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Lung cancer is a major cause of cancer-associated deaths worldwide, and lung adenocarcinoma (LUAD) is the most common lung cancer subtype. Micro RNAs (miRNAs) regulate the pattern of gene expression in multiple cancer types and have been explored as potential drug development targets. To develop an oncomiR-based panel, we identified miRNA candidates that show differential expression patterns and are relevant to the worse 5-year overall survival outcomes in LUAD patient samples. We further evaluated various combinations of miRNA candidates for association with 5-year overall survival and identified a four-miRNA panel: miR-9-5p, miR-1246, miR-31-3p, and miR-3136-5p. The combination of these four miRNAs outperformed any single miRNA for predicting 5-year overall survival (hazard ratio [HR]: 3.47, log-rank p-value = 0.000271). Experiments were performed on lung cancer cell lines and animal models to validate the effects of these miRNAs. The results showed that singly transfected antagomiRs largely inhibited cell growth, migration, and invasion, and the combination of all four antagomiRs considerably reduced cell numbers, which is twice as effective as any single miRNA-targeted transfected. The

Purpose: To investigate how single or fractionated doses of radiation change the microenvironment in transgenic adenocarcinoma of the mouse prostate (TRAMP)-C1 tumors with respect to vascularity, hypoxia, and macrophage infiltrates.Experimental Design: Murine prostate TRAMP-C1 tumors were grown in C57BL/6J mice to 4 mm tumor diameter and were irradiated with either 25 Gy in a single dose or 60 Gy in 15 fractions. Changes in vascularity, hypoxia, and macrophage infiltrates were assessed by immunohistochemistry and molecular assays.Results: Tumor growth was delayed for 1 week after both radiation schedules. Tumor microvascular density (MVD) progressively decreased over a 3-week period to nadirs of 25% and 40% of unirradiated tumors for single or fractionated treatment, respectively. In accord with the decrease in MVDs, mRNA levels of endothelial markers, such as CD31, endoglin, and TIE, decreased over the same time period after irradiation. Central dilated vessels developed surrounded by avascularized hypoxic regions that became infiltrated with aggregates of CD68+ tumor-associated macrophages, reaching a maximum at 3 weeks after irradiation. Necrotic regions decreased and were more dispersed.Conclusion: Irradiation of TRAMP-C1 tumors with either single or fractionated doses decreases MVD, leading to the development of disperse chronic hypoxic regions, which are infiltrated with CD68+ tumor-associated macrophages. Approaches to interfere in the development of these effects are promising strategies to enhance the efficacy of cancer radiotherapy.

Supplementary Figures S1-S3 from Radiotherapy Decreases Vascular Density and Causes Hypoxia with Macrophage Aggregation in TRAMP-C1 Prostate Tumors

Novel technological solutions for the design of positron emission tomography (PET) systems to be used in the in-beam beam-on monitoring of proton tracks within biological tissues represent the frontier of the instrumentation in proton therapy. We report a novel all-digital PET dual heads prototype based on the multivoltage threshold (MVT) technology, able to operate in beam-on modality. We demonstrate in phantom and animal experiments that, by using an event selection based on the energy of singles and on the delay with respect to the radio-frequency period of the proton beam bunches, the monitoring PET system is able to image the induced activity by both pristine and pencil proton beam with energy ranging between 70 and 150 MeV during irradiation.

Head and neck cancer is the leading cancer worldwide. Radiation therapy plays important role of treatment for head and neck cancer. MicroRNAs have been shown to be related to tumor progression and radiosensitivity. However, the mechanisms are still largely unknown and evidence are still limited. In the current study, we sought to identify the miRNA related the radiosensitivity of head and neck tumor cell, which leading to the disappointed prognosis of patients with head and neck cancer receiving radiation therapy. The miRNA expression profiles and clinical information of patients with head and neck cancer were obtained from The Cancer Genome Atlas. The identification of miRNA was carried out through an integrated bioinformatics analysis. The miRNA identified in previous approach was validated throughin vitroandin vivostudies. MiR-4776-5p was finally identified as the role of radio-sensitizer and predicts the prognosis of patients with head and neck cancer receiving radiotherapy. 11 of 16 genes targeted by the miR-4776-5p have been discovered to regulate the mechanisms related to radiosensitivity using functional annotation.

Radiotherapy is an important modality for the treatment of cancer, e.g., X-ray, Cs-137 γ-ray (peak energy: 662 keV). An important therapy pathway of radiation is to generate the double strand breaks of DNA to prohibit the proliferation of cancer cells. In addition, the excessive amount of reactive oxygen species (ROS) is induced to damage the organelles, which can cause cellular apoptosis or necrosis. Gold nanoparticles (GNPs) have been proven potential as a radiosensitizer due to the high biocompatibility, the low cytotoxicity and the high-Z property (Z = 79) of gold. The latter property may allow GNPs to induce more secondary electrons for generating ROS in cells as irradiated by high-energy photons. In this paper, the radiobiological effects on A431 cells with uptake of 55-nm GNPs were studied to investigate the GNPs-enhanced production of ROS on these cells as irradiated by Cs-137 γ-ray. The fluorescence-labeling image of laser scanning confocal microscopy (LSCM) shows the excessive expression of ROS in these GNPs-uptake cells after irradiation. And then, the follow-up disruption of cytoskeletons and dysfunction of mitochondria caused by the induced ROS are observed. From the curves of cell survival fraction versus the radiation dose, the radiosensitization enhancement factor of GNPs is 1.29 at a survival fraction of 30%. This demonstrates that the tumoricidal efficacy of Cs-137 radiation can be significantly raised by GNPs. Because of facilitating the production of excessive ROS to damage tumor cells, GNPs are proven to be a prospective radiosensitizer for radiotherapy, particularly for the treatment of certain radioresistant tumor cells. Through this pathway, the tumoricidal efficacy of radiotherapy can be raised.

Radiation-induced liver disease (RILD) is the major complication for cancer patients after radiation therapy. We investigated the protective effects of BPC 157 peptide in reducing RILD.Mice were irradiated with a single dose of 12 Gy to induce acute liver injury with or without oral BPC 157. Plasma levels of AST and ALT were determined. In vitro rat liver clone 9 cells and in vivo liver tissues were harvested for MTT assay, TUNEL assay, lipid staining, polypoid cell counts, Western blotting of caspase-3, PCNA, KLF-4 and HIF-2α, and immunocytochemistry for PCNA, KLF-4 and HIF-2α. SiRNAs were used to knockdown KLF-4.BPC 157 was firstly demonstrated to reduce RILD by decreasing plasma levels of AST and ALT, and inhibiting hydropic degeneration of liver. BPC 157 significantly decreased radiation-induced cell apoptosis, increased PCNA expression, promoted the expression of KLF4, decreased the radiation-induced hepatic lipid accumulation and HIF-2α expression both in mice liver and in clone 9 liver cells. The knockdown of KLF4 abolished the protective effect of BPC 157 on radiation-induced apoptosis and lipid accumulation in clone 9 liver cells, indicating that the protective effect of BPC 157 was mediated by KLF4 in liver cells.The present study provided a good model for molecular mechanism underlying the acute RILD. BPC 157, as a stable pentadecapeptide that can be chemically synthesized and purified easily for research, together with its in vivo markedly protective effect made it worth of being investigated for future clinical application for RILD.

