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Radiotherapy has become an increasingly widespread modality for treating hepatocellular cancer (HCC); however, the development of radioresistance significantly limits its effectiveness and invariably leads to tumor recurrence. Cancer stem cell (CSC) theory offers a potential explanation for tumor relapse and radioresistance, but the underlying mechanism remains unknown. Herein we investigate the role of miRNA in molecular regulation of stemness and radioresistance in HCC. Two HCC radiation-resistant cell lines (Huh7-RR and SMMC-7721-RR) were established by selecting the radioresistant subpopulation from HCC cells via clonogenic survival assays. MiRNA Sequencing was used to identify potential radiosensitivity involved miRNA in HCC-RR cells. Xenograft tumor mouse model was established for in vivo study. CSC properties were assessed using sphere formation assay and side population (SP) cells analysis. We found that miR-34a-5p was significantly downregulated in HCC-RR cells. Overexpression of miR-34a-5p counteracts CSC properties and enhances radiosensitivity in HCC. Mechanistic investigation revealed that c-MYC is the direct target of miR-34a-5p. Overexpression of miR-34a-5p reversed c-MYC-induced radioresistance. Moreover, we found that the specific molecular mechanism was that c-MYC activated CHK1 and CHK2, which are two key DNA damage checkpoint kinases, and facilitated the DNA damage response to radiation. Repression of the miR-34a-5p-c-MYC-CHK1/CHK2 axis contributes to the acquisition of radioresistance in HCC cells. In summary, the miR-34a-5p-c-MYC-CHK1/CHK2 axis counteracts cancer stem cell-like properties and enhances radiosensitivity in hepatocellular cancer through repression of the DNA damage response.

Novel biodosimetry assays for use in preparedness and response to potential malicious attacks or nuclear accidents would ideally provide accurate dose reconstruction independent of the idiosyncrasies of a complex exposure to ionizing radiation. Complex exposures will consist of dose rates spanning the low dose rates (LDR) to very high-dose rates (VHDR) that need to be tested for assay validation. Here, we investigate how a range of relevant dose rates affect metabolomic dose reconstruction at potentially lethal radiation exposures (8 Gy in mice) from an initial blast or subsequent fallout exposures compared to zero or sublethal exposures (0 or 3 Gy in mice) in the first 2 days, which corresponds to an integral time individuals will reach medical facilities after a radiological emergency. Biofluids (urine and serum) were collected from both male and female 9–10-week-old C57BL/6 mice at 1 and 2 days postirradiation (total doses of 0, 3 or 8 Gy) after a VHDR of 7 Gy/s. Additionally, samples were collected after a 2-day exposure consisting of a declining dose rate (1 to 0.004 Gy/min) recapitulating the 7:10 rule-of-thumb time dependency of nuclear fallout. Overall similar perturbations were observed in both urine and serum metabolite concentrations irrespective of sex or dose rate, with the exception of xanthurenic acid in urine (female specific) and taurine in serum (VHDR specific). In urine, we developed identical multiplex metabolite panels (N6,N6,N6-trimethyllysine, carnitine, propionylcarnitine, hexosamine-valine-isoleucine, and taurine) that could identify individuals receiving potentially lethal levels of radiation from the zero or sublethal cohorts with excellent sensitivity and specificity, with creatine increasing model performance at day 1. In serum, individuals receiving a 3 or 8 Gy exposure could be identified from their pre-irradiation samples with excellent sensitivity and specificity, however, due to a lower dose response the 3 vs. 8 Gy groups could not be distinguished from each other. Together with previous results, these data indicate that dose-rate-independent small molecule fingerprints have potential in novel biodosimetry assays.

Prediction of cancer risk from space radiation exposure is critical to ensure spaceflight crewmembers are adequately informed of the risks they face when accepting assignments to ambitious long-duration exploratory missions. Although epidemiological studies have assessed the effects of exposure to terrestrial radiation, no robust epidemiological studies of humans exposed to space radiation exist to support estimates of the risk from space radiation exposure. Mouse data derived from recent irradiation experiments provides valuable information to successfully develop mouse-based excess risks models for assessing relative biological effectiveness for heavy ions that can provide information to scale unique space radiation exposures so that excess risks estimated for terrestrial radiation can be adjusted for space radiation risk assessment. Bayesian analyses were used to simulate linear slopes for excess risk models with several different effect modifiers for attained age and sex. Relative biological effectiveness values for all-solid cancer mortality were calculated from the ratio of the heavy-ion linear slope to the gamma linear slope using the full posterior distribution and resulted in values that were substantially lower than what is currently applied in risk assessment. These analyses provide an opportunity to improve characterization of parameters used in the current NASA Space Cancer Risk (NSCR) model and generate new hypotheses for future animal experiments using out-bred mouse populations.

