Pharmacokinetics and Biodistribution of 16,16 dimethyl Prostaglandin E2 in Non-Irradiated and Irradiated Mice and Non-Irradiated Non-Human Primates.

Exposure to high-dose ionizing radiation can lead to life-threatening injuries and mortality. Bone marrow is the most sensitive organ to radiation damage, resulting in the hematopoietic acute radiation syndrome (H-ARS) with the potential sequelae of infection, hemorrhage, anemia, and death if untreated. The development of medical countermeasures (MCMs) to protect or mitigate radiation injury is a medical necessity. In our well-established murine model of H-ARS we have demonstrated that the prostaglandin E2 (PGE2) analog 16,16 dimethyl-PGE2 (dmPGE2) has survival efficacy as both a radioprotectant and radiomitigator. The purpose of this study was to investigate the pharmacokinetics (PK) and biodistribution of dmPGE2 when used as a radioprotector in irradiated and non-irradiated inbred C57BL/6J mice, PK in irradiated and non-irradiated Jackson Diversity Outbred (JDO) mice, and the PK profile of dmPGE2 in non-irradiated non-human primates (NHPs). The C57BL/6J and JDO mice each received a single subcutaneous (SC) dose of 35 ug of dmPGE2 and were randomized to either receive radiation 30 min later or remain non-irradiated. Plasma and tissue PK profiles were established. The NHP were dosed with 0.1 mg/kg by SC administration and the PK profile in plasma was established. The concentration time profiles were analyzed by standard non-compartmental analysis and the metrics of AUC0-Inf, AUC60-480 (AUC from 60-480 min), Cmax, and t1/2 were evaluated. AUC60-480 represents the postirradiation time frame and was used to assess radiation effect. Overall, AUC0-Inf, Cmax, and t1/2 were numerically similar between strains (C57BL/6J and JDO) when combined, regardless of exposure status (AUC0-Inf: 112.50 ng·h/ml and 114.48 ng·h/ml, Cmax: 44.53 ng/ml and 63.96 ng/ml; t1/2: 1.8 h and 1.1 h, respectively). PK metrics were numerically lower in irradiated C57BL/6J mice than in non-irradiated mice [irradiation ratio: irradiated values/non-irradiated values = 0.71 for AUC60-480 (i.e., 29% lower), and 0.6 for t1/2]. In JDO mice, the radiation ratio was 0.53 for AUC60-480 (i.e., 47% lower), and 1.7 h for t1/2. The AUC0-Inf, Cmax, and t1/2 of the NHPs were 29.20 ng·h/ml, 7.68 ng/ml, and 3.26 h, respectively. Despite the numerical differences seen between irradiated and non-irradiated groups in PK parameters, the effect of radiation on PK can be considered minimal based on current data. The biodistribution in C57BL/6J mice showed that dmPGE2 per gram of tissue was highest in the lungs, regardless of exposure status. The radiation ratio for the different tissue AUC60-480 in C57BL/6J mice ranged between 0.5-1.1 (50% lower to 10% higher). Spleen, liver and bone marrow showed close to twice lower exposures after irradiation, whereas heart had a 10% higher exposure. Based on the clearance values from mice and NHP, the estimated allometric scaling coefficient was 0.81 (95% CI: 0.75, 0.86). While slightly higher than the current literature estimates of 0.75, this scaling coefficient can be considered a reasonable estimate and can be used to scale dmPGE2 dosing from animals to humans for future trials.

Medical countermeasures for the hematopoietic-subsyndrome of acute radiation syndrome in space.

More than 50 years after the Apollo missions ended, the National Aeronautical and Space Administration (NASA) and other international space agencies are preparing a return to the moon as a step towards deep space exploration. At doses ranging from a fraction of a Gray (Gy) to a few Gy, crew will be at risk for developing bone marrow failure associated with the hematopoietic subsyndrome of acute radiation syndrome (H-ARS) requiring pharmacological intervention to reduce risk to life and mission completion. Four medical countermeasures (MCM) in the colony stimulating factor class of drugs are now approved for treatment of myelosuppression associated with ARS. When taken in conjunction with antibiotics, fluids, antidiarrheals, antiemetics, antipyretics, and other treatments for symptomatic illness, the likelihood for recovery and mission completion can be greatly improved. The current review describes the performance and health risks of deep space flight, ionizing radiation exposure during crewed missions to the moon and Mars, and U.S. Food and Drug Administration (FDA)-approved medical interventions to treat ARS. With an expansion of human exploration missions beyond low Earth orbit (LEO), including near-term Lunar and future Mars missions, inclusion of MCMs to counteract ARS in the spaceflight kit will be critical for preserving crew health and performance.