We aim to establish a noninvasive diagnostic platform to capture early phenotypic transformation for metastasis using 18F-FDG PET and 1H-NMR-based serum metabolomics. Mice with implantation of NCI-H460 cells grew only primary lung tumors in the localized group and had both primary and metastatic lung tumors in the metastatic group. The serum metabolites were analyzed using 1H-NMR at the time of PET/CT scan. The glycolysis status and cell proliferation were validated by Western blotting and staining. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic accuracy of SUVmean and serum metabolites in metastasis. In the metastatic mice, the SUVmean of metastatic tumors was significantly higher than that of primary lung tumors in PET images, which was supported by elevated glycolytic protein expression of HK2 and PKM2. The serum pyruvate level in the metastatic group was significantly lower than that in the localized group, corresponding to increased pyruvate-catalyzed enzyme and proliferation rates in metastatic tumors. In diagnosing localized or metastatic tumors, the areas under the ROC curves of SUVmean and pyruvate were 0.92 and 0.91, respectively, with p < 0.05. In conclusion, the combination of 18F-FDG PET and 1H-NMR-based serum metabolomics demonstrated the feasibility of a glycolytic platform for diagnosing metastatic lung cancers.

Grenz-ray therapy (GT) is commonly used for dermatological radiotherapy and has a higher linear energy transfer, relative biological effectiveness (RBE) and oxygen enhancement ratio (OER). GT is a treatment option for lentigo maligna and lentigo maligna melanoma. This study aims to calculate the RBE for DNA double-strand break (DSB) induction and cell survival under hypoxic conditions for GT. The yield of DSBs induced by GT is calculated at the aerobic and hypoxic conditions, using a Monte Carlo damage simulation (MCDS) software. The RBE value for cell survival is calculated using the repair–misrepair–fixation (RMF) model. The RBE values for cell survival for cells irradiated by 15 kV, 10 kV and 10 kVp and titanium K-shell X-rays (4.55 kV) relative to 60Co γ-rays are 1.0–1.6 at the aerobic conditions and moderate hypoxia (2% O2), respectively, but increase to 1.2, 1.4 and 1.9 and 2.1 in conditions of severe hypoxia (0.1% O2). The OER values for DSB induction relative to 60Co γ-rays are about constant and ~2.4 for GT, but the OER for cell survival is 2.8–2.0 as photon energy decreases from 15 kV to 4.55 kV. The results indicate that GT results in more DSB induction and allows effective tumor control for superficial and hypoxic tumors.

Purpose: This study evaluated the DNA double strand breaks (DSBs) induced by indirect actions and its misrepairs to estimate the relative biological effectiveness (RBE) of proton beams.Materials an...

Imaging probes/biomarkers that are correlated with molecular or microenvironmental alterations in tumors have been used not only in diagnosing cancer but also in assessing the efficacy of cancer treatment. We evaluated the early response of hepatocellular carcinoma (HCC) to radiation treatment using T2-weighted magnetic resonance imaging (MRI), diffusion-weighted (DW) MRI, and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). Orthotopic HCC tumors were established in the right liver lobe of Balb/c mice. Mice were longitudinally scanned using T2-weighted/DW MRI and 18F-FDG PET 1 day before and on days 1, 3, 6, 9 and 13 after irradiation with 15 Gy to the right liver lobe to determine tumor size, apparent diffusion coefficient (ADC) value, and maximum standardized uptake value. Immunohistochemical (IHC) staining was performed to validate the tumor microenvironment. Irradiation markedly retarded tumor growth in the orthotopic HCC model and led to increaes in ADC values as early as on day 1 after irradiation. Irradiation also resulted in increases in 18F-FDG uptake on day 1 that were sustained until the end of the observation period. IHC staining revealed a decrease in the number of proliferative cells and a continuous macrophage influx into irradiated tumors, which dramatically altered the tumor microenvironment. Lastly, in vitro coculture of HCC cells and macrophages led to interaction between the cells and enhanced the cellular uptake of 18F-FDG. ADC values and 18F-FDG uptake measured using DW MRI and 18F-FDG PET serve as potential biomarkers for early assessment of HCC tumor responses to radiation therapy.

With the continuing rise of lipidomic studies, there is an urgent need for a useful and comprehensive tool to facilitate lipidomic data analysis. The most important features making lipids different from general metabolites are their various characteristics, including their lipid classes, double bonds, chain lengths, etc. Based on these characteristics, lipid species can be classified into different categories and, more interestingly, exert specific biological functions in a group. In an effort to simplify lipidomic analysis workflows and enhance the exploration of lipid characteristics, we have developed a highly flexible and user-friendly web server called LipidSig. It consists of five sections, namely, Profiling, Differential Expression, Correlation, Network and Machine Learning, and evaluates lipid effects on cellular or disease phenotypes. One of the specialties of LipidSig is the conversion between lipid species and characteristics according to a user-defined characteristics table. This function allows for efficient data mining for both individual lipids and subgroups of characteristics. To expand the server's practical utility, we also provide analyses focusing on fatty acid properties and multiple characteristics. In summary, LipidSig is expected to help users identify significant lipid-related features and to advance the field of lipid biology. The LipidSig webserver is freely available at http://chenglab.cmu.edu.tw/lipidsig, Graphical Abstract Graphical AbstractLipidSig is the first web tool dedicated to comprehensive lipidomic data analysis.

Cervical cancer is the fourth most common cancer in women worldwide. Increasing evidence has shown that miRNAs are related to the progression of cervical cancer. However, the mechanisms that affect the prognosis of cancer are still largely unknown. In the present study, we sought to identify miRNAs associated with poor prognosis of patient with cervical cancer, as well as the possible mechanisms regulated by them. The miRNA expression profiles and relevant clinical information of patients with cervical cancer were obtained from The Cancer Genome Atlas (TCGA). The selection of prognostic miRNAs was carried out through an integrated bioinformatics approach. The most effective miRNAs with synergistic and additive effects were selected for validation through in vitro experiments. Three miRNAs (miR-216b-5p, miR-585-5p, and miR-7641) were identified as exhibiting good performance in predicting poor prognosis through additive effects analysis. The functional enrichment analysis suggested that not only pathways traditionally involved in cancer but also immune system pathways might be important in regulating the outcome of the disease. Our findings demonstrated that a synergistic combination of three miRNAs may be associated, through their regulation of specific pathways, with very poor survival rates for patients with cervical cancer.

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Shigella is one of the leading bacterial causes of diarrhea worldwide, affecting more than 165 million people annually. Among the serotypes of Shigella, Shigella sonnei is physiologically unique and endemic in human immunodeficiency virus-infected men who have sex with men. The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, a protein complex composed of NLRP3, apoptosis-associated speck-like protein, and caspase-1, recognizes, and responds to pathogen infection and diverse sterile host-derived or environmental danger signals to induce IL-1β and IL-18 production. Although the Shigella flexneri-mediated activation of the NLRP3 inflammasome has been reported, the effect of S. sonnei on NLRP3 inflammasome activation remains unclear. We found that S. sonnei induced IL-1β production through NLRP3-dependent pathways in lipopolysaccharide-primed macrophages. A mechanistic study revealed that S. sonnei induced IL-1β production through P2X7 receptor-mediated potassium efflux, reactive oxygen species generation, lysosomal acidification, and mitochondrial damage. In addition, the phagocytosis of viable S. sonnei was important for IL-1β production. Furthermore, we demonstrated that NLRP3 negatively regulated phagocytosis and the bactericidal activity of macrophages against S. sonnei. These findings provide mechanistic insight into the activation of the NLRP3 inflammasome by S. sonnei in macrophages.