This study conducted fundamental research to develop a more effective BNCT targeting cancer stem cells. We constructed plasmids that induced the overexpression of L-type amino acid transporter 1 (LAT1) tagged with tdTomato on the cytoplasmic membranes of CD133 expressing cancer cells. After transfection of the plasmids into a glioblastoma cell line (T98G), several clones overexpressing LAT1-tdTomato in the hypoxic microenvironment of the spheroids formed from each clone were obtained. Confocal laser microscopic observation confirmed that signals from LAT1-tdTomato overlapped with immunofluorescence signals from the second antibody binding to CD133 in the hypoxic microenvironment of the spheroids. As CD133-positive cells in the hypoxic microenvironment of T98G spheroids have cancer stem cell characteristics, LAT1 seems to be selectively overexpressed in cancer stem cell-like cells. An RI tracer method showed that cells overexpressing LAT1-tdTomato in the hypoxic microenvironment of spheroids incorporate 14C-BPA much more than cells that do not overexpress LAT1-tdTomato. Neutron radiation experiments showed a more significant regression in spheroids formed with clones than in spheroids formed with parental cells when spheroids were treated with 10BPA. These results suggest that BNCT combined with gene therapy targeting cancer stem cells is more effective in glioblastoma therapy.

Transdifferentiation of type II alveolar cells (AECII) is a major cause for radiation-induced lung fibrosis (RILF). Cell differentiation phenotype is determined by Lin28 (undifferentiated marker) and let-7 (differentiated marker) in a see-saw-pattern. Therefore, differentiation phenotype can be extrapolated based on Lin28/let-7 ratio. Lin28 is activated by β-catenin. To the best of our knowledge this study was the first to use the single primary AECII freshly isolated from irradiated lungs of fibrosis-resistant C3H/HeNHsd strain to further confirm RILF mechanism by comparing its differences in AECII phenotype status/state and cell differentiation regulators to fibrosis-prone C57BL/6j mice. Results showed that radiation pneumonitis and fibrotic lesions were seen in C3H/HeNHsd and C57BL/6j mouse strains, respectively. mRNAs of E-cadherin, EpCAM, HOPX and proSP-C (epithelial phenotype biomarkers) were significantly downregulated in single primary AECII isolated from irradiated lungs of both strains. Unlike C57BL/6j, α-SMA and Vimentin (mesenchymal phenotype biomarkers) were not upregulated in single AECII from irradiated C3H/HeNHsd. Profibrotic molecules, TGF-β1 mRNA was upregulated and β-catenin was significantly downregulated in AECII after irradiation (both P < 0.01). In contrast, transcriptions for GSK-3β, TGF-β1 and β-catenin were enhanced in isolated single AECII from irradiated C57BL/6j (P < 0.01–P < 0.001). The Lin28/let-7 ratios were much lower in single primary AECII from C3H/HeNHsd after irradiation vs. C57BL/6j. In conclusion, AECII from irradiated C3H/HeNHsd did not undergo epithelial-mesenchymal transition (EMT) and lower ratios of Lin28/let-7 contributed to AECII relatively higher differentiated status, leading to increased susceptibility to radiation stress and a failure in transdifferentiation in the absence of β-catenin. Reducing β-catenin expression and the ratios of Lin28/let-7 may be a promising strategy to prevent radiation fibrosis.

