Matthew D. Fountain

Soy Isoflavones Promote Radioprotection of Normal Lung Tissue by Inhibition of Radiation-Induced Activation of Macrophages and Neutrophils

Introduction: Radiation therapy for lung cancer is limited by toxicity to normal lung tissue that results from an inflammatory process, leading to pneumonitis and fibrosis. Soy isoflavones mitigate inflammatory infiltrates and radiation-induced lung injury, but the cellular immune mediators involved in the radioprotective effect are unknown. Methods: Mice received a single dose of 10 Gy radiation delivered to the lungs and daily oral treatment of soy isoflavones. At different time points, mice were either processed to harvest bronchoalveolar lavage fluid for differential cell counting and lungs for flow cytometry or immunohistochemistry studies. Results: Combined soy and radiation led to a reduction in infiltration and activation of alveolar macrophages and neutrophils in both the bronchoalveolar and lung parenchyma compartments. Soy treatment protected F4/80+CD11c− interstitial macrophages, which are known to play an immunoregulatory role and are decreased by radiation. Furthermore, soy isoflavones reduced the levels of nitric oxide synthase 2 expression while increasing arginase-1 expression after radiation, suggesting a switch from proinflammatory M1 macrophage to an anti-inflammatory M2 macrophage phenotype. Soy also prevented the influx of activated neutrophils in lung caused by radiation. Conclusions: Soy isoflavones inhibit the infiltration and activation of macrophages and neutrophils induced by radiation in lungs. Soy isoflavones-mediated modulation of macrophage and neutrophil responses to radiation may contribute to a mechanism of resolution of radiation-induced chronic inflammation leading to radioprotection of lung tissue.

Radiation-Induced Esophagitis is Mitigated by Soy Isoflavones

Introduction Lung cancer patients receiving radiotherapy present with acute esophagitis and chronic fibrosis, as a result of radiation injury to esophageal tissues. We have shown that soy isoflavones alleviate pneumonitis and fibrosis caused by radiation toxicity to normal lung. The effect of soy isoflavones on esophagitis histopathological changes induced by radiation was investigated. Methods C57BL/6 mice were treated with 10 Gy or 25 Gy single thoracic irradiation and soy isoflavones for up to 16 weeks. Damage to esophageal tissues was assessed by hematoxylin–eosin, Masson’s Trichrome and Ki-67 staining at 1, 4, 10, and 16 weeks after radiation. The effects on smooth muscle cells and leukocyte infiltration were determined by immunohistochemistry using anti-αSMA and anti-CD45, respectively. Results Radiation caused thickening of esophageal tissue layers that was significantly reduced by soy isoflavones. Major radiation alterations included hypertrophy of basal cells in mucosal epithelium and damage to smooth muscle cells in muscularis mucosae as well as disruption of collagen fibers in lamina propria connective tissue with leukocyte infiltration. These effects were observed as early as 1 week after radiation and were more pronounced with a higher dose of 25 Gy. Soy isoflavones limited the extent of tissue damage induced by radiation both at 10 and 25 Gy. Conclusion Soy isoflavones have a radioprotective effect on the esophagus, mitigating the early and late effects of radiation injury in several esophagus tissue layers. Soy could be administered with radiotherapy to decrease the incidence and severity of esophagitis in lung cancer patients receiving thoracic radiation therapy.

Innate Immune Pathways Associated with Lung Radioprotection by Soy Isoflavones

Introduction Radiation therapy for lung cancer causes pneumonitis and fibrosis. Soy isoflavones protect against radiation-induced lung injury, but the mediators of radioprotection remain unclear. We investigated the effect of radiation on myeloid-derived suppressor cells (MDSCs) in the lung and their modulation by soy isoflavones for a potential role in protection from radiation-induced lung injury. Methods BALB/c mice (5–6 weeks old) received a single 10 Gy dose of thoracic irradiation and soy isoflavones were orally administrated daily before and after radiation at 1 mg/day. Arginase-1 (Arg-1) and nuclear factor κB (NF-κB) p65 were detected in lung tissue by western blot analysis and immunohistochemistry. Lung MDSC subsets and their Arg-1 expression were analyzed by flow cytometry. Cytokine levels in the lungs were measured by ELISA. Results At 1 week after radiation, CD11b+ cells expressing Arg-1 were decreased by radiation in lung tissue yet maintained in the lungs treated with radiation and soy isoflavones. Arg-1 was predominantly expressed by CD11b+Ly6ClowLy6G+ granulocytic MDSCs (gr-MDSCs). Arg-1 expression in gr-MDSCs was reduced by radiation and preserved by supplementation with soy isoflavones. A persistent increase in Arg-1+ cells was observed in lung tissue treated with combined radiation and soy isoflavones at early and late time points, compared to radiation alone. The increase in Arg-1 expression mediated by soy isoflavones could be associated with the inhibition of radiation-induced activation of NF-κB and the control of pro-inflammatory cytokine production demonstrated in this study. Conclusion A radioprotective mechanism of soy isoflavones may involve the promotion of Arg-1-expressing gr-MDSCs that could play a role in downregulation of inflammation and lung radioprotection.