Radiation-induced bone diseases were frequently reported in radiotherapy patients. To study the diseases, microdosimeters were constructed with walls of A150-A150, A150-B100, B100-A150 and B100-B100 interfaces. Monte Carlo simulations of these microdosimeters were performed to determine the lineal energy spectra of an interface site at different depths in water for 230 MeV protons. Comparing these spectra with data of ICRU tissue and bone walls, better agreements were found at shallow depths for protons and delta-rays than deep depths for nuclear interactions.

This paper estimates the yields of DNA double-strand breaks (DSBs) induced by ultrasoft X-rays and uses the DSB yields and the repair outcomes to evaluate the relative biological effectiveness (RBE) of ultrasoft X-rays. We simulated the yields of DSB induction and predicted them in the presence and absence of oxygen, using a Monte Carlo damage simulation (MCDS) software, to calculate the RBE. Monte Carlo excision repair (MCER) simulations were also performed to calculate the repair outcomes (correct repairs, mutations, and DSB conversions). Compared to 60Co γ-rays, the RBE values for ultrasoft X-rays (titanium K-shell, aluminum K-shell, copper L-shell, and carbon K-shell) for DSB induction were respectively 1.3, 1.9, 2.3, and 2.6 under aerobic conditions and 1.3, 2.1, 2.5, and 2.9 under a hypoxic condition (2% O2). The RBE values for enzymatic DSBs were 1.6, 2.1, 2.3, and 2.4, respectively, indicating that the enzymatic DSB yields are comparable to the yields of DSB induction. The synergistic effects of DSB induction and enzymatic DSB formation further facilitate cell killing and the advantage in cancer treatment.

Alterations in metabolism following radiotherapy affect therapeutic efficacy, although the mechanism underlying such alterations is unclear. A new imaging technique—named dynamic nuclear polarization (DNP) carbon-13 magnetic resonance imaging (MRI)—probes the glycolytic flux in a real-time, dynamic manner. The [1-13C]pyruvate is transported by the monocarboxylate transporter (MCT) into cells and converted into [1-13C]lactate by lactate dehydrogenase (LDH). To capture the early glycolytic alterations in the irradiated cancer and immune cells, we designed a preliminary DNP 13C-MRI study by using hyperpolarized [1-13C]pyruvate to study human FaDu squamous carcinoma cells, HMC3 microglial cells, and THP-1 monocytes before and after irradiation. The pyruvate-to-lactate conversion rate (kPL [Pyr.]) calculated by kinetic modeling was used to evaluate the metabolic alterations. Western blotting was performed to assess the expressions of LDHA, LDHB, MCT1, and MCT4 proteins. Following irradiation, the pyruvate-to-lactate conversion rates on DNP 13C-MRI were significantly decreased in the FaDu and the HMC3 cells but increased in the THP-1 cells. Western blot analysis confirmed the similar trends in LDHA and LDHB expression levels. In conclusion, DNP 13C-MRI non-invasively captured the different glycolytic alterations among cancer and immune systems in response to irradiation, implying its potential for clinical use in the future.

Purpose To investigate the biological meaning of apparent diffusion coefficient (ADC) values in tumors following radiotherapy. Materials and Methods Five mice bearing TRAMP-C1 tumor were half-irradiated with a dose of 15 Gy. Diffusion-weighted images, using multiple b-values from 0 to 3000 s/mm2, were acquired at 7T on day 6. ADC values calculated by a two-point estimate and monoexponential fitting of signal decay were compared between the irradiated and nonirradiated regions of the tumor. Pixelwise ADC maps were correlated with histological metrics including nuclear counts, nuclear sizes, nuclear spaces, cytoplasmic spaces, and extracellular spaces. Results As compared with the nonirradiated region, the irradiated region exhibited significant increases in ADC, extracellular space, and nuclear size, and a significant decrease in nuclear counts (P < 0.001 for all). Optimal ADC to differentiate the irradiated from nonirradiated regions was achieved at a b-value of 800 s/mm2 by the two-point method and monoexponential curve fitting. ADC positively correlated with extracellular spaces (r = 0.74) and nuclear sizes (r = 0.72), and negatively correlated with nuclear counts (r = –0.82, P < 0.001 for all). Conclusion As a radiomic biomarker, ADC maps correlating with histological metrics pixelwise could be a means of evaluating tumor heterogeneity and responses to radiotherapy. Level of Evidence: 1 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;46:483–489

Purpose To quantify the differential plasma flow- (Fp-) and permeability surface area product per unit mass of tissue- (PS-) weighting in forward volumetric transfer constant (Ktrans) estimates by using a low molecular (Gd-DTPA) versus high molecular (Gadomer) weight contrast agent in dynamic contrast enhanced (DCE) MRI. Materials and methods DCE MRI was performed using a 7T animal scanner in 14 C57BL/6J mice syngeneic for TRAMP tumors, by administering Gd-DTPA (0.9 kD) in eight mice and Gadomer (35 kD) in the remainder. The acquisition time was 10 min with a sampling rate of one image every 2 s. Pharmacokinetic modeling was performed to obtain Ktrans by using Extended Tofts model (ETM). In addition, the adiabatic approximation to the tissue homogeneity (AATH) model was employed to obtain the relative contributions of Fp and PS. Results The Ktrans values derived from DCE-MRI with Gd-DTPA showed significant correlations with both PS (r2 = 0.64, p = 0.009) and Fp (r2 = 0.57, p = 0.016), whereas those with Gadomer were found only significantly correlated with PS (r2 = 0.96, p = 0.0003) but not with Fp (r2 = 0.34, p = 0.111). A voxel-based analysis showed that Ktrans approximated PS ( Conclusions The differential contributions of Fp and PS in estimating Ktrans values vary with the molecular weight of the contrast agent used. The macromolecular contrast agent resulted in Ktrans values that were much less dependent on flow. These findings support the use of macromolecular contrast agents for estimating tumor vessel permeability with DCE-MRI.

Shigella is one of the leading bacterial causes of diarrhea worldwide, affecting more than 165 million people annually. Among the serotypes of Shigella, Shigella sonnei is physiologically unique and endemic in human immunodeficiency virus-infected men who have sex with men. The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, a protein complex composed of NLRP3, apoptosis-associated speck-like protein, and caspase-1, recognizes, and responds to pathogen infection and diverse sterile host-derived or environmental danger signals to induce IL-1β and IL-18 production. Although the Shigella flexneri-mediated activation of the NLRP3 inflammasome has been reported, the effect of S. sonnei on NLRP3 inflammasome activation remains unclear. We found that S. sonnei induced IL-1β production through NLRP3-dependent pathways in lipopolysaccharide-primed macrophages. A mechanistic study revealed that S. sonnei induced IL-1β production through P2X7 receptor-mediated potassium efflux, reactive oxygen species generation, lysosomal acidification, and mitochondrial damage. In addition, the phagocytosis of viable S. sonnei was important for IL-1β production. Furthermore, we demonstrated that NLRP3 negatively regulated phagocytosis and the bactericidal activity of macrophages against S. sonnei. These findings provide mechanistic insight into the activation of the NLRP3 inflammasome by S. sonnei in macrophages.