Following our previous report on the radiation dose-response for prostate cancer incidence rates in the Life Span Study (LSS) cohort of atomic bomb survivors, we re-evaluated the radiation-related risk adjusting for differences in baseline cancer incidence rates among three subsets of the LSS cohort defined by the timing of their first participation in biennial health examinations offered to the Adult Health Study (AHS) sub-cohort members and prostate-specific-antigen (PSA) testing status for AHS participants: 1. non-AHS participants, 2. AHS participants before receiving PSA test, and 3. AHS participants after receiving PSA test. We found a 2.9-fold increase in the baseline incidence rates among AHS participants after receiving PSA test. After adjusting for the PSA-testing-status effects on the baseline rates the estimated excess relative risk (ERR) per Gy was 0.54 (95% CI: 0.15, 1.05), which was almost identical to the previously reported unadjusted ERR estimate (0.57, 95% CI: 0.21, 1.00). The current results confirmed that, while the PSA testing among AHS participants increased the baseline incidence rates, it did not impact the radiation risk estimate, strengthening the previously reported dose-response relationship for prostate cancer incidence in the LSS. As the use of PSA tests continue in screening and medical settings, analyses of possible effects of PSA testing should be an important aspect of future epidemiological studies of the association between radiation exposure and prostate cancer.

There is increasing evidence that circulatory disease incidence and mortality is associated with radiation exposure. Wake Forest School of Medicine is home to a unique cohort of total-body irradiated macaques, some with evidence of vascular end-organ disease in the brain, kidney and heart. Because there is a link between high blood pressure and vascular disease in all these sites, we undertook a retrospective study to evaluate blood pressure and radiation in this cohort of animals. In this work, we utilized a cohort of nonhuman primates (rhesus macaques, Macaca mulatta) long-term survivors of high-dose total-body irradiation (1.1–8.5 Gy, N = 129) and controls (N = 37) to evaluate the effects of radiation on blood pressure and obesity. Subjects were between 3 and 22 years of age (median 9 years). Blood pressure (BP) was measured 1–14 years postirradiation (median 4 years). Subjects were sedated with a combination of ketamine HCl (15 mg/kg body weight, IM) and midazolam (0.1 mg/kg body weight, IM) and systolic, diastolic, and mean arterial pressures were measured using a high definition oscillometer. Obesity was defined by dual energy X-ray absorptiometry as a body fat percentage >35%. Statistical analysis of the collected data indicated significant increases in blood pressure with increasing age and obesity. However, radiation did not significantly alter blood pressure in irradiated animals relative to controls, radiation dose, or age of irradiation.

The discovery of X rays in the late 19th century heralded the beginning of a new age in medicine, and the advent of channeling the power of radiation to diagnose and treat human disease. Radiation has been leveraged in medicine in a multitude of ways and is a critical element of cancer care including screening, diagnosis, surveillance, and interventional treatments. Modern radiotherapy techniques include a multitude of methodologies utilizing both externally and internally delivered radiation from a variety of approaches. This review provides a comprehensive overview of contemporary radiotherapy methodologies, the field of radiopharmaceuticals and theranostics, effects of low dose radiation and highlights the phenomena of fear of exposure to radiation and its impact in modern medicine.

Inter-laboratory exercises are important tools within the European network for biological dosimetry and physical retrospective dosimetry (RENEB) to validate and improve the performance of member laboratories and to ensure an operational network with high quality standards for dose estimations in case of a large-scale radiological or nuclear event. In addition to the RENEB inter-laboratory comparison 2021, several inter-laboratory comparisons have been performed in the frame of RENEB for a number of assays in recent years. This publication gives an overview of RENEB inter-laboratory comparisons for biological dosimetry assays in the past and a final summary of the challenges and lessons learnt from the RENEB inter-laboratory comparison 2021. In addition, the dose estimates of all RENEB inter-laboratory comparisons since 2013 that have been conducted for the dicentric chromosome assay, the most established and applied assay, are compared and discussed.

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 ± 0.03 and 1.47 ± 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 ± 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 ± 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.

