second novel social odor (novel 2, N2), whereas rats irradiated with 5 or 25 cGy ¹⁶O ions did not show a preference for the N2 odor compared to the N1 odor experienced 24 h earlier, thus displaying a memory deficit for recall of the social odor encountered 24 h prior. At six months postirradiation, rats exposed to 25 cGy showed persistent deficits in 24 h recognition memory, while the 5 cGy-exposed rats did not. Thus, 24 h recognition memory was apparently recovered at six months postirradiation for the low, but not the higher, dose of ¹⁶O ions. Both irradiated groups displayed similar numbers of Ki67 cells, a marker of cell proliferation, in the subventricular zone. These results further demonstrate that space-relevant ¹⁶O ion exposure has deleterious effects on the CNS, which are related to both radiation dose and time after exposure.

Lung exposure to radiation induces an injury response that includes the release of cytokines and chemotactic mediators; these signals recruit immune cells to execute inflammatory and wound-healing processes. However, radiation alters the pulmonary microenvironment, dysregulating the immune responses and preventing a return to homeostasis. Importantly, dysregulation is observed as a chronic inflammation, which can progress into pneumonitis and promote pulmonary fibrosis; inflammatory monocytes, which are bone marrow derived and express CCR2, have been shown to migrate into the lung after radiation exposure. Although the extent to which recruited inflammatory monocytes contribute to radiation-induced pulmonary fibrosis has not been fully investigated, we hypothesize that its pathogenesis is reliant on this population. The CC chemokine ligand, CCL2, is a chemotactic mediator responsible for trafficking of CCR2 inflammatory cells into the lung. Therefore, the contribution of this mediator to fibrosis development was analyzed. Interleukin (IL)-1β, a potent pro-inflammatory cytokine expressed during the radiation response, and its receptor, IL-1R1, were also evaluated. To this end, CCR2^–/–, IL-1β^–/– and IL-1R1^–/– chimeric mice were generated and exposed to 12.5 Gy thoracic radiation, and their response was compared to wild-type (C57BL/6) syngeneic controls. Fibrotic foci were observed in the periphery of the lungs of C57 syngeneic mice and CCR2^–/– recipient mice that received C57 bone marrow (C57 > CCR2^–/–) by 16 and 12 weeks after irradiation, respectively. In contrast, in the mice that had received bone marrow lacking CCR2 (CCR2^–/– > C57 and CCR2^–/– syngeneic mice), no pulmonary fibrosis was observed at 22 weeks postirradiation. This observation correlated with decreased numbers of infiltrating and interstitial macrophages compared to controls, as well as reduced proportions of pro-inflammatory Ly6C macrophages observed at 12–18 weeks postirradiation, suggesting that CCR2 macrophages contribute to radiation-induced pulmonary fibrosis. Interestingly, reduced proportions of CD206 lung macrophages were also present at these time points in CCR2^–/– chimeric mice, regardless of donor bone marrow type, suggesting that the phenotype of resident subsets may be influenced by CCR2. Furthermore, chimeras, in which either IL-1β was ablated from infiltrating cells or IL-1R1 from lung tissues, were also protected from fibrosis development, correlating with attenuated CCL2 production; these data suggest that IL-1β may influence chemotactic signaling after irradiation. Overall, our data suggest that CCR2 infiltrating monocyte-derived macrophages may play a critical role in the development of radiation-induced pulmonary fibrosis.

Exposure to heavy-ion radiation during cancer treatment or space travel may cause cognitive detriments that have been associated with changes in neuron morphology and plasticity. Observations in mice of reduced neuronal dendritic complexity have revealed a dependence on radiation quality and absorbed dose, suggesting that microscopic energy deposition plays an important role. In this work we used morphological data for mouse dentate granular cell layer (GCL) neurons and a stochastic model of particle track structure and microscopic energy deposition (ED) to develop a predictive model of high-charge and energy (HZE) particle-induced morphological changes to the complex structures of dendritic arbors. We represented dendrites as cylindrical segments of varying diameter with unit aspect ratios, and developed a fast sampling method to consider the stochastic distribution of ED by δ rays (secondary electrons) around the path of heavy ions, to reduce computational times. We introduce probabilistic models with a small number of parameters to describe the induction of precursor lesions that precede dendritic snipping, denoted as snip sites. Predictions for oxygen (¹⁶O, 600 MeV/n) and titanium (⁴⁸Ti, 600 MeV/n) particles with LET of 16.3 and 129 keV/μm, respectively, are considered. Morphometric parameters to quantify changes in neuron morphology are described, including reduction in total dendritic length, number of branch points and branch numbers. Sholl analysis is applied for single neurons to elucidate dose-dependent reductions in dendritic complexity. We predict important differences in measurements from imaging of tissues from brain slices with single neuron cell observations due to the role of neuron death through both soma apoptosis and excessive dendritic length reduction. To further elucidate the role of track structure, random segment excision (snips) models are introduced and a sensitivity study of the effects of the modes of neuron death in predictions of morphometric parameters is described. An important conclusion of this study is that δ rays play a major role in neuron morphological changes due to the large spatial distribution of damage sites, which results in a reduced dependence on LET, including modest difference between ¹⁶O and ⁴⁸Ti, compared to damages resulting from ED in localized damage sites.

Radiation-induced fibrosis (RIF) is a major side effect of radiotherapy in cancer patients with no effective therapeutic options. RIF involves excess deposition and aberrant remodeling of the extracellular matrix (ECM) leading to stiffness in tissues and organ failure. Development of preclinical models of RIF is crucial to elucidate the molecular mechanisms regulating fibrosis and to develop therapeutic approaches. In addition to radiation, the main molecular perpetrators of fibrotic reactions are cytokines, including transforming growth factor-β (TGF-β). We hypothesized that human oral fibroblasts would develop an in vitro fibrotic reaction in response to radiation and TGF-β. We demonstrate here that fibroblasts exposed to radiation followed by TGF-β exhibit a fibrotic phenotype with increased collagen deposition, cell proliferation, migration and invasion. In this in vitro model of RIF (RIF_iv), the early biological processes involved in fibrosis are demonstrated, along with increased levels of several molecules including collagen 1α1, collagen XIα1, integrin-α2 and cyclin D1 mRNA in irradiated cells. A clinically relevant antifibrotic agent, pentoxifylline, and a curcumin analogue both mitigated collagen deposition in irradiated fibroblast cultures. In summary, we have established an in vitro model for RIF that facilitates the elucidation of molecular mechanisms in radiation-induced fibrosis and the development of effective therapeutic approaches.