An integrative multi-omics database is needed urgently, because focusing only on analysis of one-dimensional data falls far short of providing an understanding of cancer. Previously, we presented DriverDB, a cancer driver gene database that applies published bioinformatics algorithms to identify driver genes/mutations. The updated DriverDBv3 database (http://ngs.ym.edu.tw/driverdb) is designed to interpret cancer omics’ sophisticated information with concise data visualization. To offer diverse insights into molecular dysregulation/dysfunction events, we incorporated computational tools to define CNV and methylation drivers. Further, four new features, CNV, Methylation, Survival, and miRNA, allow users to explore the relations from two perspectives in the ‘Cancer’ and ‘Gene’ sections. The ‘Survival’ panel offers not only significant survival genes, but gene pairs synergistic effects determine. A fresh function, ‘Survival Analysis’ in ‘Customized-analysis,’ allows users to investigate the co-occurring events in user-defined gene(s) by mutation status or by expression in a specific patient group. Moreover, we redesigned the web interface and provided interactive figures to interpret cancer omics’ sophisticated information, and also constructed a Summary panel in the ‘Cancer’ and ‘Gene’ sections to visualize the features on multi-omics levels concisely. DriverDBv3 seeks to improve the study of integrative cancer omics data by identifying driver genes and contributes to cancer biology.

Purpose To investigate the temporal and spatial infiltration of TRAMP-C1 tumors by myeloid-derived suppressor cells (MDSCs) after high-dose radiation therapy (RT), and to explore their effect on tumor growth. Methods and Materials TRAMP-C1 intramuscularly tumors were irradiated with a single dose of 8 Gy or 25 Gy. The dynamics of infiltrated MDSCs and their intratumoral spatial distribution were assessed by immunohistochemistry and flow cytometry. Cytokine levels in the blood and tumor were analyzed by multiplex immunoassay. Mice were injected with anti-Gr-1 antibody to determine whether MDSCs affect tumor growth after RT. Results CD11b+Gr-1+ MDSCs infiltrated TRAMP-C1 tumors irradiated with 25 Gy, but not 8 Gy, within 4 hours and recruitment persisted for at least 2 weeks. Both CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs) were involved. Tumor RT also increased the representation of both MDSC subpopulations in the spleen and peripheral blood. Levels of multiple cytokines were increased in the tumors at 2 weeks, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a; while G-CSF, IL-6, and TNF-α levels increased in the blood. PMN-MDSCs aggregated in the central necrotic region of the irradiated tumors over time, where they were associated with avascular hypoxia (CD31-PIMO+). MDSCs expressed the proangiogenic factor, matrix metalloproteinase-9, and, within the necrotic area, high levels of arginase-1 and indoleamine 2,3-dioxygenase. Depletion of PMN-MDSCs by Gr-1 antibody increased the efficacy of high-dose RT. Conclusions PMN-MDSCs infiltrate TRAMP-C1 tumors after high-dose RT. Their spatial distribution suggests they are involved in the evolution of an intratumoral state of necrosis associated with avascular hypoxia, and their phenotype is consistent with them being immunosuppressive. They appear to promote tumor growth after RT, making them a prime therapeutic target for therapeutic intervention. Assessment of MDSCs and cytokine levels in blood could be an index of the need for such an intervention.

A ketamine/xylazine (K/X) mixture is widely used before and during experiments in rodents. However, the impact of short-term use of K/X mixture and its influences on data interpretation have rarely been discussed. In this study, we administered one shot of a K/X mixture and examined acute hepatic responses using biochemical analysis, histopathological examination, and non-invasive imaging to determine the delay required prior to further assessment of the liver to avoid confounding effects triggered by anaesthesia. After the K/X injection, aspartate aminotransferase (AST) in serum was significantly elevated from 3 to 48 h. Obstructed sinusoidal circulation lasting for 24 or 36 h was revealed by DCE-MRI and drug distribution analysis, respectively. Metabolic alterations were detected at 3 h by NMR analysis and FDG-PET. Moreover, ultrasonography showed that lipid droplet accumulation increased from 1 to 16 h and declined thereafter. Taken together, our findings show that the K/X mixture induces acute hepatotoxicity and metabolic disturbance, and these disturbances cause hemodynamical disorders in the liver. The required time interval for recovery from K/X impact was dependent on the chosen assay. It took at least 16 h for metabolic recovery and 36 h for recovery of sinusoidal circulation. However, the liver was not fully recovered from the injury within 48 h.

Although nanodiamonds have long being considered as a potential tool for biomedical research, the practical in vivo application of nanodiamonds remains relatively unexplored. In this paper, we present the first application of in vivo MRI contrast enhancement using only iron embedded magnetic nanodiamonds. MR image enhancement was clearly demonstrated in the rendering of T2-weighted images of mice obtained using an unmodified commercial MRI scanner. The excellent contrast obtained using these nanodiamonds opens the door to the non-invasive in vivo tracking of NDs and image enhancement. In the future, one can apply these magnetic nanodiamonds together with surface modifications to facilitate drug delivery, targeted therapy, localized thermal treatment, and diagnostic imaging., Comment: 19 pages, 6 figures

Imaging probes/biomarkers that are correlated with molecular or microenvironmental alterations in tumors have been used not only in diagnosing cancer but also in assessing the efficacy of cancer treatment. We evaluated the early response of hepatocellular carcinoma (HCC) to radiation treatment using T2-weighted magnetic resonance imaging (MRI), diffusion-weighted (DW) MRI, andOrthotopic HCC tumors were established in the right liver lobe of Balb/c mice. Mice were longitudinally scanned using T2-weighted/DW MRI andIrradiation markedly retarded tumor growth in the orthotopic HCC model and led to increaes in ADC values as early as on day 1 after irradiation. Irradiation also resulted in increases inADC values and

We realized a prototype of a monitoring system for proton therapy based on the technique of positron emission tomography. For the first time to our knowledge, we used the Plug&Imaging (P&I)Multi Voltage Threshold (MVT) technology in this application. The sensing system includes LYSO/silicon photomultiplier (SiPM) detection elements, fast digital multi voltage threshold (MVT) readout electronics and dedicated image reconstruction algorithms. In this paper we present a new set of data collected with a water phantom and we further demonstrate that the P&I sensor system can be operated in the in-beam beamon experimental conditions of the proton therapy room, reaching a maximal count rate of 5.2 Mcps.