The Running the European Network of biological and retrospective dosimetry (RENEB) network of laboratories has a range of biological and physical dosimetry assays that can be deployed in the event of a radiation incident to provide exposure assessment. To maintain operational capability and provide training, RENEB runs regular inter-laboratory comparison (ILC) exercises. The RENEB ILC2021 was carried out with all the biological and physical dosimetry assays employed in the network. The focus of this paper is to evaluate the results from 6 laboratories that took part using the gamma-H2AX radiation-induced foci assay. For two laboratories this was their first RENEB ILC. Blood samples were homogenously exposed to 240 kVp X rays (1 Gy/min) to provide calibration data, (0–4 Gy), and a few weeks later three blind coded test samples, (0, 1.2 and 3.5 Gy) were prepared. All samples were allowed a 2 h repair time at 37°C before being transported, on ice packs, to the participating laboratories. On arrival, the samples were processed, scored either manually or automatically for gamma-H2AX foci and dose estimates for the 3 blind coded samples sent to the organizing laboratory. The temperature of samples during transit and the time taken to report the dose estimates were recorded. Subsequent examination of the data from each laboratory used the doses estimates to assign triage categories to the samples. After receipt of the samples, the quickest report of dose estimates was 4.6 h. Analysis of variance revealed that the laboratory carrying out the assay had a significant effect on the foci yield (P < 0.001) for the calibration data, but not on the dose estimates of the blind coded samples (P = 0.101). All laboratories correctly identified the unirradiated and irradiated samples, although the dose estimates for the latter tended to under-estimate the dose. Two participants seriously under-estimated the dose for the highly exposed sample, which resulted in the sample being placed in the lowest triage category not the highest. However, this under-estimation resulted from the samples not remaining cold during shipment, due to a delay in transit and was not related to the experience of the participating laboratory. Overall, the RENEB network laboratories have demonstrated it is possible to quickly identify a recent whole-body acute exposure using the gamma-H2AX assay within the conditions of the ILC. In addition, an ILC provides a useful training and harmonization exercise for laboratories.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semi-automated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

Low energy x-ray sources in the kilovoltage energy range have been shown to induce more cellular damage when compared to their megavoltage counterparts. However, low-energy x-ray sources are more susceptible to the effects of filtration on the beam spectrum. This work sought to characterize the biological effects of the Xoft Axxent(®) source, a low energy therapeutic x-ray source, both with and without the titanium vaginal applicator in place. It was hypothesized that there would be an increase in relative biological effectiveness (RBE) of the Axxent(®) source compared to (60)Co and that the source in the titanium vaginal applicator (SIA) would have decreased biological effects compared to the bare source (BS). This hypothesis was drawn from linear energy transfer (LET) simulations performed using the TOPAS Monte Carlo user code as well as reduction in dose rate of the SIA compared to the BS. A HeLa cell line was maintained and used to evaluate these effects. Clonogenic survival assays were performed to evaluate differences in the RBE between the BS and SIA using (60)Co as the reference beam quality. Neutral comet assay was used to assess induction of DNA strand damage by each beam to estimate differences in RBE. Quantification of mitotic errors was used to evaluate differences in chromosomal instability (CIN) induced by the three beam qualities. The BS was responsible for the greatest quantity of cell death due to a greater number of DNA double strand breaks (DSB) and CIN observed in the cells. The differences observed in the BS and SIA surviving fractions and RBE values were consistent with the 13% difference in LET as well as the factor of 3.5 reduction in dose rate of the SIA. Results from the comet and CIN assays were consistent with these results as well. The use of the titanium applicator results in a reduction in the biological effects observed with these sources, but still provides an advantage over megavoltage beam qualities.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0–1 Gy), moderately (1–2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semi-automatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

We studied the effects of neutrons, neutrons and γ rays, and γ rays exposures on the transcription spectrum in human peripheral blood of three healthy adult men. Samples were irradiated with 1.42 Gy 2.5-MeV neutrons, 0.71 Gy neutrons and 0.71 Gy 137Cs γ rays, and 1.42 Gy 137Cs γ rays. Transcriptome sequencing identified 56 differentially co-expressed genes and enriched 26 KEGG pathways. There are 97, 45 and 30 differentially expressed genes in neutron, neutron and γ ray combined treatment, and γ rays, respectively, and 21, 3 and 8 KEGG pathways with significant differences are enriched. Fluorescence quantitative polymerase chain reaction (qPCR) verified differential co-expression of AEN, BAX, DDB2, FDXR, and MDM2. Additionally, irradiation of AHH-1 human lymphocytes with a 252Cf neutron source at 0, 0.14, 0.35, and 0.71 Gy, fluorescence qPCR revealed a dose-response relationship for BAX, DDB2, and FDXR at dose ranges of 0–0.71 Gy, with R2 of 0.803, 0.999, and 0.999, respectively. Thus, neutrons can induce more differentially expressed genes and enrich more pathways. Combined treatment of neutrons and γ-rays may incorporate damage of both high and low LET, the genes activated by neutrons and γ rays combined are almost the combination of genes activated by neutron and γ rays combined treatment. BAX, DDB2 and FDXR are differentially expressed after irradiation by Deuterium-Deuterium (D-D) neutron source and 252Cf neutron source, so they are expected to be molecular targets of neutron damage.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8–10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

Gomes, N. P., Frederick, B., Jacobsen, J. R., Chapnick. D. and Su, T. T. A high throughput screen with a clonogenic endpoint to identify radiation modulators of cancer. Radiat. Res.Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter ‘clonogenic HTS’, exhibits excellent robustness (Z’ values >0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators of approved therapies.

