Genetic influence on scar vascularity after burn injury in individuals of European ancestry: A prospective cohort study.

After burn injury there is considerable variation in scar outcome, partially due to genetic factors. Scar vascularity is one characteristic that varies between individuals, and this study aimed to identify genetic variants contributing to different scar vascularity outcomes. An exome-wide array association study and gene pathway analysis was performed on a prospective cohort of 665 patients of European ancestry treated for burn injury, using their scar vascularity (SV) sub-score, part of the modified Vancouver Scar Scale (mVSS), as an outcome measure. DNA was genotyped using the Infinium HumanCoreExome-24 BeadChip, imputed to the Haplotype Reference Consortium panel. Associations between genetic variants (single nucleotide polymorphisms) and SV were estimated using an additive genetic model adjusting for sex, age, % total body surface area and number of surgical procedures, utilising linear and multinomial logistic regression. No individual genetic variants achieved the cut-off threshold for significance. Gene sets were also analysed using the Functional Mapping and Annotation (FUMA) platform, in which biological processes indirectly related to angiogenesis were significantly represented. This study suggests that SNPs in genes associated with angiogenesis may influence SV, but further studies with larger datasets are essential to validate these findings.

Non-severe thermal burn injuries induce long-lasting downregulation of gene expression in cortical excitatory neurons and microglia.

Burn injuries are devastating traumas, often leading to life-long consequences that extend beyond the observable burn scar. In the context of the nervous system, burn injury patients commonly develop chronic neurological disorders and have been suggested to have impaired motor cortex function, but the long-lasting impact on neurons and glia in the brain is unknown. Using a mouse model of non-severe burn injury, excitatory and inhibitory neurons in the primary motor cortex were labelled with fluorescent proteins using adeno-associated viruses (AAVs). A total of 5 weeks following the burn injury, virus labelled excitatory and inhibitory neurons were isolated using fluorescence-activated cell sorting (FACS). In addition, microglia and astrocytes from the remaining cortical tissue caudal to the motor cortex were immunolabelled and isolated with FACS. Whole transcriptome RNA-sequencing was used to identify any long-lasting changes to gene expression in the different cell types. RNA-seq analysis showed changes to the expression of a small number of genes with known functions in excitatory neurons and microglia, but not in inhibitory neurons or astrocytes. Specifically, genes related to GABA-A receptors in excitatory neurons and several cellular functions in microglia were found to be downregulated in burn injured mice. These findings suggest that non-severe burn injuries lead to long lasting transcriptomic changes in the brain, but only in specific cell types. Our findings provide a broad overview of the long-lasting impact of burn injuries on the central nervous system which may help identify potential therapeutic targets to prevent neurological dysfunction in burn patients.

Non-severe burn injury causes sustained platelet hyperreactivity.

Individuals who present to a hospital for treatment of a burn of any magnitude are more frequently hospitalised for ischemic heart disease, even decades after injury. Blood platelets are key mediators of cardiovascular disease. To investigate platelet involvement in post-burn cardiovascular risk, platelet reactivity was assessed in patients at 2- and 6-weeks after non-severe (TBSA < 20%) burn injury, and in a murine model 30 days after 8% TBSA full-thickness burn injury. Platelets were stimulated with canonical agonists and function reported by GPIIb/IIIa PAC1-binding site, CD62P expression, and formation of monocyte-platelet aggregates. In vivo thrombosis in a modified Folts model of vascular injury was assessed. Burn survivors had elevated frequencies of circulating monocyte-platelet aggregates, and platelets were hyperreactive, primarily to collagen stimulation. Burn plasma did not cause hyper-reactivity when incubated with control platelets. Platelets from burn injured mice also demonstrated increased response to collagen peptides but did not show any change in thrombosis following vascular injury. This study demonstrates the persistence of a small but significant platelet hyperreactivity following burn injury. Although our data does not suggest this heightened platelet sensitivity modulates thrombosis following vascular injury, the contribution of sub-clinical platelet hyperreactivity to accelerating atherogenesis merits further investigation.