One of the most challenging areas of sensor development for nuclear medicine is the design of proton therapy monitoring systems. Sensors are operated in a high detection rate regime in beam-on conditions. We realized a prototype of a monitoring system for proton therapy based on the technique of positron emission tomography. We used the Plug and Imaging (P&amp, I) technology in this application. This sensing system includes LYSO/silicon photomultiplier (SiPM) detection elements, fast digital multi voltage threshold (MVT) readout electronics and dedicated image reconstruction algorithms. In this paper, we show that the P&amp, I sensor system has a uniform response and is controllable in the experimental conditions of the proton therapy room. The prototype of PET monitoring device based on the P&amp, I sensor system has an intrinsic experimental spatial resolution of approximately 3 mm (FWHM), obtained operating the prototype both during the beam irradiation and right after it. The count-rate performance of the P&amp, I sensor approaches 5 Mcps and allows the collection of relevant statistics for the nuclide analysis. The measurement of both the half life and the relative abundance of the positron emitters generated in the target volume through irradiation of 10 10 protons in approximately 15 s is performed with 0.5% and 5 % accuracy, respectively.

Background Radiotherapy (RT) is a common approach that accounts for nearly 50% of cancer patient treatment and has the potential for long-term tumor control. Recently, we published a research article on gene expression profiling of tumor-associated macrophages (TAM) that were exposed to ionizing radiation (IR). Single-dose irradiation of tumors could initiate differentially expressed genes in TAM as a time series from irradiated tumors that are associated with the immune response. It is also well known that human cancers are associated with microRNA (miRNA) alterations that are involved in cancer progression. However, the role of miRNA on TAM after exposure to irradiation remains unclear. Results In this study, miRNA expression profiles from microarrays were used to identify the key miRNAs and correlating pathways involved in the role of TAMs in tumor progression and recurrence after RT. Using a mouse tumor model, we identified miRNA pattern changes over time in response to irradiation. Based on our results, we hypothesize that miRNA expression in the irradiated tumor may be used as a distinguishing marker to indicate the best time for therapeutic intervention to prevent tumor recurrence after RT. Conclusions We established a murine model irradiated with a single dose of 25 Gy that could initiate temporal changes in the expression of miRNAs associated with cell proliferation and the immune response, as evidenced by macrophages directly extracted from irradiated tumors after two weeks of IR. Statistical analyses were conducted by comparing the miRNA expression in macrophages from non-irradiated versus irradiated tumors. Thus, our study could lead to a better understanding of the function of miRNA expressions, which changed temporally in an irradiated tumor microenvironment.

Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/μm) to (60)Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1-4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing.

Radiotherapy (RT) is a common cancer treatment approach that accounts for nearly 50% of patient treatment; however, tumor relapse after radiotherapy is still a major issue. To study the crucial role of tumor-associated macrophages (TAMs) in the regulation of tumor progression post-RT, microarray experiments comparing TAM gene expression profiles between unirradiated and irradiated tumors were conducted to discover possible roles of TAMs in initiation or contribution to tumor recurrence following RT, taking into account the relationships among gene expression, tumor microenvironment, and immunology. A single dose of 25Gy was given to TRAMP C-1 prostate tumors established in C57/B6 mice. CD11b-positive macrophages were extracted from the tumors at one, two and three weeks post-RT. Gene ontology (GO) term analysis using the DAVID database revealed that genes that were differentially expressed at one and two weeks after irradiation were associated with biological processes such as morphogenesis of a branching structure, tube development, and cell proliferation. Analysis using Short Time-Series Expression Miner (STEM) revealed the temporal gene expression profiles and identified 13 significant patterns in four main groups of profiles. The genes in the upregulated temporal profile have diverse functions involved in the intracellular signaling cascade, cell proliferation, and cytokine-mediated signaling pathway. We show that tumor irradiation with a single 25-Gy dose can initiate a time-series of differentially expressed genes in TAMs, which are associated with the immune response, DNA repair, cell cycle arrest, and apoptosis. Our study helps to improve our understanding of the function of the group of genes whose expression changes temporally in an irradiated tumor microenvironment.

Purpose: To examine whether brain tumors grown in pre-irradiated (PreIR) thigh have a similar tumor bed effect (TBE) as in PreIR brain tissue.Material and methods: Tumor growth delay and immunohistochemical (IHC) staining for CD31, an endothelial surface marker, and PIMO, a hypoxia marker, were used to study the TBE of a murine astrocytoma, ALTS1C1, or a stromal-derived factor-1 (SDF-1) gene-silenced astrocytoma, ALTS1C1-SDFkd, growing in different PreIR stroma beds.Results: ALTS1C1 tumors growing in both PreIR brain and PreIR thigh had reduced microvascular density (MVD) and more chronic hypoxia, but tumor growth delay was only seen in PreIR brain tissue. In contrast, ALTS1C1-SDFkd tumors showed tumor growth delay in PreIR thigh, with little effect in PreIR brain tissue.Conclusions: This study cautions that both the tumor and the nature of the PreIR stromal bed are important when using pre-irradiation as a model of recurrent brain tumors after radiation therapy.

Ion implantation is used to create nanodiamonds (NDs) with embedded magnetic ions for use in a wide range of biological and medical applications; however, the effectiveness of this process depends heavily on separating magnetic NDs from nonmagnetic ones. In this study, we use secondary ion mass spectrometry to verify the implantation of magnetic ions in NDs and the success of separation. When applied to a series of NDs with embedded iron or manganese ions, the sorting tool used in this study proved highly effective in selecting magnetic NDs. Besides, multienergy ion implantation and precise thickness control of NDs coating on the silicon wafer were suggested to improve this technology.

To investigate the biological meaning of apparent diffusion coefficient (ADC) values in tumors following radiotherapy.Five mice bearing TRAMP-C1 tumor were half-irradiated with a dose of 15 Gy. Diffusion-weighted images, using multiple b-values from 0 to 3000 s/mmAs compared with the nonirradiated region, the irradiated region exhibited significant increases in ADC, extracellular space, and nuclear size, and a significant decrease in nuclear counts (P0.001 for all). Optimal ADC to differentiate the irradiated from nonirradiated regions was achieved at a b-value of 800 s/mmAs a radiomic biomarker, ADC maps correlating with histological metrics pixelwise could be a means of evaluating tumor heterogeneity and responses to radiotherapy.1 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;46:483-489.

To quantify the differential plasma flow- (FDCE MRI was performed using a 7T animal scanner in 14 C57BL/6J mice syngeneic for TRAMP tumors, by administering Gd-DTPA (0.9kD) in eight mice and Gadomer (35kD) in the remainder. The acquisition time was 10min with a sampling rate of one image every 2s. Pharmacokinetic modeling was performed to obtain KThe KThe differential contributions of F

Purpose: To investigate the effects of single and fractionated doses of radiation on tumors and tumor-associated macrophages (TAMs), and to elucidate the potential of TAMs to influence tumor growth. Methods and Materials: A murine prostate cell line, TRAMP-C1, was grown in C57Bl/6J mice to 4-mm tumor diameter and irradiated with either 25 Gy in a single dose, or 60 Gy in 15 fractions. The tumors were removed at the indicated times and assessed for a variety of markers related to TAM content, activation status, and function. Results: In tumors receiving a single radiation dose, arginase (Arg-I), and cycloxygenase-2 (COX-2) mRNA expression increased as a small transient wave within 24 h and a larger persistent wave starting after 3 days. Inducible nitric oxide synthase (iNOS) mRNA was elevated only after 3 days and continued to increase up to 3 weeks. After fractionated irradiation, Arg-1 and COX-2 mRNA levels increased within 5 days, whereas iNOS was increased only after 10 fractions of irradiation had been given. Increased levels of Arg-I, COX-2, and, to a lesser extent, iNOS protein were found to associate with TAMs 1–2 weeks after tumor irradiation. Function of TAMs were compared by mixing them with TRAMP-C1 cells and injecting them into mice; TRAMP-C1 cells mixed with TAMs from irradiated tumors appeared earlier and grew significantly faster than those mixed with TAMs from unirradiated tumors or TRAMP-C1 alone. Conclusions: Tumor-associated macrophages in the postirradiated tumor microenvironment express higher levels of Arg-1, COX-2, and iNOS, and promote early tumor growth in vivo .