This study aims to dosimetrically compare multi-leaf collimator (MLC)-based and cone-based 3D LATTICE radiotherapy (LRT) plans. Valley-peak ratios were evaluated using seven different 3D LATTICE designs. Target volumes of 8 cm and 12 cm were defined on the RANDO phantom. Valley-peak dose patterns were obtained by creating high-dose vertices in the target volumes. By changing the vertex diameter, vertices separation, and volume ratio, seven different LATTICE designs were generated. Treatment plans were implemented using CyberKnife and Varian RapidArc. Thermoluminescent dosimeter (TLD), EBT3 films, and electronic portal-imaging device (EPID) were employed for dosimetric treatment verification, and measured doses were compared to calculated doses. By changing the vertex diameter and vertices separation, the valley-peak ratio was exhibited little difference between the two systems. By changing the vertex diameter and volume ratio, the valley-peak ratio was observed nearly the same for the two systems. The film, TLD, and EPID dosimetry showed good agreement between the calculated and measured doses. Based on the results, we concluded that although smaller valley-peak ratios were obtained with cone-based plans, the dose-volume histograms were comparable in both systems. Also, when we evaluated the treatment duration, the MLC-based plans were more appropriate to apply the treatment in a single fraction.

The linear, non-threshold (LNT) hypothesis of cancer induction derived from studies of populations exposed to moderate-to-high acute radiation doses may not be indicative of cancer risks associated with lifetime radiation exposures less than 100 mSv. The objective of this study was to examine risks and dose-response patterns of lymphohematopoietic cancer (LHC) and its types associated with low radiation exposure while adjusting for possible confounding factors. A retrospective cohort of 437,937 U.S. nuclear shipyard workers (153,930 radiation and 284,007 non-radiation workers) was followed from 1957 to 2011, with 3,699 LHC deaths observed. The risk of LHC in radiation workers was initially compared to the risk in non-radiation workers. Time dependent accumulated radiation dose, lagged 2 years, was used in categorical and continuous dose analysis among radiation workers to examine the LHC risks and possible dose-response relationships based on Poisson regression models. These analyses controlled for sex, race, time dependent age, calendar time, socioeconomic status, solvent-related last job, and age at first hire. The median lifetime radiation dose for the radiation worker population was 0.82 mSv and the 95th percentile dose was 83.63 mSv. The study shows: 1. LHC mortality for radiation workers was significantly lower than non-radiation workers relative risk: 0.927; 95% confidence intervals (95% CI): 0.865, 0.992; P = 0.030]. Among LHC types, the risks for lymphoid leukemia and lymphomas in radiation workers were lower than the risk in non-radiation workers with statistical significance, while the risk for the rest of LHC types did not show any statistically significant difference. 2. In categorical dose analysis among radiation workers, sample size weighted linear trend of relative risk (RRs) for LHC and its types in five dose categories (>0– = 200 mSv) vs. 0 mSv were not statistically significant, although there was an elevation of RR for chronic myeloid leukemia only in the 50