Interspecies Comparison and Radiation Effect on Pharmacokinetics of BIO 300, a Nanosuspension of Genistein, after Different Routes of Administration in Mice and Non-Human Primates.

BIO 300, a suspension of synthetic genistein nanoparticles, is being developed for mitigating the delayed effects of acute radiation exposure (DEARE). The purpose of the current study was to characterize the pharmacokinetic (PK) profile of BIO 300 administered as an oral or parenteral formulation 24 h after sham-irradiation, total-body irradiation (TBI) with 2.5-5.0% bone marrow sparing (TBI/BMx), or in nonirradiated sex-matched C57BL/6J mice and non-human primates (NHP). C57BL/6J mice were randomized to the following arms in two consecutive studies: sham-TBI [400 mg/kg, oral gavage (OG)], TBI/BM2.5 (400 mg/kg, OG), sham-TBI [200 mg/kg, subcutaneous (SC) injection], TBI/BM2.5 (200 mg/kg, SC), sham-TBI (100 mg/kg, SC), or nonirradiated [200 mg/kg, intramuscular (IM) injection]. The PK profile was also established in NHP exposed to TBI/BM5.0 (100 mg/kg, BID, OG). Genistein-aglycone serum concentrations were measured in all groups using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. The PK profile demonstrates 11% and 19% reductions in Cmax and AUC0-inf, respectively, among mice administered 400 mg/kg, OG, after TBI/BM2.5 compared to the sham-TBI control arm. Administration of 200 mg/kg SC in mice exposed to TBI/BM2.5 showed a 53% increase in AUC0-inf but a 28% reduction in Cmax compared to the sham-TBI mice. The relative bioavailability of the OG route compared to the SC and IM routes in mice was 9% and 7%, respectively. After the OG route, the dose-normalized AUC0-inf was 13.37 (ng.h/mL)/(mg/kg) in TBI/BM2.5 mice compared to 6.95 (ng.h/mL)/(mg/kg) in TBI/BM5.0 NHPs. Linear regression of apparent clearances and weights of mice and NHPs yielded an allometric coefficient of 1.06. Based on these data, the effect of TBI/BMx on BIO 300 PK is considered minimal. Future studies should use SC and IM routes to maximize drug exposure when administered postirradiation. The allometric coefficient is useful in predicting therapeutic drug dose regimens across species for drug approval under the FDA animal rule.

Best Practices for Authentication of Cell Lines to Ensure Data Reproducibility and Integrity.

Cell line misidentification and contamination are major contributors to the reproducibility crisis in academic research. Authentication of cell lines provides assurances of the data generated; however, commercially available cells are often not subjected to rigorous identification testing. In this study, commercially available cell lines underwent testing to confirm cell identity and purity. The methods reported here outline the best practices for cell line authentication. Briefly, a commercially available primary rabbit aortic endothelial cell line was purchased for the intent of producing target proteins necessary for generating species-specific recombinant antibodies. These rabbit-specific antibodies would then be utilized for the development of in-house enzyme-linked immunosorbent assays (ELISA) to evaluate blood-based biomarkers of vascular injury after total-body irradiation. To authenticate the cell line, cell identity and purity were determined by single tandem repeat (STR) testing, flow cytometry, polymerase chain reaction (PCR), and cytochrome c oxidase subunit 1 (CO1) DNA Barcoding in-house and/or through commercial vendors. Fresh cells obtained from a New Zealand White rabbit (Charles River, Wilmington, DE) were used as a positive control. The results of STR and flow cytometry analyses indicated the cells were not contaminated with human or mouse cells, and that the cells were not of endothelial origin. PCR demonstrated that cells were also not of rabbit origin, which was further confirmed by a third-party vendor. An unopened vial of cells was submitted to another vendor for CO1 DNA Barcoding analysis, which identified the cells as being purely of bovine origin. Results revealed that despite purchase through a commercial vendor, the cell line marketed as primary rabbit aortic endothelial cells were of bovine origin. Purity analysis found cells were misidentified rather than contaminated. Further investigation to determine the cell type was not performed. The most cost-effective and efficient methodology for confirming cell line identity was found to be CO1 DNA Barcoding performed by a commercial vendor.

All for one, though not one for all: team players in normal tissue radiobiology.