Purpose To investigate whether changes in the volume transfer coefficient (K trans ) in a growing tumor could be used as a surrogate marker for predicting tumor responses to radiation therapy (RT) and chemotherapy (CT). Methods and Materials Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was consecutively performed on tumor-bearing mice, and temporal and spatial changes of K trans values were measured along with tumor growth. Tumor responses to RT and CT were studied before and after observed changes in K trans values with time. Results Dynamic changes with an initial increase and subsequent decline in K trans values were found to be associated with tumor growth. When each tumor was divided into core and peripheral regions, the K trans decline was greater in core, although neither vascular structure or necrosis could be linked to this spatial difference. Tumor responses to RT were worse if applied after the decline of K trans , and there was less drug distribution and cell death in the tumor core after CT. Conclusion The K trans value in growing tumors, reflecting the changes of tumor microenvironment and vascular function, is strongly associated with tumor responses to RT and CT and could be a potential surrogate marker for predicting the tumor response to these treatments.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Radiotherapy (RT) is one of modalities for HCC therapy in clinics, but the therapeutic effect is unsatisfied due to high radiosensitivity of normal liver tissues that limits the optimal dose. This study is to develop a new theraprutic approach to enhance the efficiency of RT on HCC tumors. Suberoylanilide hydroxamic acid (SAHA), a FDA approved chemotherapeutic drug, is an inhibitor of histone deacetylase (HDACs) and has been reported to enhance radiosensitivity in many cell lines. This study hypothesized that combination of SAHA and RT could improve tumor control on HCC tumors. HCC-834 cell, a primary culture of human HCC, had a dose-dependent response to RT when analyzed by clonogenic assay. SAHA had mild cytotocixity to HCC-834 cell (IC50=1μM). Combination of SAHA and RT reached a synergestic effect to prevent colony formation. In detailed, SAHA treatment did not induce DNA damage on HCC-834 cells when assayed by the expression of γH2AX. RT of 2Gy induced highest γH2AX expression within 10min and quickly returned to baseline after 24hrs. The expression of γH2AX was sustained at 24hrs when treated by SAHA and RT, and gradually recovered after 48hrs. This data indicated that SAHA inhibited the DNA repair process in HCC-834 cell lines after RT. An orthotopic tumor model was developed for further exploring the role of SAHA on HCC tumor radiotherapy. Mice were orally fed with SAHA (100mg/kg) 3hrs and 24hrs before RT of 25Gy. Mice were sacrificed at day10 after RT and maximum tumor area was examined by H&E staining. Either RT (38.81 ± 15.6mm2) or SAHA alone (45.09 ± 4.493mm2) inhibited tumor growth significantly compared with control tumors (99.1 ± 0.732mm2). Combined SAHA with RT had an additive effect on tumor control (28.03±12.25mm2). These results supported our hypothsis and demonstrated that SAHA combined with local RT was effective on inhibiting DNA repair and anti-tumor growth. This study provided a new strategy on HCC tumor therapy that SAHA could act as a radiosensitizer to improve the therapeutic efficiency of RT on HCC tumors. Citation Format: Fang-Hsin Chen, Ching-Fang Yu, Ji-Hong Hong. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, improved radiosensitivity of human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5844. doi:10.1158/1538-7445.AM2017-5844

Immunotherapy such as anti-PD-1/PD-L1 is effective in several types of tumors, but in general the response rate is less than 30%. Systemic administration of cytokine such as IL-12 could create anti-tumor immunity but has unacceptable side effects. This study is to test our hypothesis that combination of high-dose local radiotherapy (RT) and local production of IL-12 inside tumors within short period could have potent anti-tumor effects but low systemic toxicity. TRAMP-C1 tumors derived from a murine prostate cancer were either no treatment, or irradiated with 25 Gy, or intra-tumorally injected with Ad-sc-IL12 virus (1×10sup8 pfu), or treated with both modalities at the tumor size of 4mm. RT alone and Ad-sc-IL12 alone could not shrink tumors and only caused tumor growth delay around 7 and 12 days, respectively. The combination of RT with Ad-sc-IL12 virus could shrink the tumors to the sizes of < 2mm and further extended the tumor growth delay more than 20 days. The percentage of lymphocytes and IFN-γproduction were evaluated by flow cytometry and ELISA. The percentages of CD4 cells in tumors were too low to be reliably measured in all groups, but the percentages of CD8 cells were increased in Ad-sc-IL12 virus-treated groups and even much higher in those combined with RT. For mice treated with Ad-sc-IL12 virus injection, the serum IL-12 levels were around 4.5 times increase at day7, but those combined with with RT had significantly lower levels than those treated with virus alone. The serum IFN-γ(Th1 response) levels were around 12 times higher in Ad-sc-IL12 groups at day7 as compared to control or RT only group, but had no significant differences between those treated with combined modality and with Ad-sc-IL12 virus alone. Liver damage is an index for IL-12 toxicity and the levels of GOT and GTP were also lower in those treated IL-12 combined with RT than those treated with IL-12 alone. This study supported our hypothesis and showed that combination of local IL-12 production and local RT are effective in increasing CD8 cells, exerting Th1 response, and enhancing tumor growth delay. The most important is that this combination has less systemic toxicity than local IL-12 production alone. This study provides evidence to develop new strategy for producing a strong anti-tumor and low toxicity for radioresistant tumors by combining RT and IL-12 immunotherapy. Citation Format: Ji-Hong Hong, Ching-Fang Yu, Fang-Hsin Chen, Chi-Shiun Chiang. Combination of high-dose irradiation and local interleukin-12 treatment enhance tumor killing and have less toxicities than either treatment alone. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4014.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that leads to the production of GM-CSF, G-CSF, IL-1, IL-6, TNF-α. It can be secreted by various cells, including T cells, NKT cells, fibroblasts, and even tumor cells. In addition, reports have shown that IL-17 could also promote tumor growth by recruiting and expanding granulocytic myeloid suppressor cells (G-MDSCs). In our preliminary study, we found that Tramp-C1, a murine prostate cancer cell line, had remarkable IL-17 level when it grew in pre-irradiated (PreIR) stroma. In contrast, B16F0, a murine melanoma cell line, had much less IL-17 and stronger tumor bed effect, in terms of tumor growth delay, but lung metastasis were formed gradually as B16F0 developed. When the recombinant IL-17 was directly injected into the B16F0 tumors growing in PreIR stroma, we found that the probability of lung metastasis was decreased despite a slight increase of tumor growth at primary site. This indicates that IL-17 may have different effects on irradiated tumor bed and distant metastatic site. The influence of IL-17 on tumor microenvironments of distant metastatic sites and irradiated tumor bed is currently under investigation. In conclusion, we propose a new therapeutic approach of reducing distant metastasis following radiation therapy.. This work was kindly supported by MOST-104-2314-B-182 -024 grant to Fang-Hsin Chen. Citation Format: Yi-Nan Li, Fang-Hsin Chen, Ji-Hong Hong, Chi-Shiun Chiang. Intratumoral injection of interleukin-17 inhibits distant metastasis of radiation-induced recurrent tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1575.

