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Purpose: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR., Methods and Materials: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed., Results: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors., Conclusions: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Radiation therapy is the most effective cytotoxic therapy for localized tumors. However, normal tissue toxicity limits the radiation dose and the curative potential of radiation therapy when treating larger target volumes. In particular, the highly radiosensitive intestine limits the use of radiation for patients with intra-abdominal tumors. In metastatic ovarian cancer, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of abdominal metastases. However, TAI fell out of favor due to high toxicity of the intestine. Here we utilized an innovative preclinical irradiation platform to compare the safety and efficacy of TAI ultra-high dose rate FLASH irradiation to conventional dose rate (CONV) irradiation in mice. We demonstrate that single high dose TAI-FLASH produced less mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and spared cell death in crypt base columnar cells compared to TAI-CONV irradiation. Importantly, TAI-FLASH and TAI-CONV irradiation had similar efficacy in reducing tumor burden while improving intestinal function in a preclinical model of ovarian cancer metastasis. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of abdominal radiotherapy, with potential application to metastatic ovarian cancer.

Introduction: Stereotactic ablative radiotherapy (SABR) is highly effective at controlling early stage primary lung cancer and lung metastases. Although previous studies have suggested that treating multiple lung tumors with SABR is safe, post-treatment changes in respiratory function have not been analyzed in detail., Patients and Methods: We retrospectively identified patients with 2 or more primary lung cancers or lung metastases treated with SABR and analyzed clinical outcomes and predictors of toxicity. We defined a composite respiratory decline endpoint to include increased oxygen requirement, increased dyspnea scale, or death from respiratory failure not owing to disease progression., Results: A total of 86 patients treated with SABR to 203 lung tumors were analyzed. A total of 21.8% and 41.8% of patients developed composite respiratory decline at 2 and 4 years, respectively. When accounting for intrathoracic disease progression, 12.7% of patients developed composite respiratory decline at 2 years. Of the patients, 7.9% experienced grade 2 or greater radiation pneumonitis. No patient- or treatment-related factor predicted development of respiratory decline. The median overall survival was 46.9 months, and the median progression-free survival was 14.8 months. The cumulative incidence of local failure was 9.7% at 2 years., Conclusion: Although our results confirm that SABR is an effective treatment modality for patients with multiple lung tumors, we observed a high rate of respiratory decline after treatment, which may be owing to a combination of treatment and disease effects. Future studies may help to determine ways to avoid pulmonary toxicity from SABR., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aim: To evaluate the impact of ultra-rapid FLASH mouse whole brain irradiation on hippocampal dendritic spines and neuroinflammation, factors associated with cognitive impairment after brain irradiation., Methods: We administered 30 Gy whole brain irradiation to C57BL6/J mice in sub-second (FLASH) vs. 240 s conventional delivery time keeping all other parameters constant, using a custom configured clinical linac. Ten weeks post-irradiation, we evaluated spatial and non-spatial object recognition using novel object location and object recognition testing. We measured dendritic spine density by tracing Golgi-stained hippocampal neurons and evaluated neuroinflammation by CD68 immunostaining, a marker of activated microglia, and expression of 10 pro-inflammatory cytokines using a multiplex immunoassay., Results: At ten weeks post-irradiation, compared to unirradiated controls, conventional delivery time irradiation significantly impaired novel object location and recognition tasks whereas the same dose given in FLASH delivery did not. Conventional delivery time, but not FLASH, was associated with significant loss of dendritic spine density in hippocampal apical dendrites, with a similar non-significant trend in basal dendrites. Conventional delivery time was associated with significantly increased CD68-positive microglia compared to controls whereas FLASH was not. Conventional delivery time was associated with significant increases in 5 of 10 pro-inflammatory cytokines in the hippocampus (and non-significant increases in another 3), whereas FLASH was associated with smaller increases in only 3., Conclusion: Reduced cognitive impairment and associated neurodegeneration were observed with FLASH compared to conventional delivery time irradiation, potentially through decreased induction of neuroinflammation, suggesting a promising approach to increasing therapeutic index in radiation therapy of brain tumors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background and Purpose: A major challenge in CT screening for lung cancer is limited specificity when distinguishing between malignant and non-malignant pulmonary nodules (PN). Malignant nodules have different mechanical properties and tissue characteristics ('stiffness') from non-malignant nodules. This study seeks to improve CT specificity by demonstrating in rats that measurements of volumetric ratios in PNs with varying composition can be determined by respiratory-gated dynamic CT imaging and that these ratios correlate with direct physical measurements of PN stiffness., Methods and Materials: Respiratory-gated MicroCT images acquired at extreme tidal volumes of 9 rats with PNs from talc, matrigel and A549 human lung carcinoma were analyzed and their volumetric ratios (δ) derived. PN stiffness was determined by measuring the Young's modulus using atomic force microscopy (AFM) for each nodule excised immediately after MicroCT imaging., Results: There was significant correlation (p=0.0002) between PN volumetric ratios determined by respiratory-gated CT imaging and the physical stiffness of the PNs determined from AFM measurements., Conclusion: We demonstrated proof of concept that PN volume changes measured non-invasively correlate with direct physical measurements of stiffness. These results may translate clinically into a means of improving the specificity of CT screening for lung cancer and/or improving individual prognostic assessments based on lung tumor stiffness., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Using [(18) F]PBR06 positron emission tomography (PET) to characterize the time course of stroke-associated neuroinflammation (SAN) in mice, to evaluate whether brain microglia influences motor function after stroke, and to demonstrate the use of [(18) F]PBR06 PET as a therapeutic assessment tool., Procedures: Stroke was induced by transient middle cerebral artery occlusion (MCAO) in Balb/c mice (control, stroke, and stroke with poststroke minocycline treatment). [18 F]PBR06 PET/CT imaging, rotarod tests, and immunohistochemistry (IHC) were performed 3, 11, and 22 days poststroke induction (PSI)., Results: The stroke group exhibited significantly increased microglial activation, and impaired motor function. Peak microglial activation was 11 days PSI. There was a strong association between microglial activation, motor function, and microglial protein expression on IHC. Minocycline significantly reduced microglial activation and improved motor function by day 22 PSI., Conclusion: [18 F]PBR06 PET imaging noninvasively characterizes the time course of SAN, and shows increased microglial activation is associated with decreased motor function.