Topical application of an irreversible small molecule inhibitor of lysyl oxidases ameliorates skin scarring and fibrosis.

Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases.

Dark-field tomography of an attenuating object using intrinsic x-ray speckle tracking.

Purpose: We investigate how an intrinsic speckle tracking approach to speckle-based x-ray imaging is used to extract an object's effective dark-field (DF) signal, which is capable of providing object information in three dimensions. Approach: The effective DF signal was extracted using a Fokker-Planck type formalism, which models the deformations of illuminating reference beam speckles due to both coherent and diffusive scatter from the sample. Here, we assumed that (a) small-angle scattering fans at the exit surface of the sample are rotationally symmetric and (b) the object has both attenuating and refractive properties. The associated inverse problem of extracting the effective DF signal was numerically stabilized using a "weighted determinants" approach. Results: Effective DF projection images, as well as the DF tomographic reconstructions of the wood sample, are presented. DF tomography was performed using a filtered back projection reconstruction algorithm. The DF tomographic reconstructions of the wood sample provided complementary, and otherwise inaccessible, information to augment the phase contrast reconstructions, which were also computed. Conclusions: An intrinsic speckle tracking approach to speckle-based imaging can tomographically reconstruct an object's DF signal at a low sample exposure and with a simple experimental setup. The obtained DF reconstructions have an image quality comparable to alternative x-ray DF techniques.

A Methylome and Transcriptome Analysis of Normal Human Scar Cells Reveals a Role for FOXF2 in Scar Maintenance.

Scars are maintained for life and increase in size during periods of growth such as puberty. Epigenetic changes in fibroblasts after injury may underpin the maintenance and growth of scars. In this study, we combined methylome and transcriptome data from normotrophic mature scar and contralateral uninjured normal skin fibroblasts to identify potential regulators of scar maintenance. In total, 219 significantly differentially expressed and 1,199 significantly differentially methylated promoters were identified, of which there were 12 genes both significantly differentially methylated and expressed. Of these, the two transcription factors, FOXF2 and MKX, were selected for further analysis. Immunocytochemistry and qPCR suggested that FOXF2 but not MKX had elevated expression in scar fibroblasts. Using RNA sequencing, FOXF2 knockdown was shown to significantly reduce the expression of extracellular matrix‒related genes, whereas MKX did not appear to affect similar pathways. Finally, FOXF2 knockdown was also shown to significantly decrease collagen I production in scar and keloid fibroblasts. This study provides insights into the maintenance of normotrophic scar, suggesting that FOXF2 is an important regulator of this process. Targeting genes responsible for maintenance of scar phenotype may ameliorate scar appearance and improve patient outcomes in the future.

Keloid fibroblasts have elevated and dysfunctional mechanotransduction signaling that is independent of TGF-ß.

BACKGROUND: Fibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood. OBJECTIVES: Investigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression. METHODS: We used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-ß (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay. RESULTS: KFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-ß. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-ß. TGF-ß1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype. CONCLUSIONS: These data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.

The epigenetics of keloids.

Keloid scarring is a fibroproliferative disorder of the skin with unknown pathophysiology, characterised by fibrotic tissue that extends beyond the boundaries of the original wound. Therapeutic options are few and commonly ineffective, with keloids very commonly recurring even after surgery and adjunct treatments. Epigenetics, defined as alterations to the DNA not involving the base-pair sequence, is a key regulator of cell functions, and aberrant epigenetic modifications have been found to contribute to many pathologies. Multiple studies have examined many different epigenetic modifications in keloids, including DNA methylation, histone modification, microRNAs and long non-coding RNAs. These studies have established that epigenetic dysregulation exists in keloid scars, and successful future treatment of keloids may involve reverting these aberrant modifications back to those found in normal skin. Here we summarise the clinical and experimental studies available on the epigenetics of keloids, discuss the major open questions and future perspectives on the treatment of this disease.

Synchrotron X-Ray Radiation-Induced Bystander Effect: An Impact of the Scattered Radiation, Distance From the Irradiated Site and p53 Cell Status.