Recent studies indicate that sunitinib, a tyrosine kinase inhibitor, could decease accumulation of myeloid-derived suppressor cells (MDSC) in tumors and also in peripheral blood. This study aims to investigate the behavior and dynamic profile of myeloid-derived suppressor cells in tumor tissues and peripheral blood of sunitinib-treated mice bearing prostate adenocarcinoma TRAMP-C1 tumors. To this end, 3×106 TRAMP-C1 tumor cells were inoculated at shank muscle of C57BL6/J mouse and sunitinib was administered by intraperitoneal injection of a daily dose of 20mg/kg into mice bearing 4 mm diameter tumor. Comparing to tumors in untreated group, sunitinib administration slightly delayed tumor growth delay for 2 days, but significantly decreased micro-vascular density and induced chronic hypoxia in tumors, resulting in the specific accumulation of CD11b+Gr-1+ MDSCs at the tumor necrotic region within CA-IX positive chronic hypoxic area. Flow cytometry was used to analyze the change of CD11b+ myeloid cells within tumor tissues and peripheral blood. Results showed that sunitinib treatment decreased the percentage of CD11b+Ly6G-LyC- tumor-associated macrophages (TAMs), but increased the percentage of CD11b+Ly6G-Ly6C+ monocytic MDSCs (M-MDSCs) while had no effect on CD11b+Ly6G+Ly6C+ neutrophilic MDSCs (N-MDSCs) within tumor tissues. In contrast, both the percentage of N-MDSCs and M-MDSC were expanded in peripheral blood of sunitinib-treated mice. Multiplex immunoassay showed significant increase of CCL2, CCL3, CCL5, CXCL5, IL-17a, GM-CSF, G-CSF and VEGF-A in tumor tissues, but only CXCL5, IL-á, IL-6 and G-CSF in the peripheral blood of sunitinib-treated mice were affected. In conclusion, this study shows that sunitinib has anti-vascular effect and could disrupt the recruitment of CD11b+Ly6G-Ly6C- TAMs into tumors. We also propose that sunitinib might exert different effects on tumor microenvironment and peripheral blood. Citation Format: Fang-Hsin Chen, Sheng-Yung Fu, Chun-Chieh Wang, Chi-Shiun Chiang, Ji-Hong Hong. Sunitinib has opposite roles to regulate the myeloid-derived suppressor cells in tumors and peripheral blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 759.

Purpose To investigate vascular responses during fractionated radiation therapy (F-RT) and the effects of targeting pericytes or bone marrow-derived cells (BMDCs) on the efficacy of F-RT. Methods and Materials Murine prostate TRAMP-C1 tumors were grown in control mice or mice transplanted with green fluorescent protein-tagged bone marrow (GFP-BM), and irradiated with 60 Gy in 15 fractions. Mice were also treated with gefitinib (an epidermal growth factor receptor inhibitor) or AMD3100 (a CXCR4 antagonist) to examine the effects of combination treatment. The responses of tumor vasculatures to these treatments and changes of tumor microenvironment were assessed. Results After F-RT, the tumor microvascular density (MVD) was reduced; however, the surviving vessels were dilated, incorporated with GFP-positive cells, tightly adhered to pericytes, and well perfused with Hoechst 33342, suggesting a more mature structure formed primarily via vasculogenesis. Although the gefitinib+F-RT combination affected the vascular structure by dissociating pericytes from the vascular wall, it did not further delay tumor growth. These tumors had higher MVD and better vascular perfusion function, leading to less hypoxia and tumor necrosis. By contrast, the AMD3100+F-RT combination significantly enhanced tumor growth delay more than F-RT alone, and these tumors had lower MVD and poorer vascular perfusion function, resulting in increased hypoxia. These tumor vessels were rarely covered by pericytes and free of GFP-positive cells. Conclusions Vasculogenesis is a major mechanism for tumor vessel survival during F-RT. Complex interactions occur between vessel-targeting agents and F-RT, and a synergistic effect may not always exist. To enhance F-RT, using CXCR4 inhibitor to block BM cell influx and the vasculogenesis process is a better strategy than targeting pericytes by epidermal growth factor receptor inhibitor.

Purpose To investigate vasculatures and microenvironment in tumors growing from preirradiated tissues (pre-IR tumors) and study the vascular responses of pre-IR tumors to radiation and antiangiogenic therapy. Methods and Materials Transgenic adenocarcinoma of the mouse prostate C1 tumors were implanted into unirradiated or preirradiated tissues and examined for vascularity, hypoxia, and tumor-associated macrophage (TAM) infiltrates by immunohistochemistry. The origin of tumor endothelial cells was studied by green fluorescent protein–tagged bone marrow (GFP-BM) transplantation. The response of tumor endothelial cells to radiation and antiangiogenic agent was evaluated by apoptotic assay. Results The pre-IR tumors had obvious tumor bed effects (TBE), with slower growth rate, lower microvascular density (MVD), and more necrotic and hypoxic fraction compared with control tumors. The vessels were dilated, tightly adhered with pericytes, and incorporated with transplanted GFP-BM cells. In addition, hypoxic regions became aggregated with TAM. As pre-IR tumors developed, the TBE was overcome at the tumor edge where the MVD increased, TAM did not aggregate, and the GFP-BM cells did not incorporate into the vessels. The vessels at tumor edge were more sensitive to the following ionizing radiation and antiangiogenic agent than those in the central low MVD regions. Conclusions This study demonstrates that vasculatures in regions with TBE are mainly formed by vasculogenesis and resistant to radiation and antiangiogenic therapy. Tumor bed effects could be overcome at the edge of larger tumors, but where vasculatures are formed by angiogenesis and sensitive to both treatments. Vasculatures formed by vasculogenesis should be the crucial target for the treatment of recurrent tumors after radiotherapy.

