A workshop entitled “Radiation-Induced Fibrosis: Mechanisms and Opportunities to Mitigate” (held in Rockville, MD, September 19, 2016) was organized by the Radiation Research Program and Radiation Oncology Branch of the Center for Cancer Research (CCR) of the National Cancer Institute (NCI), to identify critical research areas and directions that will advance the understanding of radiation-induced fibrosis (RIF) and accelerate the development of strategies to mitigate or treat it. Experts in radiation biology, radiation oncology and related fields met to identify and prioritize the key areas for future research and clinical translation. The consensus was that several known and newly identified targets can prevent or mitigate RIF in pre-clinical models. Further, basic and translational research and focused clinical trials are needed to identify optimal agents and strategies for therapeutic use. It was felt that optimally designed preclinical models are needed to better study biomarkers that predict for development of RIF, as well as to understand when effective therapies need to be initiated in relationship to manifestation of injury. Integrating appropriate endpoints and defining efficacy in clinical trials testing treatment of RIF were felt to be critical to demonstrating efficacy. The objective of this meeting report is to (a) highlight the significance of RIF in a global context, (b) summarize recent advances in our understanding of mechanisms of RIF, (c) discuss opportunities for pharmacological mitigation, intervention and modulation of specific molecular pathways, (d) consider the design of optimal clinical trials for mitigation and treatment and (e) outline key regulatory nonprescriptive frameworks for approval.

The increased threat of terrorism across the globe has raised fears that certain groups will acquire and use radioactive materials to inflict maximum damage. In the event that an improvised nuclear device (IND) is detonated, a potentially large population of victims will require assessment for radiation exposure. While photons will contribute to a major portion of the dose, neutrons may be responsible for the severity of the biologic effects and cellular responses. We investigated differences in response between these two radiation types by using metabolomics and lipidomics to identify biomarkers in urine and blood of wild-type C57BL/6 male mice. Identification of metabolites was based on a 1 Gy dose of radiation. Compared to X rays, a neutron spectrum similar to that encountered in Hiroshima at 1–1.5 km from the epicenter induced a severe metabolic dysregulation, with perturbations in amino acid metabolism and fatty acid β-oxidation being the predominant ones. Urinary metabolites were able to discriminate between neutron and X rays on day 1 as well as day 7 postirradiation, while serum markers showed such discrimination only on day 1. Free fatty acids from omega-6 and omega-3 pathways were also decreased with 1 Gy of neutrons, implicating cell membrane dysfunction and impaired phospholipid metabolism, which should otherwise lead to release of those molecules in circulation. While a precise relative biological effectiveness value could not be calculated from this study, the results are consistent with other published studies showing higher levels of damage from neutrons, demonstrated here by increased metabolic dysregulation. Metabolomics can therefore aid in identifying global perturbations in blood and urine, and effectively distinguishing between neutron and photon exposures.

Exposure of the lung to ionizing radiation that occurs in radiotherapy, as well as after accidental or intentional mass casualty incident can result in pulmonary fibrosis, which has few treatment options. Pulmonary fibrosis is characterized by an accumulation of extracellular matrix proteins that create scar tissue. Although the mechanisms leading to radiation-induced pulmonary fibrosis remain poorly understood, one frequent observation is the activation of the profibrotic cytokine transforming growth factor-beta (TGF-β). Our laboratory has shown that the metabolite lactate activates latent TGF-β by a reduction in extracellular pH. We recently demonstrated that lactate dehydrogenase-A (LDHA), the enzyme that produces lactate, is upregulated in patients with radiation-induced pulmonary fibrosis. Furthermore, genetic silencing of LDHA or pharmacologic inhibition using the LDHA inhibitor gossypol prevented radiation-induced extracellular matrix secretion in vitro through inhibition of TGF-β activation. In the current study, we hypothesized that LDHA inhibition in vivo prevents radiation-induced pulmonary fibrosis. To test this hypothesis, C57BL/6 mice received 5 Gy total-body irradiation plus 10 Gy thoracic irradiation from a ¹³⁷Cs source to induce pulmonary fibrosis. Starting at 4 weeks postirradiation, mice were treated with 5 mg/kg of the LDHA inhibitor gossypol or vehicle daily until sacrifice at 26 weeks postirradiation. Exposure to radiation resulted in pulmonary fibrosis, characterized by an increase in collagen content, fibrosis area, extracellular matrix gene expression and TGF-β activation. Irradiated mice treated with gossypol had significantly reduced fibrosis outcomes, including reduced collagen content in the lungs, reduced expression of active TGF-β, LDHA and the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). These findings suggest that inhibition of LDHA protects against radiation-induced pulmonary fibrosis, and may be a novel therapeutic strategy for radiation-induced pulmonary fibrosis.