Synchrotron radiation, especially microbeam radiotherapy (MRT), has a great potential to improve cancer radiotherapy, but non-targeted effects of synchrotron radiation have not yet been sufficiently explored. We have previously demonstrated that scattered synchrotron radiation induces measurable γ-H2AX foci, a biomarker of DNA double-strand breaks, at biologically relevant distances from the irradiated field that could contribute to the apparent accumulation of bystander DNA damage detected in cells and tissues outside of the irradiated area. Here, we quantified an impact of scattered radiation to DNA damage response in "naïve" cells sharing the medium with the cells that were exposed to synchrotron radiation. To understand the effect of genetic alterations in naïve cells, we utilised p53-null and p53-wild-type human colon cancer cells HCT116. The cells were grown in two-well chamber slides, with only one of nine zones (of equal area) of one well irradiated with broad beam or MRT. γ-H2AX foci per cell values induced by scattered radiation in selected zones of the unirradiated well were compared to the commensurate values from selected zones in the irradiated well, with matching distances from the irradiated zone. Scattered radiation highly impacted the DNA damage response in both wells and a pronounced distance-independent bystander DNA damage was generated by broad-beam irradiations, while MRT-generated bystander response was negligible. For p53-null cells, a trend for a reduced response to scattered irradiation was observed, but not to bystander signalling. These results will be taken into account for the assessment of genotoxic effects in surrounding non-targeted tissues in preclinical experiments designed to optimise conditions for clinical MRT and for cancer treatment in patients.

Abscopal Gene Expression in Response to Synchrotron Radiation Indicates a Role for Immunological and DNA Damage Response Genes.

Abscopal effects are an important aspect of targeted radiation therapy due to their implication in normal tissue toxicity from chronic inflammatory responses and mutagenesis. Gene expression can be used to determine abscopal effects at the molecular level. Synchrotron microbeam radiation therapy utilizing high-intensity X rays collimated into planar microbeams is a promising cancer treatment due to its reported ability to ablate tumors with less damage to normal tissues compared to conventional broadbeam radiation therapy techniques. The low scatter of synchrotron radiation enables microbeams to be delivered to tissue effectively, and is also advantageous for out-of-field studies because there is minimal interference from scatter. Mouse legs were irradiated at a dose rate of 49 Gy/s and skin samples in the out-of-field areas were collected. The out-of-field skin showed an increase in Tnf expression and a decrease in Mdm2 expression, genes associated with inflammation and DNA damage. These expression effects from microbeam exposure were similar to those found with broadbeam exposure. In immune-deficient Ccl2 knockout mice, we identified a different gene expression profile which showed an early increase in Mdm2, Tgfb1, Tnf and Ccl22 expression in out-of-field skin that was not observed in the immune-proficient mice. Our results suggest that the innate immune system is involved in out-of-field tissue responses and alterations in the immune response may not eliminate abscopal effects, but could change them.

The Role of IL-6 in Skin Fibrosis and Cutaneous Wound Healing.

The timely resolution of wound healing is critical for restoring the skin as a protective barrier. The switch from a proinflammatory to a reparative microenvironment must be tightly regulated. Interleukin (IL)-6 is a key modulator of the inflammatory and reparative process: it is involved in the differentiation, activation, and proliferation of leukocytes, endothelial cells, keratinocytes, and fibroblasts. This review examines the role of IL-6 in the healing of cutaneous wounds, and how dysregulation of IL-6 signaling can lead to either fibrosis or a failure to heal. The role of an IL-6/TGF-ß feedback loop is discussed in the context of fibrogenesis, while IL-6 expression and responses in advanced age, diabetes, and obesity is outlined regarding the development of chronic wounds. Current research on therapies that modulate IL-6 is explored. Here, we consider IL-6's diverse impact on cutaneous wound healing.

Validation of a Monte Carlo simulation for Microbeam Radiation Therapy on the Imaging and Medical Beamline at the Australian Synchrotron.