Macrophages display different phenotypes that can switch in response to their micro-environment. In our earlier study (Chiang, C. S., Liu, W. C. and Jung, S. M., 2005. Compartmental responses after thoracic irradiation of mice: strain differences. Int. J. Radiat. Oncol. Biol. Phys. 62:862) on radiation-induced cytokine expression in lung lavage samples, there was a suggestion that the procedures used to harvest lung macrophages affected the profiles they expressed. To further explore this issue, we examined gene expression by cell populations, mainly macrophages, isolated by lavage from lung and peritoneal cavity following either in vivo or in vitro stimulation with LPS, IFN-gamma or irradiation. We found that expression of mRNA for tumor necrosis factor-alpha, IL-1 alpha/beta and IL-6 varied several fold depending on whether the assay was performed on cells immediately after isolation or after in vitro manipulation. The relative level of inducible nitric oxide synthase (iNOS) to arginase I (Arg I), which is frequently used as index of the M1 versus M2 functional macrophage phenotype, also varied. LPS stimulation in vivo was able to change the profile from Arg I expression to one where the iNOS pathway became dominant, but was unable to do this in vitro. This contrasts with the ability of IFN-gamma to generate an iNOS-dominant pathway in vitro, but not in vivo. This study cautions that the expression of inflammatory cytokines and the iNOS to Arg I ratio, which is often used as an index of their functional capacity, varies with the experimental conditions.

Purpose: To examine and compare the molecular and cellular processes leading to radiation fibrosis and pneumonitis in C57BL/6J and C3H/HeN mice. Methods and Materials: At indicated times after various doses of thoracic irradiation, the cell populations obtained by bronchoalveolar lavage of C57BL/6J mice were differentially analyzed by cytology and assessed by RNase protection (RPA) assay for levels of cytokines and related genes. The molecular responses in bronchial alveolar lavage (BAL) populations were compared with those in whole lung of C57BL/6J mice and with those of C3H/HeN mice. The former strain develops late radiation fibrosis, whereas the latter develop subacute radiation pneumonitis. Results: In C57BL/6J mice, a decrease in the total number of BAL cells was found 1 week after 6, 12, or 20 Gy thoracic irradiation with a subsequent dose-dependent increase up to 6 months. After 12 and 20 Gy, large, foamy macrophages and multinucleated cells became evident in BAL at 3 weeks, only to disappear at 4 months and reappear at 6 months. This biphasic response was mirrored by changes expression of mRNA for proinflammatory cytokines and the Mac-1 macrophage-associated antigen. As with BAL, whole lung tissue also showed biphasic cytokine and Mac-1 mRNA responses, but there were striking temporal differences between the two compartments, with changes in whole lung tissue correlating better than BAL with the onset of fibrosis in this strain. The radiation-induced proinflammatory mRNA responses had strain-dependent and strain-independent components. Thoracic irradiation of C3H/HeN induced similar increases in tumor necrosis factor (TNF)-α, interleukin (IL)-1α/β, and interferon (IFN)-γ mRNA expression in lung as it did in C57BL/6J mice during the “presymptom” phase at 1–2 months. However, immediately preceding and during the pneumonitic time period at 3–4 months, TNF-α and IL-1α/β mRNAs were highly upregulated in C3H/HeN mice, which develop pneumonitis, but not in C57BL/6J mice, which do not. At the onset of radiation fibrosis in C57BL/6J mice (5–6 months), irradiated lungs had increased levels of IL-1α/β and IFN-γ mRNA expression, but the TNF-α response was, notably, still muted. Conclusions: The major molecular and cellular events in lungs of C57BL/6J and C3H/HeN mice, which develop late fibrosis and subacute pneumonitis after thoracic irradiation respectively, take place within the interstitium and are not reflected within BAL populations. The initial proinflammatory responses are similar in the two strains, but later responses reflect the latent time to lesion development. TNF-α expression at 3–4 months may be important in radiation-induced pneumonitis, and its downregulation is important in avoiding this radiation-induced complication.

To determine the contribution of intra-alveolar cells as opposed to cells fixed in the interstitium in the development of radiation-induced lung injury.C3H/HeN mice were irradiated to the thorax with various doses of radiation. The cellular composition and cytokine production were assessed in the two sites by histological staining and RNase protection assay.Following thoracic irradiation, there was an initial decrease in the number of bronchial alveolar lavage (BAL) cells that was followed after 2 months by a dose-dependent increase up to 4 months. Foamy Mac-1 positive macrophages were present early in the BAL populations, which also expressed the pro-inflammatory cytokines TNF-alpha, IL-1alpha and IL-1beta, but this response subsided by the time of onset of pneumonitis (3 months). In contrast, in whole lung tissue there was a steady increase in Mac-1 positive cells and increased expression of TNF-alpha, IL-1alpha and IL-1beta mRNAs to maximum levels at 3-4 months.These data indicate distinct temporal and spatial changes in pro-inflammatory cytokine gene expression in different cellular compartments of the irradiated lung. BAL cells became inflammatory early on, but interstitial cells became involved later and were probably more involved in contributing to the pneumonitis.

Tumor-associated macrophages (TAMs) are a major non-cancer component in solid tumors and actively participate in remodeling tumor microenvironment after a variety of treatment; however their functions in irradiated tumors are not fully clarified. Our previous studies showed that high-dose irradiation to TRAMP-C1 tumor (a mouse prostate cancer cell line) decreased micro-vascular density (MVD) and induced CD68+ TAMs aggregation at avascular hypoxia region. These findings drive us to examine if these aggregated CD68+ TAMs promote regrowth of irradiated tumors. The TRAMP-C1 tumor cells were i.m. injection into shank muscle and irradiated with single-dose 25Gy at size of 4mm diameter. Sutent, a tyrosine kinase inhibitor, was i.p. injected (20mg/kg per day) to some mice and served as a model for hypoxia induced by anti-angiogenesis treatment. Tumors treated with Sutent alone also had decreased MVD and large amount of avascular hypoxia, but the aggregation of CD68+ TAMs in avascular hypoxia region was only observed in irradiated tumors, not in sutent-treated tumors; this finding suggests this aggregation of CD68+ TAMs in hypoxic region is not a universal finding and could be specific for irradiated tumor microenvironment. The phenotype of TAMs in regions with and without aggregation was studied and the differences existed. Arg-1+iNOS− TAMs were the dominant phenotype outside the avascular hypoxia regions and randomly distributed, but presence and accumulation of Arg-1+iNOS+ TAMs in avascular hypoxia regions was detected. The NOS inhibitor, L-NAME (500mg/L in drinking water), was therefore used to block the effects of nitric oxide produced from specific Arg-1+iNOS+ TAMs accumulated in avascular hypoxia region. The blockade of nitric oxide delayed tumor regrowth in irradiated but not in control tumors. Aggregation of TAMs and presence of Arg-1+ iNOS+ TAMs in avascular hypoxia regions were totally vanished in tumors treated with NOS inhibitor. In addition, the MVD was further decreased and percentages of avascular hypoxia regions were increased in the irradiated tumor treated with NOS inhibitor. These findings suggested that Arg-1+iNOS+TAMs accumulated in avascular hypoxia regions promote tumor regrowth after irradiation, and nitric oxide blockage is a potential strategy to improve the effects of radiotherapy. (This work is supported by grants 101-2314-B-182A-129-MY3 from National Science Council, Taiwan, CMRPG390943 from Chang Gung Memorial Hospital) Citation Format: Ji-Hong Hong, Sheng-Yung Fu, Fang-Hsin Chen, Chun-Chieh Wang, Chi-Shiun Chiang. The role of Arg-1+iNOS+ tumor-associated macrophages and nitric oxide on tumor microenviroment after high-dose irradiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1532. doi:10.1158/1538-7445.AM2013-1532