The contribution of radiation oncology to the future of cancer treatment depends significantly on our continued clinical progress and future research advancements. Such progress relies on multidisciplinary collaboration among radiation oncologists, medical physicists and radiobiologists. Cultivating collaborative educational and research opportunities among these three disciplines and further investing in the infrastructure used to train both clinicians and researchers will therefore help us improve the future of cancer care. This article evaluates the success of a short-term educational environment to foster multidisciplinary collaboration. The NIH-funded educational course developed at Wayne State University, called “Integration of Biology and Physics into Radiation Oncology” (IBPRO), was designed to facilitate the engagement of radiation oncologists, medical physicists and radiobiologists in activities that enhance collaborative investigation. Having now been delivered to nearly 200 participants over the past four years, the relative success of IBPRO in fostering productive interdisciplinary collaboration and producing tangible research outcomes can be evaluated. The 140 IBPRO participants from the first three years were surveyed to quantify the effectiveness of the course. In total, 62 respondents reported developing 23 institutional protocols, submitting more than 25 research grants (nine of which have been funded thus far), and publishing more than 30 research manuscripts attributable to participation in IBPRO. Nearly one-half (45%) of respondents reported generating at least one of these research metrics attributable to participation in IBPRO and these participants reported an average of over four such quantitative research metrics per respondent. This represents a very substantial contribution to radiation oncology research by a relatively small number of researchers within a relatively short time. Nearly one-half of respondents reported ongoing collaborative working relationships generated by IBPRO. In addition, approximately one-half of respondents stated that specific information presented at IBPRO changed the way they practice, and over 80% of respondents practicing in a clinical setting stated that, since participation in IBPRO, they have approached clinical dilemmas more collaboratively. We believe that educational opportunities such as IBPRO can have a significant impact on interdisciplinary collaborative research. In addition, such interventions have the ability to effect significant clinical change. Both of these should have a positive impact on future advancements in radiation oncology and affect the future contribution of radiation oncology to the treatment of cancer.

Vascular injury after radiation exposure contributes to multiple types of tissue injury through a cascade of events. Some of the earliest consequences of radiation damage include increased vascular permeability and promotion of inflammation, which is partially manifested by increased leukocyte-endothelial (L/E) interactions. We describe herein a novel intravital imaging method to evaluate L/E interactions, as a function of shear stress, and vascular permeability at multiple time points after local irradiation to the ear. This model permitted analysis of quiescent vasculature that was not perturbed by any surgical manipulation prior to imaging. To evaluate the effects of radiation on vascular integrity, fluorescent dextran was injected intravenously and its extravasation in the extravascular space surrounding the ear vasculature was measured at days 3 and 7 after 6 Gy irradiation. The vascular permeability rate increased approximately twofold at both days 3 and 7 postirradiation (P < 0.05). Leukocyte rolling, which is indicative of L/E interactions, was significantly increased in mice at 24 h postirradiation compared to that of nonirradiated mice. To assess our model, as a means for assessing vascular radioprotectants, we treated additional cohorts of mice with a thrombopoietin mimetic, TPOm (RWJ-800088). In addition to stimulating platelet formation, thrombopoietin can protect vasculature after several forms of injury. Thus, we hypothesized that TPOm would reduce vascular permeability and L/E adhesion after localized irradiation to the ear vasculature of mice. If TPOm reduced these consequences of radiation, it would validate the utility of our intravital imaging method. TPOm reduced radiation-induced vascular leakage to control levels at day 7. Furthermore, L/E cell interactions were also reduced in irradiated mice treated with TPOm, compared with mice receiving irradiation alone, particularly at high shear stress (P = 0.03, Kruskal-Wallis). We conclude that the ear model is useful for monitoring quiescent normal tissue vascular injury after radiation exposure. Furthermore, the application of TPOm, for preventing early inflammatory response created by damage to vascular endothelium, suggests that this drug may prove useful in reducing toxicities from radiotherapy, which damage microvasculature that critically important to tissue function.