Microbeam Radiation Therapy (MRT) is an emerging cancer treatment modality characterised by the use of high-intensity synchrotron-generated x-rays, spatially fractionated by a multi-slit collimator (MSC), to ablate target tumours. The implementation of an accurate treatment planning system, coupled with simulation tools that allow for independent verification of calculated dose distributions are required to ensure optimal treatment outcomes via reliable dose delivery. In this article we present data from the first Geant4 Monte Carlo radiation transport model of the Imaging and Medical Beamline at the Australian Synchrotron. We have developed the model for use as an independent verification tool for experiments in one of three MRT delivery rooms and therefore compare simulation results with equivalent experimental data. The normalised x-ray spectra produced by the Geant4 model and a previously validated analytical model, SPEC, showed very good agreement using wiggler magnetic field strengths of 2 and 3 T. However, the validity of absolute photon flux at the plane of the Phase Space File (PSF) for a fixed number of simulated electrons was unable to be established. This work shows a possible limitation of the G4SynchrotronRadiation process to model synchrotron radiation when using a variable magnetic field. To account for this limitation, experimentally derived normalisation factors for each wiggler field strength determined under reference conditions were implemented. Experimentally measured broadbeam and microbeam dose distributions within a Gammex RMI457 Solid Water® phantom were compared to simulated distributions generated by the Geant4 model. Simulated and measured broadbeam dose distributions agreed within 3% for all investigated configurations and measured depths. Agreement between the simulated and measured microbeam dose distributions agreed within 5% for all investigated configurations and measured depths.

Impact of magnetic fields on dose measurement with small ion chambers illustrated in high-resolution response maps.

PURPOSE: Dosimetry of ionizing radiation in the presence of strong magnetic fields is gaining increased relevance in light of advances for MRI-guided radiation therapy. While the impact of strong magnetic fields on the overall response of ionization chambers has been simulated and measured before, this work investigates the local impact of the magnetic field on dose response in an ion chamber. High-resolution 1D and 2D response maps have been created for two small clinical thimble ionization chambers, the PinPoint chambers 31006 and 31014 (Physikalisch Technische Werkstaetten Freiburg, Germany). METHODS: Working on the Imaging and Medical Beam Line of the Australian Synchrotron an intense kilovoltage radiation beam with very low divergence, collimated to 0.1 mm was used to scan the chambers by moving them on a 2D motion platform. Measured current and beam position were correlated to create the response maps. Small neodymium magnets were used to create a field of about 0.25 T. Chamber axis, magnetic field, and beam direction were perpendicular to each other. Measurements were performed with both orientations of the magnetic field as well as without it. Chamber biases of 5 and 250 V in both polarities were used. RESULTS: The local distribution of the response of small thimble-type ionization chambers was found to be impacted by a magnetic field. Depending on the orientation of the magnetic field, the chamber response near the stem was either enhanced or reduced with the response near the tip behaving the opposite way. Local changes were in the order of up to 40% compared to measurements without the magnetic field present. Bending of the central electrode was observed for the chamber with the steel electrode. The size of the volume of reduced collection near the guard electrode was impacted by the magnetic field. As the here investigated beam and field parameters differ from those of clinical systems, quantitatively different results would be expected for the latter. However, the gyroradii encountered here were similar to those of a 6-7 MV MRI linac with a 1.5 T magnet. CONCLUSIONS: Magnetic fields impact the performance of ionization chambers also on a local level. For practical measurements this might mean a change in the effective point of measurement, in addition to any global corrections. Further knowledge about the local response will help in selecting or constructing optimized chambers for use in magnetic fields.

Synchrotron microbeam radiotherapy evokes a different early tumor immunomodulatory response to conventional radiotherapy in EMT6.5 mammary tumors.

BACKGROUND: Synchrotron microbeam radiation therapy (MRT) is a new, evolving form of radiotherapy that has potential for clinical application. Several studies have shown in preclinical models that synchrotron MRT achieves equivalent tumor control to conventional radiotherapy (CRT) but with significantly reduced normal tissue damage. METHODS: To explore differences between these two modalities, we assessed the immune cell infiltrate into EMT6.5 mammary tumors after CRT and MRT. RESULTS: CRT induced marked increases in tumor-associated macrophages and neutrophils while there were no increases in these populations following MRT. In contrast, there were higher numbers of T cells in the MRT treated tumors. There were also increased levels of CCL2 by immunohistochemistry in tumors subjected to CRT, but not to MRT. Conversely, we found that MRT induced higher levels of pro-inflammatory genes in tumors than CRT. CONCLUSION: Our data are the first to demonstrate substantial differences in macrophage, neutrophil and T cell numbers in tumors following MRT versus CRT, providing support for the concept that MRT evokes a different immunomodulatory response in tumors compared to CRT.