Purpose: The purpose of this study is to evaluate the 18 kDa translocator protein (TSPO) radioligand [(18)F]N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline ([(18)F]PBR06) as a positron emission tomography (PET) imaging biomarker of stroke-induced neuroinflammation in a rodent model., Procedures: Stroke was induced by transient middle cerebral artery occlusion in Balb/c mice. Dynamic PET/CT imaging with displacement and preblocking using PK111195 was performed 3 days later. PET data were correlated with immunohistochemistry (IHC) for the activated microglial markers TSPO and CD68 and with autoradiography., Results: [(18)F]PBR06 accumulation peaked within the first 5 min postinjection, then decreased gradually, remaining significantly higher in infarct compared to noninfarct regions. Displacement or preblocking with PK11195 eliminated the difference in [(18)F]PBR06 uptake between infarct and noninfarct regions. Autoradiography and IHC correlated well spatially with uptake on PET., Conclusions: [(18)F]PBR06 PET specifically images TSPO in microglial neuroinflammation in a mouse model of stroke and shows promise for imaging and monitoring microglial activation/neuroinflammation in other disease models.

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G(2)/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.

Experiments were performed to investigate the activity of hepatic Phe hydroxylase (PAH) and plasma amino acid concentrations under conditions of Phe imbalance or toxicity in chicks fed on experimental diets from 7 to 14 or 16 d of age. In experiment 1, Phe imbalance was created by adding 10% of a mixture of indispensable amino acids lacking Phe (IAA - Phe) to a basal diet containing 0.46% Phe. The activity of PAH was not significantly affected by the imbalance. Correcting the imbalance by adding 1.12% Phe to the diet prevented the growth impairment and increased the activity of PAH. In experiment 2, growth was reduced by the addition of excess (2%) Phe to the basal diet. Correcting the excess by adding the IAA - Phe to the diet prevented the growth reduction. The activity of PAH was not significantly affected by 2% Phe, but it increased in chicks fed the corrected diet. The levels of PAH mRNA were not affected by the dietary treatments. A factorial arrangement of treatments with 3 dietary levels of Phe (0.46, 1.58, and 2.46%) with or without the IAA - Phe was used in experiment 3. The effects on growth were similar to those of the same treatments in experiments 1 and 2. The addition of Phe significantly increased hepatic PAH activity, but there was no detectable main effect of the IAA - Phe and no interaction. Plasma Phe concentration was increased by dietary Phe and decreased by the IAA - Phe mixture. We conclude that hepatic PAH activity in chicks variably increases in response to Phe or a 10% dietary supplement of indispensable amino acids including Phe but does not increase in response to IAA - Phe when the amino acids are added to a diet that is marginally adequate in Phe. The increased activity does not involve changes in PAH mRNA. The effects of IAA - Phe on plasma Phe concentrations appear to be independent of hepatic PAH activity as measured in vitro.

Two experiments were performed to study Phe imbalance and toxicity in 1-wk-old Babcock B380 chicks resulting from the addition of either a mixture of indispensable amino acids lacking Phe (IAA - Phe) or excess Phe to a diet that was nutritionally adequate in Phe. Chicks received a preexperimental semipurified diet for 1 wk and experimental diets from 7 to 14 d of age. In the first experiment, the chicks were given diets with Phe levels at 0.24, 0.29, 0.34, 0.39, 0.44, and 0.49% of the diet to determine the Phe requirement. The requirement of the chicks for Phe, based on weight gain and feed efficiency, was determined to be 0.39% of the diet. In experiment 2, the IAA - Phe (10% of the diet) or excess Phe (2% of the diet) was added to a diet containing 0.44% Phe. Chicks given the IAA - Phe or excess Phe had significantly slower growth rates than chicks given the basal diet (P > or = 0.05). The activities of the major hepatic enzyme of Phe catabolism, Phe hydroxylase (PAH), were significantly higher than that of chicks fed the basal diet when the chicks were fed the diets containing IAA - Phe plus 1.1% Phe (P > or = 0.05) but not when chicks were fed the diet containing IAA - Phe alone. The activity of PAH in chicks given the excess (2%) Phe was nearly 4 times the activity of PAH in chicks given the basal diet. Adding IAA - Phe to the diet containing excess Phe also resulted in higher PAH activity than was observed in chicks fed the basal diet, although the activity was significantly lower than observed for chicks receiving the diet containing excess Phe alone (P > or = 0.05). It is concluded that hepatic PAH activity in chicks increases primarily in response to its substrate, Phe. A dietary amino acid load without Phe reduces this response to excess Phe.