PURPOSE: As part of the special issue on 'Women in Science', this review offers a perspective on past and ongoing work in the field of normal (non-cancer) tissue radiation biology, highlighting the work of many of the leading contributors to this field of research. We discuss some of the hypotheses that have guided investigations, with a focus on some of the critical organs considered dose-limiting with respect to radiation therapy, and speculate on where the field needs to go in the future. CONCLUSIONS: The scope of work that makes up normal tissue radiation biology has and continues to play a pivotal role in the radiation sciences, ensuring the most effective application of radiation in imaging and therapy, as well as contributing to radiation protection efforts. However, despite the proven historical value of preclinical findings, recent decades have seen clinical practice move ahead with altered fractionation scheduling based on empirical observations, with little to no (or even negative) supporting scientific data. Given our current appreciation of the complexity of normal tissue radiation responses and their temporal variability, with tissue- and/or organ-specific mechanisms that include intra-, inter- and extracellular messaging, as well as contributions from systemic compartments, such as the immune system, the need to maintain a positive therapeutic ratio has never been more urgent. Importantly, mitigation and treatment strategies, whether for the clinic, emergency use following accidental or deliberate releases, or reducing occupational risk, will likely require multi-targeted approaches that involve both local and systemic intervention. From our personal perspective as five 'Women in Science', we would like to acknowledge and applaud the role that many female scientists have played in this field. We stand on the shoulders of those who have gone before, some of whom are fellow contributors to this special issue.

A New Zealand White rabbit model of thrombocytopenia and coagulopathy following total body irradiation across the dose range to induce the hematopoietic-subsyndrome of acute radiation syndrome.

PURPOSE: The purpose if this study was to develop a rabbit model of total body irradiation (TBI) -induced thrombocytopenia and coagulopathy across the dose-range which induces the hematopoietic subsyndrome of the acute radiation syndrome (H-ARS). METHODS: Twenty male New Zealand White rabbits were assigned to arms to receive 6-MV of TBI at a dose of 6.5, 7.5, 8.5 or 9.5 Gy. Animals were treated with moderate levels of supportive care including buprenorphine for pain management, antibiotics, antipyretics for rectal body temperature >104.8 °F, and fluids for signs of dehydration. Animals were closelyfollowed for up to 45 days after TBI for signs of major morbidity/mortality. Hematology and serum chemistry parameters were routinely monitored. Hemostasis parameters were analyzed prior to TBI, 2 and 6 hours post-TBI, and at the time of euthanasia. RESULTS: Animals developed the characteristic signs and symptoms of H-ARS during the first-week post TBI. Animals became thrombocytopenic with signs of severe acute anemia during the second week post TBI. Moribund animals presented with petechia and ecchymosis of the skin and generalized internal hemorrhage. Multiorgan dysfunction characterized by bone marrow failure, gastric ileus, acute renal toxicity, and liver abnormalities were common. Severe abnormalities in coagulation parameters were observed. CONCLUSIONS: The presentation of bone marrow failure and multiorogan injury associated with ARS in the New Zealand White rabbit model is consistent with that described in the canine, swine, non-human primate, and in humans. The hemorrhagic syndrome associated with the ARS in rabbits is characterized by thrombocytopenia and hemostasis dysfunction, which appear to underlie the development of multiorgan dysfunction following TBI to rabbits. Taken together, the rabbit recapitulates the pathogenesis of ARS in humans, and may present an alternative small animal model for medical countermeasure pilot efficacy screening, dose-finding and schedule optimization studies prior to moving into large animal models of TBI-induced ARS.

Use of CT simulation and 3-D radiation therapy treatment planning system to develop and validate a total-body irradiation technique for the New Zealand White rabbit.

PURPOSE: Well-controlled ionizing radiation injury animal models for testing medical countermeasure efficacy require robust radiation physics and dosimetry to ensure accuracy of dose-delivery and reproducibility of the radiation dose-response relationship. The objective of this study was to establish a simple, convenient, robust and accurate technique for validating total body irradiation (TBI) exposure of the New Zealand White rabbit. METHODS: We use radiotherapy techniques such as computed tomography simulation and a 3 D-conformal radiation therapy treatment planning system (TPS) on three animals to comprehensively design and preplan a TBI technique for rabbits. We evaluate the requirement for bolus, treatment geometry, bilateral vs anterior-posterior treatment delivery, the agreement between monitor units calculated using the TPS vs a traditional hand calculation to the mid-plane, and resulting individual organ doses. RESULTS: The optimal technique irradiates animals on the left-decubitus position using two isocentric bilateral parallel-opposed 6 MV x-ray beams. Placement of a 5 mm bolus and 8.5 mm beam spoiler was shown to increase the dose to within ≤5 mm of the surface, improving dose homogeneity throughout the body of the rabbit. A simple hand calculation formalism, dependent only on mid-abdominal separation, could be used to calculate the number of monitor units (MUs) required to accurately deliver the prescribed dose to the animal. For the representative animal, the total body volume receiving > 95% of the dose, V95% > 99%, V100% > 95%, and V107% < 20%. The area of the body receiving >107% of the prescribed dose was mainly within the limbs, head, and around the lungs of the animal, where the smaller animal width reduces the x-ray attenuation. Individual organs were contoured by an experienced dosimetrist, and each received doses within 95-107% of the intended dose, with mean values ∼104%. Only the bronchus showed a maximal dose >107% (113%) due to the decreased attenuation of the lungs. To validate the technique, twenty animals were irradiated with four optically-stimulated luminescence dosimeters (OSLDs) placed on the surface of each animal (two on each side in the center of the radiation beam). The average dose over all animals was within <0.1% from intended values, with no animal receiving an average dose more than ±3.1% from prescription. CONCLUSION: The TBI technique developed in this pilot study was successfully used to establish the dose-response relationship for 45-day lethality across the dose-range to induce the hematopoietic-subsyndrome of the acute radiation syndrome (ARS).