Radiological and especially nuclear accidents and incidents pose a threat to populations. In such events, gene expression (GE) analysis of a set of 4 genes (FDXR, DDB2, POU2AF1, WNT3) is an emerging approach for early and high-throughput prediction of the later manifesting severity degrees of the hematological acute radiation syndrome (H-ARS). Validation of this gene set on radiation victims is difficult since these events are rare. However, chemotherapy (CTX) is widely used e.g., breast cancer patient treatment and pathomechanisms, as well as blood cell count changes are comparable among both exposure types. We wondered whether GE changes are similarly deregulated after CTX, which would be interpreted as a confirmation of our already identified gene set for H-ARS prediction after irradiation. We examined radiation-induced differential GE (DGE) of our gene set as a positive control using in vitro whole blood samples from ten healthy donors (6 females, 4 males, aged: 24-40 years). Blood was incubated in vitro for 8 h after X irradiation with 0 and 4 Gy (1 Gy/min). These data were compared with DGE measured in vivo in blood samples of 10 breast tumor CTX patients (10 females, aged: 39-71 years) before and 4 days after administration of cyclophosphamide and epirubicin. RNA was isolated, reverse transcribed and quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to assess DGE of FDXR, DDB2, POU2AF1 and WNT3 relative to the unexposed samples using TaqMan assays. After X irradiation, we found a significant upregulation (irrespective of sex) with mean fold changes of 21 (P0.001) and 7 (P0.001) for FDXR and DDB2 and a significant down-regulation with mean fold changes of 2.5 (P0.001) and 2 (P = 0.005) for POU2AF1 and WNT3, respectively. After CTX, a similar pattern was observed, although mean fold changes of up-regulated FDXR (6-fold, P0.001) and DDB2 (3-fold, P0.001) as well as down-regulated POU2AF1 (1.2-fold, P = 0.270) and WNT3 (1.3-fold, P = 0.069) appeared lower corresponding to less altered blood cell count changes observed after CTX compared to historic radiation exposure data. However, a subpopulation of CTX patients (n = 6) showed on average a significant down-regulation of POU2AF1 (1.8-fold, P = 0.04) and WNT3 (2.1-fold, P = 0.008). In summary, the pattern of up-regulated GE changes observed in all CTX patients and down-regulated GE changes observed in a subgroup of CTX patients appeared comparable with an already identified gene set predictive for the radiation-induced H-ARS. This underlines the significance of in vivo GE measurements in CTX patients, employed as a surrogate model to further validate already identified radiation-induced GE changes predictive for the H-ARS.

Radiation-induced gene expression (GE) changes can be used for early and high-throughput biodosimetry within the first three days postirradiation. However, is the method applicable in situations such as the Alexander Litvinenko case or the Goiania accident, where diagnosis occurred in a prefinal health stage? We aimed to characterize gene expression changes in a prefinal health stage of lethally irradiated male and female rhesus macaques. Peripheral blood was drawn pre-exposure and at the prefinal stage of male and female animals, which did not survive whole-body exposure with 700 cGy (LD66/60). RNA samples originated from a blinded randomized Good Laboratory Practice study comprising altogether 142 irradiated rhesus macaques of whom 60 animals and blood samples (15 samples for both time points and sexes) were used for this analysis. We evaluated GE on 34 genes widely used in biodosimetry and prediction of the hematological acute radiation syndrome severity (H-ARS) employing quantitative real-time polymerase chain reaction (qRT-PCR). These genes were run in duplicate and triplicate and altogether 96 measurements per time point and sex could be performed. In addition, 18S ribosomal RNA (rRNA) was measured to depict the ribosome/transcriptome status as well as for normalization purposes and 16S rRNA was evaluated as a surrogate for bacteremia. Mean differential gene expression (DGE) was calculated for each gene and sex including all replicate measurements and using pre-exposure samples as the reference. From 34 genes, altogether 27 genes appeared expressed. Pre-exposure samples revealed no signs of bacteremia and 18S rRNA GE was in the normal range in all 30 samples. Regarding prefinal samples, 46.7% and 40% of animals appeared infected in females and males, respectively, and for almost all males this was associated with out of normal range 18S rRNA values. The total number of detectable GE measurements was sixfold (females) and 15-fold (males) reduced in prefinal relative to pre-exposure samples and about tenfold lower in 80% of prefinal compared to pre-exposure samples (P0.0001). An overall 11-fold (median) downregulation in prefinal compared to pre-exposure samples was identified for most of the 27 genes and even FDXR appeared 4-14-fold downregulated in contrast to a pronounced up-regulation according to cited work. This pattern of overall downregulation of almost all genes and the rapid reduction of detectable genes at a prefinal stage was found in uninfected animals with normal range 18S rRNA as well. In conclusion, in a prefinal stage after lethal radiation exposure, the ribosome/transcriptome status remains present (based on normal range 18S rRNA values) in 60-67% of animals, but the whole transcriptome activity in general appears silenced and cannot be used for biodosimetry purposes, but probably as an indicator for an emerging prefinal health stage.