Circulating tumor DNA (ctDNA) analysis has been shown to aid in both the detection of cancer and evaluation of somatic mutations in tumors. CtDNA concentration in plasma increases in proportion to tumor volume and/or metabolic activity and growth; however, this principle has yet to be applied to cell culture. We hypothesized that cell line-specific cell-free DNA (cfDNA) can be used to measure cell viability and cell survival in cell culture. Clonogenic assays on non-small cell lung cancer (NSCLC) cell lines H322, A549 and H322 were exposed to radiation doses of 0, 4 and 8 Gy. Prior to colony fixation and counting, cfDNA was extracted and quantified from cell culture media. The correlation between cell line-specific cfDNA and number of colonies grown on culture plates was examined. An H1299:A549 coculture model was used to evaluate the differential release of cell line-specific cfDNA. The results of this work indicate a strong correlation between CfDNA quantification from cell culture media and clonogenic survival at all radiation doses and in all cell lines tested (R² range = 0.77–0.99). Cell survival curves derived from cfDNA were virtually indistinguishable from matched traditional clonogenic survival data (P > 0.05; no significant difference exists between clonogenic curves). CfDNA quantification also accurately estimates colony count in a two-cell-line coculture model. In conclusion, cell-free DNA quantification from cell culture media can be used to measure cell survival, and appears suitable for development in a high-throughput clonogenic assay and radiosensitizer screening platform.

A hybrid of radiotherapy and photodynamic therapy (PDT) has been proposed in previously reported studies. This approach utilizes scintillating nanoparticles to transfer energy to attached photosensitizers, thus generating singlet oxygen for local killing of malignant cells. Its effectiveness strongly depends upon the scintillation yield of the nanoparticles. Using a liquid scintillator as a reference standard, we estimated the scintillation yield of Ce_0.1La_0.9F₃/LaF₃ core/shell nanoparticles at 28.9 mg/ml in water to be 350 photons/MeV under orthovoltage X-ray irradiation. The subsequent singlet oxygen production for a 60 Gy cumulative dose to cells was estimated to be four orders of magnitude lower than the “Niedre killing dose,” used as a target value for effective cell killing. Without significant improvements in the radioluminescence properties of the nanoparticles, this approach to “deep PDT” is likely to be ineffective. Additional considerations and alternatives to singlet oxygen are discussed.

3′-Deoxy-3-[¹⁸F]fluorothymidine, or [¹⁸F]FLT, is a positron emission tomography (PET) tracer used in clinical studies for noninvasive assessment of proliferation activity in several types of cancer. Although the use of this PET tracer is expanding, to date, few studies concerning its dosimetry have been published. In this work, new [¹⁸F]FLT dosimetry estimates are determined for human and mice using Monte Carlo simulations. Modern voxelized male and female phantoms and [¹⁸F]FLT biokinetic data, both published by the ICRP, were used for simulations of human cases. For most human organs/tissues the absorbed doses were higher than those reported in ICRP Publication 128. An effective dose of 1.70E-02 mSv/MBq to the whole body was determined, which is 13.5% higher than the ICRP reference value. These new human dosimetry estimates obtained using more realistic human phantoms represent an advance in the knowledge of [¹⁸F]FLT dosimetry. In addition, mice biokinetic data were obtained experimentally. These data and a previously developed voxelized mouse phantom were used for simulations of animal cases. Concerning animal dosimetry, absorbed doses for organs/tissues ranged from 4.47 ± 0.75 to 155.74 ± 59.36 mGy/MBq. The obtained set of organ/tissue radiation doses for healthy Swiss mice is a useful tool for application in animal experiment design.

There is concern that degradation of vision as a result of space flight may compromise both mission goals and long-term quality of life after space travel. The visual disturbances may be due to a combination of intracerebral pressure changes and exposure to ionizing radiation. The retina and the retinal vasculature play important roles in vision, yet have not been studied extensively in relationship to space travel and space radiation. The goal of the current study was to characterize oxidative damage and apoptosis in retinal endothelial cells after whole-body gamma-ray, proton and oxygen (¹⁶O) ion radiation exposure at 0.1 to 1 Gy. Six-month-old male C57Bl/6J mice were whole-body irradiated with 600 MeV/n ¹⁶O ions (0, 0.1, 0.25, 1 Gy), solar particle event (SPE)-like protons (0, 0.1, 0.25, 0.5 Gy) or ⁶⁰Co gamma rays (0, 0.1, 0.25, 0.5 Gy). Eyes were isolated for examining endothelial nitric oxide synthase (eNOS) expression and characterization of apoptosis in retina and retinal endothelial cells at two weeks postirradiation. The expression of eNOS was significantly increased in the retina after proton and ¹⁶O ion exposure. ¹⁶O ions induced over twofold increase in eNOS expression compared to proton exposure at two weeks postirradiation (P < 0.05). TUNEL assays showed dose-dependent increases in apoptosis in the retina after irradiation. Low doses of ¹⁶O ions elicited apoptosis in the mouse retinal endothelial cells with the most robust changes observed after 0.1 Gy irradiation (P < 0.05) compared to controls. Data also showed that ¹⁶O ions induced a higher frequency of apoptosis in retinal endothelial cells compared to protons (P < 0.05). In summary, our study revealed that exposure to low-dose ionizing radiation induced oxidative damage and apoptosis in the retina. Significant changes in retinal endothelial cells occur at doses as low as 0.1 Gy. There were significant differences in the responses of endothelial cells among the radiation types examined here.