Characterization of the hemorrhagic syndrome in the New Zealand white rabbit model following total body irradiation.

PURPOSE: The hemorrhagic syndrome is a major cause of morbidity and mortality associated with the acute radiation syndrome (ARS). We previously characterized the dose-response relationship for total body irradiation (TBI)-induced ARS in the New Zealand White (NZW) rabbit. Thrombocytopenia, hemorrhage, and anemia were strongly associated with morbidity/mortality during the first three weeks post-TBI. The objective of the current study was to further characterize the natural history of thrombocytopenia, hemostatic dysfunction and hemorrhage in the rabbit model at a TBI dose range to induce ARS. METHODS: Fifty male NZW rabbits were randomized to receive 7.0 or 7.5 Gy of 6 MV-derived TBI. Sham-irradiated controls (n = 6) were included as a comparator. Animals were treated with minimal supportive care including pain medication, antibiotics, antipyretics for temperature >104.8 °F, and fluids for signs of dehydration. Animals were culled at pre-determined timepoints post-TBI, or for signs of imminent mortality based on pre-defined euthanasia criteria. Hematology parameters, serum chemistry, viscoelasticity of whole blood, coagulation tests, and coagulation factor activities were measured. A gross exam of vital organs was performed at necropsy. RESULTS: Findings in this study include severe neutropenia during the first week post-TBI followed by thrombocytopenia and severe acute anemia with petechial hemorrhages of the skin and hemorrhage of the vital organs during the second to third weeks post-TBI. Abnormalities in whole blood viscoelastometry were observed concurrent with thrombocytopenia and hemorrhage. Antithrombin activity was significantly elevated in animals after exposure to 7.5 Gy, but not 7.0 Gy TBI. CONCLUSIONS: The hemorrhagic syndrome in the rabbit model of TBI recapitulates the pathogenesis described in humans following accidental or deliberate exposures. The rabbit may present an alternative to the rodent model as a small animal species for characterization of the full spectrum of multiorgan injury following TBI and early testing of promising medical countermeasures.

The Impact of Radiation Energy on Dose Homogeneity and Organ Dose in the Göttingen Minipig Total-Body Irradiation Model.

Animal models of total-body irradiation (TBI) are used to elucidate normal tissue damage and evaluate the efficacy of medical countermeasures (MCM). The accuracy of these TBI models depends on the reproducibility of the radiation dose-response relationship for lethality, which in turn is highly dependent on robust radiation physics and dosimetry. However, the precise levels of radiation each organ absorbs can change dramatically when different photon beam qualities are used, due to the interplay between their penetration and the natural variation of animal sizes and geometries. In this study, we evaluate the effect of varying the radiation energy, namely cobalt-60 (Co-60); of similar penetration to a 4-MV polyenergetic beam), 6 MV and 15 MV, in the absorbed dose delivered by TBI to individual organs of eight Göttingen minipigs of varying weights (10.3-24.1 kg) and dimensions (17.5-25 cm width). The main organs, i.e. heart, lungs, esophagus, stomach, bowels, liver, kidneys and bladder, were contoured by an experienced radiation oncologist, and the volumetric radiation dose distribution was calculated using a commercial treatment planning system commissioned and validated for Co-60, 6-MV and 15-MV teletherapy units. The dose is normalized to the intended prescription at midline in the abdomen. For each animal and each energy, the body and organ dose volume histograms (DVHs) were computed. The results show that more penetrating photon energies produce dose distributions that are systematically and consistently more homogeneous and more uniform, both within individual organs and between different organs, across all animals. Thoracic organs (lungs, heart) received higher dose than prescribed while pelvic organs (bowel, bladder) received less dose than prescribed, due to smaller and wider separations, respectively. While these trends were slightly more pronounced in the smallest animals (10.3 kg, 19 cm abdominal width) and largest animals (>20 kg, ∼25 cm abdominal width), they were observed in all animals, including those in the 9-15 kg range typically used in MCM models. Some organs received an average absorbed dose representing <80% of prescribed dose when Co-60 was used, whereas all organs received average doses of >87% and >93% when 6 and 15 MV were used, respectively. Similarly, average dose to the thoracic organs reached as high as 125% of the intended dose with Co-60, compared to 115% for 15 MV. These results indicate that Co-60 consistently produces less uniform dose distributions in the Göttingen minipig compared to 6 and 15 MV. Moreover, heterogeneity of dose distributions for Co-60 is accentuated by anatomical and geometrical variations across various animals, leading to different absorbed dose delivered to organs for different animals. This difference in absorbed radiation organ doses, likely caused by the lower penetration of Co-60 and 6 MV compared to 15 MV, could potentially lead to different biological outcomes. While the link between the dose distribution and variation of biological outcome in the Göttingen minipig has never been explicitly studied, more pronounced dose heterogeneity within and between organs treated with Co-60 teletherapy units represents an additional confounding factor which can be easily mitigated by using a more penetrating energy.

Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.

A Systematic Review of Metabolomic and Lipidomic Candidates for Biomarkers in Radiation Injury.

A large-scale nuclear event has the ability to inflict mass casualties requiring point-of-care and laboratory-based diagnostic and prognostic biomarkers to inform victim triage and appropriate medical intervention. Extensive progress has been made to develop post-exposure point-of-care biodosimetry assays and to identify biomarkers that may be used in early phase testing to predict the course of the disease. Screening for biomarkers has recently extended to identify specific metabolomic and lipidomic responses to radiation using animal models. The objective of this review was to determine which metabolites or lipids most frequently experienced perturbations post-ionizing irradiation (IR) in preclinical studies using animal models of acute radiation sickness (ARS) and delayed effects of acute radiation exposure (DEARE). Upon review of approximately 65 manuscripts published in the peer-reviewed literature, the most frequently referenced metabolites showing clear changes in IR induced injury were found to be citrulline, citric acid, creatine, taurine, carnitine, xanthine, creatinine, hypoxanthine, uric acid, and threonine. Each metabolite was evaluated by specific study parameters to determine whether trends were in agreement across several studies. A select few show agreement across variable animal models, IR doses and timepoints, indicating that they may be ubiquitous and appropriate for use in diagnostic or prognostic biomarker panels.

Down-Regulation of miR-23a-3p Mediates Irradiation-Induced Neuronal Apoptosis.

Radiation-induced central nervous system toxicity is a significant risk factor for patients receiving cancer radiotherapy. Surprisingly, the mechanisms responsible for the DNA damage-triggered neuronal cell death following irradiation have yet to be deciphered. Using primary cortical neuronal cultures in vitro, we demonstrated that X-ray exposure induces the mitochondrial pathway of intrinsic apoptosis and that miR-23a-3p plays a significant role in the regulation of this process. Primary cortical neurons exposed to irradiation show the activation of DNA-damage response pathways, including the sequential phosphorylation of ATM kinase, histone H2AX, and p53. This is followed by the p53-dependent up-regulation of the pro-apoptotic Bcl2 family molecules, including the BH3-only molecules PUMA, Noxa, and Bim, leading to mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c, which activates caspase-dependent apoptosis. miR-23a-3p, a negative regulator of specific pro-apoptotic Bcl-2 family molecules, is rapidly decreased after neuronal irradiation. By increasing the degradation of PUMA and Noxa mRNAs in the RNA-induced silencing complex (RISC), the administration of the miR-23a-3p mimic inhibits the irradiation-induced up-regulation of Noxa and Puma. These changes result in an attenuation of apoptotic processes such as MOMP, the release of cytochrome c and caspases activation, and a reduction in neuronal cell death. The neuroprotective effects of miR-23a-3p administration may not only involve the direct inhibition of pro-apoptotic Bcl-2 molecules downstream of p53 but also include the attenuation of secondary DNA damage upstream of p53. Importantly, we demonstrated that brain irradiation in vivo results in the down-regulation of miR-23a-3p and the elevation of pro-apoptotic Bcl2-family molecules PUMA, Noxa, and Bax, not only broadly in the cortex and hippocampus, except for Bax, which was up-regulated only in the hippocampus but also selectively in isolated neuronal populations from the irradiated brain. Overall, our data suggest that miR-23a-3p down-regulation contributes to irradiation-induced intrinsic pathways of neuronal apoptosis. These regulated pathways of neurodegeneration may be the target of effective neuroprotective strategies using miR-23a-3p mimics to block their development and increase neuronal survival after irradiation.