A Functional Immune System Is Required for the Systemic Genotoxic Effects of Localized Irradiation.

PURPOSE: Nontargeted effects of ionizing radiation, by which unirradiated cells and tissues are also damaged, are a relatively new paradigm in radiobiology. We recently reported radiation-induced abscopal effects (RIAEs) in normal tissues; namely, DNA damage, apoptosis, and activation of the local and systemic immune responses in C57BL6/J mice after irradiation of a small region of the body. High-dose-rate, synchrotron-generated broad beam or multiplanar x-ray microbeam radiation therapy was used with various field sizes and doses. This study explores components of the immune system involved in the generation of these abscopal effects. METHODS AND MATERIALS: The following mice with various immune deficiencies were irradiated with the microbeam radiation therapy beam: (1) SCID/IL2γR-/- (NOD SCID gamma, NSG) mice, (2) wild-type C57BL6/J mice treated with an antibody-blocking macrophage colony-stimulating factor 1 receptor, which depletes and alters the function of macrophages, and (3) chemokine ligand 2/monocyte chemotactic protein 1 null mice. Complex DNA damage (ie, DNA double-strand breaks), oxidatively induced clustered DNA lesions, and apoptotic cells in tissues distant from the irradiation site were measured as RIAE endpoints and compared with those in wild-type C57BL6/J mice. RESULTS: Wild-type mice accumulated double-strand breaks, oxidatively induced clustered DNA lesions, and apoptosis, enforcing our RIAE model. However, these effects were completely or partially abrogated in mice with immune disruption, highlighting the pivotal role of the immune system in propagation of systemic genotoxic effects after localized irradiation. CONCLUSIONS: These results underline the importance of not only delineating the best strategies for tumor control but also mitigating systemic radiation toxicity.

Synchrotron radiation external beam rotational radiotherapy of breast cancer: proof of principle.

The principle of rotational summation of the absorbed dose for breast cancer treatment with orthovoltage X-ray beams was proposed by J. Boone in 2012. Here, use of X-ray synchrotron radiation for image guided external beam rotational radiotherapy treatment of breast cancer is proposed. Tumor irradiation occurs with the patient in the prone position hosted on a rotating bed, with her breast hanging from a hole in the bed, which rotates around a vertical axis passing through the tumor site. Horizontal collimation of the X-ray beam provides for whole breast or partial breast irradiation, while vertical translation of the bed and successive rotations allow for irradiation of the full tumor volume, with dose rates which permit also hypofractionated treatments. In this work, which follows a previous preliminary report, results are shown of a full series of measurements on polyethylene and acrylic cylindrical phantoms carried out at the Australian Synchrotron, confirmed by Geant4 Monte Carlo simulations, intended to demonstrate the proof of principle of the technique. Dose measurements were carried out with calibrated ion chambers, radiochromic films and thermoluminescence dosimeters. The photon energy investigated was 60 keV. Image guidance may occur with the transmitted beam for contrast-enhanced breast computed tomography. For a horizontal beam collimation of 1.5 cm and rotation around the central axis of a 14 cm-diameter polyethylene phantom, a periphery-to-center dose ratio of 14% was measured. The simulations showed that under the same conditions the dose ratio decreases with increasing photon energy down to 10% at 175 keV. These values are comparable with those achievable with conventional megavoltage radiotherapy of breast cancer with a medical linear accelerator. Dose painting was demonstrated with two off-center `cancer foci' with 1.3 Gy and 0.6 Gy target doses. The use of a radiosensitizing agent for dose enhancement is foreseen.