Aside from the generally accepted potential to cause DNA damage, it is becoming increasingly recognized that ionizing radiation has the capability to target the cellular epigenome. Epigenetics unifies the chemical marks and molecules that collectively facilitate the proper reading of genetic material. Among the epigenetic mechanisms of regulation, methylation of DNA is known to be the key player in the postirradiation response by controlling the expression of genetic information and activity of transposable elements. Radiation-induced alterations to DNA methylation may lead to cellular epigenetic reprogramming that, in turn, can substantially compromise the genomic integrity and has been proposed as one of the mechanisms of radiation-induced carcinogenesis. DNA methylation is strongly dependent on the one-carbon metabolism. This metabolic pathway is central to the support of DNA methylation by means of providing the donor of methyl groups, as well as for the synthesis of amino acids. To better understand the mechanisms of radiation-induced health effects, we study how exposure to radiation affects DNA methylation and one-carbon metabolism. Also, a tight interaction that exists between DNA methylation and one-carbon metabolism allows us to simultaneously manipulate both cellular epigenetic and metabolic profiles to modulate the normal and cancerous tissue response to radiotherapy.

There is a current interest in the development of biodosimetric methods for rapidly assessing radiation exposure in the wake of a large-scale radiological event. This work was initially focused on determining the exposure dose to an individual using biological indicators. Gene expression signatures show promise for biodosimetric application, but little is known about how these signatures might translate for the assessment of radiological injury in radiosensitive individuals, who comprise a significant proportion of the general population, and who would likely require treatment after exposure to lower doses. Using Parp1^–/– mice as a model radiation-sensitive genotype, we have investigated the effect of this DNA repair deficiency on the gene expression response to radiation. Although Parp1 is known to play general roles in regulating transcription, the pattern of gene expression changes observed in Parp1^–/– mice 24 h postirradiation to a LD_50/30 was remarkably similar to that in wild-type mice after exposure to LD_50/30. Similar levels of activation of both the p53 and NFκB radiation response pathways were indicated in both strains. In contrast, exposure of wild-type mice to a sublethal dose that was equal to the Parp1^–/– LD_50/30 resulted in a lower magnitude gene expression response. Thus, Parp1^–/– mice displayed a heightened gene expression response to radiation, which was more similar to the wild-type response to an equitoxic dose than to an equal absorbed dose. Gene expression classifiers trained on the wild-type data correctly identified all wild-type samples as unexposed, exposed to a sublethal dose or exposed to an LD_50/30. All unexposed samples from Parp1^–/– mice were also correctly classified with the same gene set, and 80% of irradiated Parp1^–/– samples were identified as exposed to an LD_50/30. The results of this study suggest that, at least for some pathways that may influence radiosensitivity in humans, specific gene expression signatures have the potential to accurately detect the extent of radiological injury, rather than serving only as a surrogate of physical radiation dose.