Psychological stress enhances tumor growth and diminishes radiation response in preclinical model of lung cancer.

BACKGROUND AND PURPOSE: Patients with life-threatening illnesses, such as cancer, experience emotional distress. This study was to investigate the molecular and cellular mechanisms of relevant psychological stressor on tumor growth and therapeutic resistance. MATERIALS AND METHODS: Stress was induced in C57BL/6J mice bearing LLC lung tumors by exposure to a conspecific mice receiving inescapable foot shocks. Mice were irradiated at 7 Gy for 3 consecutive days. Behaviors were monitored by open field test (OFT), elevated plus maze (EPM), sucrose preference test (SPT), and learned helplessness (LH) test. Protein expression in tissues and cultured cells were measured by Western blot. RESULTS: This study in animals showed that observing a conspecific mouse receiving foot shocks induced depression like behaviors with increased plasma corticosterone and adrenaline levels which increased tumor growth and radioresistance. Stress increased Wnt1, Drosha, and vimentin expression and decreased E-cadherin expression in tumor tissues. The combination of stress and irradiation enhanced radioresistance along with the increase in vimentin expression. The in vitro study showed that a ß2-adrenergic receptor (ß2-AR) agonist blocked irradiation-induced cell apoptosis and decreased cell viability, while silencing ß2-AR expression reduced the protective effects of ß2-AR agonist. ß2-AR agonist obviously increased Wnt1 and Drosha expression in LLC-1 cells. CONCLUSION: Psychological stress increased tumor growth and enhanced radioresistance associated with the activation of epithelial-mesenchymal transition by stress hormone-stimulated adrenergic receptors.

Manufacturing biological medicines on demand: Safety and efficacy of granulocyte colony-stimulating factor in a mouse model of total body irradiation.

Protein therapeutics, also known as biologics, are currently manufactured at centralized facilities according to rigorous protocols. The manufacturing process takes months and the delivery of the biological products needs a cold chain. This makes it less responsive to rapid changes in demand. Here, we report on technology application for on-demand biologics manufacturing (Bio-MOD) that can produce safe and effective biologics from cell-free systems at the point of care without the current challenges of long-term storage and cold-chain delivery. The objective of the current study is to establish proof-of-concept safety and efficacy of Bio-MOD-manufactured granulocyte colony-stimulating factor (G-CSF) in a mouse model of total body irradiation at a dose estimated to induce 30% lethality within the first 30 days postexposure. To illustrate on-demand Bio-MOD production feasibility, histidine-tagged G-CSF was manufactured daily under good manufacturing practice-like conditions prior to administration over a 16-day period. Bio-MOD-manufactured G-CSF improved 30-day survival when compared with saline alone (p = .073). In addition to accelerating recovery from neutropenia, the platelet and hemoglobin nadirs were significantly higher in G-CSF-treated animals compared with saline-treated animals (p < .05). The results of this study demonstrate the feasibility of consistently manufacturing safe and effective on-demand biologics suitable for real-time release.

ASTRO Journals' Data Sharing Policy and Recommended Best Practices.

Transparency, openness, and reproducibility are important characteristics in scientific publishing. Although many researchers embrace these characteristics, data sharing has yet to become common practice. Nevertheless, data sharing is becoming an increasingly important topic among societies, publishers, researchers, patient advocates, and funders, especially as it pertains to data from clinical trials. In response, ASTRO developed a data policy and guide to best practices for authors submitting to its journals. ASTRO's data sharing policy is that authors should indicate, in data availability statements, if the data are being shared and if so, how the data may be accessed.

BIO 300, a Nanosuspension of Genistein, Mitigates Radiation-Induced Erectile Dysfunction and Sensitizes Human Prostate Cancer Xenografts to Radiation Therapy.

