Antiangiogenic effects of noscapine enhance radioresponse for GL261 tumors.

PURPOSE: To assess the effects of noscapine, a tubulin-binding drug, in combination with radiation in a murine glioma model. 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, and 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 terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, and a decrease in tumor vessel density compared with tumors treated with radiation alone. CONCLUSION: Noscapine enhanced the sensitivity of GL261 glioma tumors to radiation, resulting in a significant tumor growth delay. An antiangiogenic mechanism contributed to the effect. These findings are clinically relevant, particularly in view of the mild toxicity profile of this drug.

Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth factor-beta, prevents radiation-induced lung fibrosis.

RATIONALE: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta 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 alpha(v)beta6. METHODS: Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. MEASUREMENTS AND MAIN RESULTS: Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 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-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. CONCLUSIONS: Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy.

Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer.

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.

Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated.

PURPOSE: Ionizing radiation can reduce tumor growth outside the field of radiation, known as the abscopal effect. Although it has been reported in multiple malignancies, the abscopal effect remains a rare and poorly understood event. Ionizing radiation generates inflammatory signals and, in principle, could provide both tumor-specific antigens from dying cells and maturation stimuli that are necessary for dendritic cells' activation of tumor-specific T cells. We therefore tested the hypothesis that the abscopal effect elicited by radiation is immune mediated. This was directly tested by enhancing the number of available dendritic cells using the growth factor Flt3-Ligand (Flt3-L). METHODS AND MATERIALS: Mice bearing a syngeneic mammary carcinoma, 67NR, in both flanks were treated with Flt3-L daily for 10 days after local radiation therapy (RT) to only 1 of the 2 tumors at a single dose of 2 or 6 Gy. The second nonirradiated tumor was used as indicator of the abscopal effect. Data were analyzed using repeated measures regression. RESULTS: RT alone led to growth delay exclusively of the irradiated 67NR tumor, as expected. Surprisingly, growth of the nonirradiated tumor was also impaired by the combination of RT and Flt3-L. As control, Flt3-L had no effect without RT. Importantly, the abscopal effect was shown to be tumor specific, because growth of a nonirradiated A20 lymphoma in the same mice containing a treated 67NR tumor was not affected. Moreover, no growth delay of nonirradiated 67NR tumors was observed when T cell deficient (nude) mice were treated with RT plus Flt3-L. CONCLUSIONS: These results demonstrate that the abscopal effect is in part immune mediated and that T cells are required to mediate distant tumor inhibition induced by radiation.