The effects of ionizing radiation to human health are of great concern in the field of space exploration and for patients considering radiotherapy. However, to date, the effect of high-dose radiation on metabolism in the liver has not been clearly defined. In this study, ¹H nuclear magnetic resonance (NMR)-based metabolomics combined with multivariate data analysis was applied to study the changes of metabolism in the liver of C57BL/6 mouse after whole-body gamma (3.0 and 7.8 Gy) or proton (3.0 Gy) irradiation. Principal component analysis (PCA) and orthogonal projection to latent structures analysis (OPLS) were used for classification and identification of potential biomarkers associated with exposure to gamma and proton radiation. The results show that the radiation exposed groups can be well separated from the control group. Where the same dose was received, the proton exposed group was nevertheless well separated from the gamma-exposed group, indicating that different radiation sources induce different alterations in the metabolic profile. Common among all high-dose gamma and proton exposed groups were the statistically decreased concentrations of choline, O-phosphocholine and trimethylamine N-oxide, while the concentrations of glutamine, glutathione, malate, creatinine, phosphate, betaine and 4-hydroxyphenylacetate were statistically and significantly elevated. Since these altered metabolites are associated with multiple biological pathways, the results suggest that radiation induces abnormality in multiple biological pathways. In particular, metabolites such as 4-hydroxyphenylacetate, betaine, glutamine, choline and trimethylamine N-oxide may be prediagnostic biomarkers candidates for ionizing exposure of the liver.

The abscopal effect is the response to radiation at sites that are distant from the irradiated site of an organism, and it is thought to play a role in bone marrow (BM) recovery by initiating responses in the unirradiated bone marrow. Understanding the mechanism of this effect has applications in treating BM failure (BMF) and BM transplantation (BMT), and improving survival of nuclear disaster victims. Here, we investigated the use of multimodality imaging as a translational tool to longitudinally assess bone marrow recovery. We used positron emission tomography/computed tomography (PET/CT), magnetic resonance imaging (MRI) and optical imaging to quantify bone marrow activity, vascular response and marrow repopulation in fully and partially irradiated rodent models. We further measured the effects of radiation on serum cytokine levels, hematopoietic cell counts and histology. PET/CT imaging revealed a radiation-induced increase in proliferation in the shielded bone marrow (SBM) compared to exposed bone marrow (EBM) and sham controls. T₂-weighted MRI showed radiation-induced hemorrhaging in the EBM and unirradiated SBM. In the EBM and SBM groups, we found alterations in serum cytokine and hormone levels and in hematopoietic cell population proportions, and histological evidence of osteoblast activation at the bone marrow interface. Importantly, we generated a BMT mouse model using fluorescent-labeled bone marrow donor cells and performed fluorescent imaging to reveal the migration of bone marrow cells from shielded to radioablated sites. Our study validates the use of multimodality imaging to monitor bone marrow recovery and provides evidence for the abscopal response in promoting bone marrow recovery after irradiation.

One of the most concerning side effects of exposure to radiation are the carcinogenic risks. To reduce the negative effects of radiation, both cytoprotective and radioprotective agents have been developed. However, little is known regarding their potential for suppressing carcinogenesis. Andrographis paniculata, a plant, with multiple medicinal uses that is commonly used in traditional medicine, has three major constituents known to have cellular antioxidant activity: andrographolide (AP1); 14-deoxy-11,12-didehydroandrographolide (AP3); and neoandrographolide (AP4). In our study, we tested these elements for their radioprotective properties as well as their anti-neoplastic effects on transformation using the BALB/3T3 cell model. All three compounds were able to reduce radiation-induced DNA damage. However, AP4 appeared to have superior radioprotective properties compared to the other two compounds, presumably by protecting mitochondrial function. The compound was able to suppress radiation-induced cellular transformation through inhibition of STAT3. Treatment with AP4 also reduced expressions of MMP-2 and MMP-9. These results suggest that AP4 could be further studied and developed into an anti-transformation/carcinogenic drug as well as a radioprotective agent.