PURPOSE: To assess whether BIO 300, a synthetic genistein nanosuspension, improves the therapeutic index in prostate cancer treatment by preventing radiation-induced erectile dysfunction (ED) without reducing tumor radiosensitivity. METHODS AND MATERIALS: Male Sprague-Dawley rats were exposed to 25 Gy of 220-kV prostate-confined x-rays. Animals were randomized to receive sham radiation therapy (RT), RT alone, RT with daily BIO 300 at 2 experimental dosing regimens, or RT with daily genistein. Erectile response was evaluated over time. Penile shaft tissue was harvested for histologic analyses. Murine xenograft studies using prostate cancer cell lines determined the effects of BIO 300 dosing on RT efficacy. RESULTS: Prostate-confined RT significantly decreased apomorphine-induced erectile response (P < .05 vs sham RT). Erection frequency in animals receiving prophylactic treatment with BIO 300 starting 3 days before RT was similar to sham controls after RT. Treatment with synthetic genistein did not mitigate loss in erectile frequency. At week 14, post-RT treatment with BIO 300 resulted in significantly higher quality of erectile function compared with both the RT arm and the RT arm receiving genistein starting 3 days before irradiation (P < .05). In hormone-sensitive and insensitive prostate tumor-bearing mice, BIO 300 administration did not negatively affect radiation-induced tumor growth delay. CONCLUSIONS: BIO 300 prevents radiation-induced ED, measured by erection frequency, erectile function, and erection quality, when administered 3 days before RT and continued daily for up to 14 weeks. Data also suggest that BIO 300 administered starting 2 hours after RT mitigates radiation-induced ED. Data provide strong nonclinical evidence to support clinical translation of BIO 300 for mitigation of ED while maintaining treatment response to RT.

Hematological Effects of Non-Homogenous Ionizing Radiation Exposure in a Non-Human Primate Model.

Detonation of a radiological or nuclear device in a major urban area will result in heterogenous radiation exposure, given to the significant shielding of the exposed population due to surrounding structures. Development of biodosimetry assays for triage and treatment requires knowledge of the radiation dose-volume effect for the bone marrow (BM). This proof-of-concept study was designed to quantify BM damage in the non-human primate (NHP) after exposure to one of four radiation patterns likely to occur in a radiological/nuclear attack with varying levels of BM sparing. Rhesus macaques (11 males, 12 females; 5.30-8.50 kg) were randomized by weight to one of four arms: 1. bilateral total-body irradiation (TBI); 2. unilateral TBI; 3. bilateral upper half-body irradiation (UHBI); and 4. bilateral lower half-body irradiation (LHBI). The match-point for UHBI vs. LHBI was set at 1 cm above the iliac crest. Animals were exposed to 4 Gy of 6 MV X rays. Peripheral blood samples were drawn 14 days preirradiation and at days 1, 3, 5, 7 and 14 postirradiation. Dosimetric measurements after irradiation indicated that dose to the mid-depth xiphoid was within 6% of the prescribed dose. No high-grade fever, weight loss >10%, dehydration or respiratory distress was observed. Animals in the bilateral- and unilateral TBI arms presented with hematologic changes [e.g., absolute neutrophil count (ANC) <500/ll; platelets <50,000/ll] and clinical signs/symptoms (e.g., petechiae, ecchymosis) characteristic of the acute radiation syndrome. Animals in the bilateral UHBI arm presented with myelosuppression; however, none of the animals developed severe neutropenia or thrombocytopenia (ANC remained >500/µl; platelets >50,000/µl during 14-day follow-up). In contrast, animals in the LHBI arm (1 cm above the ilieac crest to the toes) were protected against BM toxicity with no marked changes in hematological parameters and only minor gross pathology [petechiae (1/5), splenomegaly (1/5) and mild pulmonary hemorrhage (1/5)]. The model performed as expected with respect to the dose-volume effect of total versus partial-BM irradiation, e.g., increased shielding resulted in reduced BM toxicity. Shielding of the major blood-forming organs (e.g., skull, ribs, sternum, thoracic and lumbar spine) spared animals from bone marrow toxicity. These data suggest that the biological consequences of the absorbed dose are dependent on the total volume and pattern of radiation exposure.

Agglutination testing for human erythrocyte product in the rhesus macaque.

INTRODUCTION: There has been interest in using human blood products in nonhuman primate models of trauma to supplement human studies and to provide evidence to guide novel trauma resuscitation strategies. The compatibility of human RBCs has not been extensively studied in nonhuman primate species. METHODS: Whole blood samples were collected from five healthy, nontransfused, not previously pregnant Chinese-bred rhesus macaques. The whole blood was centrifuged, and the plasma was decanted from each sample. Group O-negative human RBCs were mixed with the plasma from the rhesus macaque monkeys. Compatibility testing was performed by an immediate spin test and polyspecific and monospecific anti-human globulin (AHG) tests in glass tubes. RESULTS: Immediate spin testing revealed three out of five plasma samples (60%) from rhesus macaques caused at least 1+ agglutination with the human RBCs. Polyspecific anti-human globulin (AHG) tests demonstrated that two of five plasma samples (40%) from rhesus macaques caused at least 1+ agglutination with the human RBC, while the monospecific AHG testing revealed that the incompatibility was caused by C3d, not IgG. CONCLUSION: Human RBCs are not compatible with the plasma of some, but not all, Chinese-bred rhesus macaques.