Stereotactic body radiation therapy (SBRT) is associated with an increased risk of vertebral compression fracture. While bone is typically considered radiation resistant, fractures frequently occur within the first year of SBRT. The goal of this work was to determine if rapid deterioration of bone occurs in vertebrae after irradiation. Sixteen male rhesus macaque non-human primates (NHPs) were analyzed after whole-chest irradiation to a midplane dose of 10 Gy. Ages at the time of exposure varied from 45–134 months. Computed tomography (CT) scans were taken 2 months prior to irradiation and 2, 4, 6 and 8 months postirradiation for all animals. Bone mineral density (BMD) and cortical thickness were calculated longitudinally for thoracic (T) 9, lumbar (L) 2 and L4 vertebral bodies; gross morphology and histopathology were assessed per vertebra. Greater mortality (related to pulmonary toxicity) was noted in NHPs <50 months at time of exposure versus NHPs >50 months (P = 0.03). Animals older than 50 months at time of exposure lost cortical thickness in T9 by 2 months postirradiation (P = 0.0009), which persisted to 8 months. In contrast, no loss of cortical thickness was observed in vertebrae out-of-field (L2 and L4). Loss of BMD was observed by 4 months postirradiation for T9, and 6 months postirradiation for L2 and L4 (P < 0.01). For NHPs younger than 50 months at time of exposure, both cortical thickness and BMD decreased in T9, L2 and L4 by 2 months postirradiation (P < 0.05). Regions that exhibited the greatest degree of cortical thinning as determined from CT scans also exhibited increased porosity histologically. Rapid loss of cortical thickness was observed after high-dose chest irradiation in NHPs. Younger age at time of exposure was associated with increased pneumonitis-related mortality, as well as greater loss of both BMD and cortical thickness at both in- and out-of-field vertebrae. Older NHPs exhibited rapid loss of BMD and cortical thickness from in-field vertebrae, but only loss of BMD in out-of-field vertebrae. Bone is sensitive to high-dose radiation, and rapid loss of bone structure and density increases the risk of fractures.

Hypoxia in tumors has many well-characterized effects that are known to prevent optimal cancer treatment. Despite the existence of a large number of assays that have supported hypoxia as an important diagnostic, there is no routine clinical assay in use, and anti-hypoxia therapies have often not included parallel hypoxia measurements. Even with a functioning hypoxia assay, it is difficult to match the oxygen dependence of treatment resistance to that of the assay, and this mismatch can vary substantially from assay to assay and even from tumor to tumor [e.g., caused by endogenous variations in non-protein sulfhydryls (NPSH)]. An underlying concern is the current inability to measure the three types of hypoxia; in particular, cycling hypoxia can affect all aspects of detection and treatment strategy. Here we present data that help validate a new two-component hypoxia assay recently suggested by our laboratory. This assay incorporates the long-term bioreduction of the 2-nitroimidazole, EF5, and the short-term production of γ-H2AX (e.g., time of ionizing radiation exposure). The former can be calibrated to provide the average tissue pO₂ over the EF5 exposure time while the latter provides the combined sum of microenvironmental radiation response modifiers (e.g., oxygen and NPSH) at the time of irradiation. Importantly, formation of γ-H2AX is not dependent on blood flow, while EF5 binding is only minimally so, due to the rapid and extensive diffusion characteristics of lipophilic compounds. While both individual assays have their limitations, which are addressed in this article, their combination can dissect the type of hypoxia present. In particular, a mismatch between the two assays can directly detect cycling hypoxia in a therapeutically relevant manner. Preliminary use of this two-component assay in small PC3 tumors showed essentially no binding of EF5. Similarly, there were no tumor regions (for uniform irradiation with 12 Gy) with the low levels of γ-H2AX expected for a condition of cycling hypoxia. Thus, both assays were consistent with an essentially aerobic, radiation-responsive tumor. In a larger PC3 tumor, all regions of high EF5 binding had low levels of γ-H2AX.

Chromosome rearrangements are large-scale structural variants that are recognized drivers of oncogenic events in cancers of all types. Cytogenetics allows for their rapid, genome-wide detection, but does not provide gene-level resolution. Massively parallel sequencing (MPS) promises DNA sequence-level characterization of the specific breakpoints involved, but is strongly influenced by bioinformatics filters that affect detection efficiency. We sought to characterize the breakpoint junctions of chromosomal translocations and inversions in the clonal derivatives of human cells exposed to ionizing radiation. Here, we describe the first successful use of DNA paired-end analysis to locate and sequence across the breakpoint junctions of a radiation-induced reciprocal translocation. The analyses employed, with varying degrees of success, several well-known bioinformatics algorithms, a task made difficult by the involvement of repetitive DNA sequences. As for underlying mechanisms, the results of Sanger sequencing suggested that the translocation in question was likely formed via microhomology-mediated non-homologous end joining (mmNHEJ). To our knowledge, this represents the first use of MPS to characterize the breakpoint junctions of a radiation-induced chromosomal translocation in human cells. Curiously, these same approaches were unsuccessful when applied to the analysis of inversions previously identified by directional genomic hybridization (dGH). We conclude that molecular cytogenetics continues to provide critical guidance for structural variant discovery, validation and in “tuning” analysis filters to enable robust breakpoint identification at the base pair level.