Saliva, a biological fluid, is a promising candidate for novel approaches to prognosis, clinical diagnosis, monitoring and management of patients with both oral and systemic diseases. However, to date, saliva has not been widely investigated as a biomarker for radiation exposure. Since white blood cells are also present in saliva, it should theoretically be possible to investigate the transcriptional biomarkers of radiation exposure classically studied in whole blood. Therefore, we collected whole blood and saliva samples from eight head and neck cancer patients before the start of radiation treatment, at mid-treatment and after treatment. We then used a panel of five genes: BAX, BBC3, CDKN1A, DDB2 and MDM2, designated for assessing radiation dose in whole blood to evaluate gene expression changes that can occur during radiotherapy. The results revealed that the expression of the five genes did not change in whole blood. However, in saliva, CDKN1A and DDB2 were significantly overexpressed at the end, compared to the start, of radiotherapy, and MDM2 was significantly underexpressed between mid-treatment and at the end of treatment. Interestingly, CDKN1A and DDB2 expressions also showed an increasing monotonic relationship with total radiation dose received during radiotherapy. To our knowledge, these results show for the first time the ability to detect gene expression changes in saliva after head and neck cancer radiotherapy, and pave the way for further promising studies validating saliva as a minimally invasive means of biofluid collection to directly measure radiation dose escalation during treatment.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

Normal tissue damage after head and neck radiotherapy involves a continuum of pathologic events to the mucosa, tongue and salivary glands. We examined the radioprotective effects of MnBuOE, a redox-active manganese porphyrin, at three stages of normal tissue damage: immediate (leukocyte endothelial cell [L/E] interactions), early (mucositis) and late (xerostomia and fibrosis) after treatment. In this study, mice received 0 or 9 Gy irradiation to the oral cavity and salivary glands ± MnBuOE treatment. Changes in leukocyte-endothelial cell interactions were measured 24 h postirradiation. At 11 days postirradiation, mucositis was assessed with a cathepsin-sensitive near-infrared optical probe. Stimulated saliva production was quantified at 11 weeks postirradiation. Finally, histological analyses were conducted to assess the extent of long-term effects in salivary glands at 12 weeks postirradiation. MnBuOE reduced oral mucositis, xerostomia and salivary gland fibrosis after irradiation. Additionally, although we have previously shown that MnBuOE does not interfere with tumor control at high doses when administered with radiation alone, most head and neck cancer patients will be treated with the combinations of radiotherapy and cisplatin. Therefore, we also evaluated whether MnBuOE would protect tumors against radiation and cisplatin using tumor growth delay as an endpoint. Using a range of radiation doses, we saw no evidence that MnBuOE protected tumors from radiation and cisplatin. We conclude that MnBuOE radioprotects normal tissue at both early and late time points, without compromising anti-tumor effects of radiation and cisplatin.

While radiotherapy continues to be a major cancer treatment option, its dose-limiting side effects, such as pulmonary fibrosis, severely impair the quality of life in these patients. In this study, we evaluated the radioprotective effects of metformin, a commonly used biguanide antidiabetic medication, in a murine model of pulmonary damage. Sprague Dawley^® rats received whole lung 20 Gy irradiation with or without metformin treatment. Computed tomography (CT) was performed and Hounsfield units (HU) were determined during the observation period. Histological analysis and evaluation of fibrosis/inflammatory markers by Western blot were performed at 12 weeks postirradiation. CCK-8 and colony formation assays were used to explore the effects of metformin on non-small cell lung cancer cells A549 and H460. Results of this study showed that metformin reduced radiological and histological signs of fibrosis, inflammatory infiltration, alterations to alveolar structures and radiation-induced HU lung density. In addition, metformin was found to significantly decrease collagen 1a and TGF-β expression and inhibit p-Smad2 and p-Smad3 expression compared to that of the irradiated group alone. Moreover, metformin reduced A549 and H460 cell growth and clonogenic survival. In conclusion, metformin exerted a protective effect on normal tissues from radiation-induced pulmonary injury, and shows promise as a radioprotective agent in the treatment of lung cancer.

The quantitative analysis of foci plays an important role in various cell biological methods. In the fields of radiation biology and experimental oncology, the effect of ionizing radiation, chemotherapy or molecularly targeted drugs on DNA damage induction and repair is frequently performed by the analysis of protein clusters or phosphorylated proteins recruited to so called repair foci at DNA damage sites, involving for example γ-H2A.X, 53BP1 or RAD51. We recently developed “The Focinator” as a reliable and fast tool for automated quantitative and qualitative analysis of nuclei and DNA damage foci. The refined software is now even more user-friendly due to a graphical interface and further features. Thus, we included an R-script-based mode for automated image opening, file naming, progress monitoring and an error report. Consequently, the evaluation no longer required the attendance of the operator after initial parameter definition. Moreover, the Focinator v2-0 is now able to perform multi-channel analysis of four channels and evaluation of protein–protein colocalization by comparison of up to three foci channels. This enables for example the quantification of foci in cells of a specific cell cycle phase.