Experimental optimisation of the X-ray energy in microbeam radiation therapy.

Microbeam radiation therapy has demonstrated superior normal tissue sparing properties compared to broadbeam radiation fields. The ratio of the microbeam peak dose to the valley dose (PVDR), which is dependent on the X-ray energy/spectrum and geometry, should be maximised for an optimal therapeutic ratio. Simulation studies in the literature report the optimal energy for MRT based on the PVDR. However, most of these studies have considered different microbeam geometries to that at the Imaging and Medical Beamline (50 µm beam width with a spacing of 400 µm). We present the first fully experimental investigation of the energy dependence of PVDR and microbeam penumbra. Using monochromatic X-ray energies in the range 40-120 keV the PVDR was shown to increase with increasing energy up to 100 keV before plateauing. PVDRs measured for pink beams were consistently higher than those for monochromatic energies similar or equivalent to the average energy of the spectrum. The highest PVDR was found for a pink beam average energy of 124 keV. Conversely, the microbeam penumbra decreased with increasing energy before plateauing for energies above 90 keV. The effect of bone on the PVDR was investigated at energies 60, 95 and 120 keV. At depths greater than 20 mm beyond the bone/water interface there was almost no effect on the PVDR. In conclusion, the optimal energy range for MRT at IMBL is 90-120 keV, however when considering the IMBL flux at different energies, a spectrum with 95 keV weighted average energy was found to be the best compromise.

Spatial response of synthetic microDiamond and diode detectors measured with kilovoltage synchrotron radiation.

PURPOSE: To map the spatial response of four solid-state radiation detectors of types commonly used for radiotherapy dosimetry. METHODS: PTW model 60016 Diode P, 60017 Diode E, 60018 Diode SRS, and 60019 microDiamond detectors were radiographed using a high resolution conventional X-ray system. Their spatial response was then investigated using a 0.1 mm diameter beam of 95 keV average energy photons generated by a synchrotron. The detectors were scanned through the beam while their signal was recorded as a function of position, to map the response. These 2D response maps were created in both the end-on and side-on orientations. RESULTS: The results show the location and size of the active region. End-on, the active area was determined to be centrally located and within 0.2 mm of the manufacturer's specified diameter. The active areas of the 60016 Diode P, 60017 Diode E, 60018 Diode SRS detectors are uniform to within approximately 5%. The 60019 microDiamond showed local variations up to 30%. The extra-cameral signal in the microDiamond was calculated from the side-on scan to be approximately 8% of the signal from the active element. CONCLUSIONS: The spatial response of four solid-state detectors has been measured. The technique yielded information about the location and uniformity of the active area, and the extra-cameral signal, for the beam quality used.

Localized Synchrotron Irradiation of Mouse Skin Induces Persistent Systemic Genotoxic and Immune Responses.

The importance of nontargeted (systemic) effects of ionizing radiation is attracting increasing attention. Exploiting synchrotron radiation generated by the Imaging and Medical Beamline at the Australian Synchrotron, we studied radiation-induced nontargeted effects in C57BL/6 mice. Mice were locally irradiated with a synchrotron X-ray broad beam and a multiplanar microbeam radiotherapy beam. To assess the influence of the beam configurations and variations in peak dose and irradiated area in the response of normal tissues outside the irradiated field at 1 and 4 days after irradiation, we monitored oxidatively induced clustered DNA lesions (OCDL), DNA double-strand breaks (DSB), apoptosis, and the local and systemic immune responses. All radiation settings induced pronounced persistent systemic effects in mice, which resulted from even short exposures of a small irradiated area. OCDLs were elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, there was a trend for elevated apoptotic cell death under most irradiation conditions; however, DSBs were elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in plasma concentrations of macrophage-derived cytokine, eotaxin, IL10, TIMP1, VEGF, TGFß1, and TGFß2, along with changes in tissues in frequencies of macrophages, neutrophils, and T lymphocytes. Overall, our findings have implications for the planning of therapeutic and diagnostic radiation treatments to reduce the risk of radiation-related adverse systemic effects. Cancer Res; 77(22); 6389-99. ©2017 AACR.