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Rationale: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-b signaling. TGF-b is secreted in a latent complex with its propeptide, and TGF-b activators release TGF-b from this complex. Because the integrin avb6 is a major TGF-b activator in the lung, inhibition of avb6-mediated TGF-b activation is a logical strategy to treat lung fibrosis. Objectives: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on avb6. Methods: Wild-type mice, avb6-deficient (Itgb6 2/2 ) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1 1/RGE ) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-avb6 monoclonal antibody or a soluble TGF-b receptor fusion protein. avb6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. Measurements and Main Results: b6 Integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6 2/2 mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-avb6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6 2/2 mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. Conclusions: avb6-Mediated TGF-b activation is required for radiationinduced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for avb6 in distinct fibrosis models. Inhibition of avb6-mediated TGF-b activation is a promising new approach for antifibrosis therapy.

Purpose: Ionizing radiation therapy (RT) is an important component in the management of breast cancer. Although the primary tumor can be successfully treated by surgery and RT, metastatic breast cancer remains a therapeutic challenge. Here we tested the hypothesis that the combination of RT to the primary tumor with CTLA-4 blockade can elicit antitumor immunity inhibiting the metastases.Experimental Design: The poorly immunogenic metastatic mouse mammary carcinoma 4T1 was used as a model. Mice were injected s.c. with 4T1 cells, and treatment was started 13 days later when the primary tumors measured 5 mm in average diameter. Mice were randomly assigned to four treatment groups receiving: (1) control IgG (IgG), (2) RT + IgG, (3) 9H10 monoclonal antibody against CTLA-4, (4) RT + 9H10. RT was delivered to the primary tumor by one or two fractions of 12 Gy. 9H10 and IgG were given i.p. thrice after RT.Results: Consistent with the fact that 4T1 is poorly immunogenic, 9H10 alone did not have any effect on primary tumor growth or survival. RT was able to delay the growth of the primary irradiated tumor, but in the absence of 9H10 survival was similar to that of control mice. In contrast, mice treated with RT + 9H10 had a statistically significant survival advantage. The increased survival correlated with inhibition of lung metastases formation and required CD8+ but not CD4+ T cells.Conclusions: The combination of local RT with CTLA-4 blockade is a promising new immunotherapeutic strategy against poorly immunogenic metastatic cancers.

Purpose-To determine whether the tubulin-binding drug noscapine could enhance radiosensitivity of GL261 glioma tumors by inhibiting tumor angiogenesis.Methods and Materials-The human T98G and murine GL261 glioma cell lines treated with noscapine, radiation, or both were assayed for clonogenic survival. Mice with established GL261 hind limb tumors were treated with noscapine, radiation or both to evaluate the effect of noscapine on radioresponse. In a separate experiment with the same treatment groups, 7 days after radiation tumors were resected and immunostained to measure proliferation rate, apoptosis and angiogenic activity.Results-Noscapine reduced clonogenic survival without enhancement of radiosensitivity in vitro. Noscapine combined with radiation significantly increased tumor growth delay: 5, 8, 13, 18 days for control, noscapine alone, radiation alone, and the combination treatment, respectively (p < 0.001). To assess the effect of the combination of noscapine plus radiation on the tumor vasculature, tubule formation by the murine endothelial 2H11 cells was tested. Noscapine with radiation significantly inhibited tubule formation compared with radiation alone. By immunohistochemistry, tumors treated with the combination of noscapine plus radiation showed a decrease in BrdU incorporation, an increase in apoptosis by TUNEL and a decrease in tumor vessel density compared with tumors treated with radiation alone. Conflict of interest: none.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conclusion-Noscapine enhanced the sensitivity of GL261 glioma tumors to radiation resulting in a significant tumor growth delay. These findings are clinically relevant, particularly in view of the mild toxicity profile of this drug. NIH Public Access

We have used in vivo 19F NMR spectroscopy to study the metabolism of 5-fluorouracil (FUra) in tumors with and without pretreatment with methotrexate (MTX). Using the CD8F1 murine mammary tumor as an in vivo model, we observed signals from FUra, alpha-fluoro-beta-alanine (F beta ALA), alpha-fluoro-beta-ureidopropionic acid (FUPA), and 5-fluorouracil-nucleotides (FUN) after intravenous or intraperitoneal injection of 150 mg/kg FUra. Formation of FUN was increased about 1.7-fold in CD8F1 tumor with methotrexate pretreatment as determined by acid extraction and HPLC analysis. A comparison of in vivo NMR spectra from FUra and sequential MTX + FUra-treated tumors showed a significantly higher ratio of the FUN signal to the initial total 19F signal in the MTX + FUra-treated tumors (p less than 0.001) for animals receiving FUra either intravenously or intraperitoneally. In addition, tumors treated with MTX + FUra had significantly longer time durations during which FUN was detected, independent of the mode of administration. These experiments indicate that in vivo 19F NMR spectroscopy can be used to noninvasively monitor alterations of 5-fluorouracil metabolism that occur with administration of modulating agents such as methotrexate. Further studies, in both murine tumor models and patients, are indicated to determine if these results can be correlated with tumor response.