Mild hyperthermia as a localized radiosensitizer for deep-seated tumors: investigation in an orthotopic prostate cancer model in mice.

OBJECTIVE:: Non-ablative or mild hyperthermia (HT) has been shown in preclinical (and clinical) studies as a localized radiosensitizer that enhances the tumoricidal effects of radiation. Most preclinical in vivo HT studies use subcutaneous tumor models which do not adequately represent clinical conditions (e.g. proximity of normal/critical organs) or replicate the tumor microenvironment-both of which are important factors for eventual clinical translation. The purpose of this work is to demonstrate proof-of-concept of locoregional radiosensitization with superficially applied, radiofrequency (RF)-induced HT in an orthotopic mouse model of prostate cancer. METHODS:: In a 4-arm study, 40 athymic male nude mice were inoculated in the prostate with luciferase-transfected human prostate cancer cells (PC3). Tumor volumes were allowed to reach 150-250 mm3 (as measured by ultrasound) following which, mice were randomized into (i) control (no intervention); (ii) HT alone; (iii) RT alone; and (iv) HT + RT. RF-induced HT was administered (Groups ii and iv) using the Oncotherm LAB EHY-100 device to achieve a target temperature of 41 °C in the prostate. RT was administered ~30 min following HT, using an image-guided small animal radiotherapy research platform. In each case, a dual arc plan was used to deliver 12 Gy to the target in a single fraction. One animal from each cohort was euthanized on Day 10 or 11 after treatment for caspase-9 and caspase-3 Western blot analysis. RESULTS:: The inoculation success rate was 89%. Mean tumor size at randomization (~16 days post-inoculation) was ~189 mm3 . Following the administration of RT and HT, mean tumor doubling times in days were: control = 4.2; HT = 4.5; RT = 30.4; and HT + RT = 33.4. A significant difference (p = 0.036) was noted between normalized nadir volumes for the RT alone (0.76) and the HT + RT (0.40) groups. Increased caspase-3 expression was seen in the combination treatment group compared to the other treatment groups. CONCLUSION:: These early results demonstrate the successful use of external mild HT as a localized radiosensitizer for deep-seated tumors. ADVANCES IN KNOWLEDGE:: We successfully demonstrated the feasibility of administering external mild HT in an orthotopic tumor model and demonstrated preclinical proof-of-concept of HT-based localized radiosensitization in prostate cancer radiotherapy.

Cavernous Nerve Injury by Radiation Therapy May Potentiate Erectile Dysfunction in Rats.

PURPOSE/OBJECTIVES: Radiation-induced erectile-dysfunction (RiED) is one of the most common side effects of radiation therapy (RT) and significantly reduces the quality of life (QoL) of cancer patients. Approximately 50% of prostate cancer patients experience RiED within 3 to 5 years after completion of RT. A series of vascular, muscular, and neurogenic injuries after prostate RT lead to RiED; however, the precise role of RT-induced neurogenic injury in RiED has not been fully established. The cavernous nerves (CN) are postganglionic parasympathetic nerves located beside the prostate gland that assist in penile erection. This study was designed to investigate the role of CN injury, tissue damage, and altered signaling pathways in an RiED rat model. METHODS AND MATERIALS: Male rats were exposed to a single dose of 25 Gy prostate-confined RT. Erectile function was evaluated by intracavernous pressure (ICP) measurements conducted both 9 and 14 weeks after RT. Neuronal injury was evaluated in the CN using quantitative polymerase chain reaction, conduction studies, transmission electron microscopy, and immunoblotting. Masson trichrome staining was performed to elucidate fibrosis level in penile tissues. RESULTS: There were significant alterations in the ICP (P<.0001) of RT rats versus non-RT rats. TEM analysis showed decreased myelination, increased microvascular damage, and progressive axonal atrophy of the CN fibers after RT. Electrophysiologic analysis showed significant impairment of the CN conduction velocity after RT. RT also significantly increased RhoA/Rho-associated protein kinase 1 (ROCK1) mRNA and protein expression. In addition, penile tissue showed increased apoptosis and fibrosis 14 weeks after RT. CONCLUSIONS: RT-induced CN injury may contribute to RiED; this is therefore a rationale for developing novel therapeutic strategies to mitigate CN and tissue damage. Moreover, further investigation of the RhoA/ROCK pathway's role in mitigating RiED is necessary.