Abstract B185: Induction of specific immunity to MUC1 antigen by tumor irradiation and cancer vaccines in murine tumor models

We have previously demonstrated that tumor irradiation potentiates cancer vaccines using genetic modification of tumor cells in murine tumor models. We showed that prior tumor irradiation enhanced the response of mice to intratumoral gene therapy with an IL-2 adenovector for non-immunogenic tumors. To investigate whether tumor irradiation augments the immune response to a specific tumor antigen, we have now tested the efficacy of tumor irradiation to enhance the therapeutic effect of MVA-MUC1-IL2 cancer vaccine (Transgene TG4010) for the treatment of murine renal adenocarcinoma Renca cells transfected with MUC1. Established subcutaneous (s.c.) Renca-MUC1 tumors (18mm3) were irradiated with 8 Gy on day 11 and peritumoral s.c. injections of MVA-MUC1-IL2 vector were administered at 106- 107 PFU on day 12 and 20. Tumor growth delays were monitored by tumor measurements and histological responses were evaluated following treatment with radiation alone, vector alone, radiation + MVA-MUC1-IL2 vector or radiation + MVA empty vector. Histological evaluation of tumors at an early time point, by 2-3 weeks after radiation and cancer vaccine, revealed that tumors treated with radiation and vaccine showed extensive areas of necrosis due to complete tumor destruction. Intense hemorrhages were observed due to disruption of tumor vasculature. Small areas of remaining tumor showed apoptotic tumor cells and degenerating giant tumor cells typical of radiation-induced changes. At the periphery of the tumor and infiltrating the tumor, we observed large bands of inflammatory cells including lymphocytes and macrophages as well as fibroblasts spindled shaped cells. Immuno-histochemical staining with CD45 leukocyte marker and F4/80 macrophage marker confirmed extensive infiltration of leukocytes and macrophages at the periphery and inside of areas of tumor destruction. These alterations were not as pronounced with radiation alone suggesting a drastic effect of the combined radiation + vaccine therapy on the tumor microenvironment. Radiation induced tumor growth delays for about 15 days but longer tumor growth delays of 30-35 days were observed with radiation + MVA-MUC1-IL2. By day 55, over 30% of the mice treated with radiation + MVA-MUC1-IL2 had a complete response with no sign of tumor whereas no responders were observed with either radiation alone or cancer vaccine alone. Complete responders were immune to rechallenge with Renca-MUC1 cells. These findings suggest that tumor irradiation given prior to cancer vaccine augments a specific immune response targeted at a specific tumor antigen that results in specific tumor immunity. The mechanisms of interaction between tumor irradiation and gene-mediated immunotherapy could include radiation-induced alterations in the tumor microenvironment, as those observed histologically in our studies, which could facilitate a more effective anti-tumor immune response. These include radiation-induced apoptosis and necrosis of tumor cells causing tumor-debulking and release of tumor antigens for APC presentation. Inflammatory cells mobilized in the tumor by radiation-induced tissue damage could subsequently be activated by the immune response triggered by the cancer vaccine. These studies support investing further pre-clinical and clinical efforts to combine radiotherapy with cancer vaccines for the treatment of cancer. Citation Format: Gilda Gali Hillman, Matthew D. Fountain, Shoshana E. Rothstein, Lyndsey Reich, Lisa Abernathy, Christopher K. Yunker, Fulvio Lonardo, Philippe Slos. Induction of specific immunity to MUC1 antigen by tumor irradiation and cancer vaccines in murine tumor models. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B185.

Abstract 277: Radioprotection of lung tissue involves modulation of radiation-induced macrophage activation by soy isoflavones

Radiation therapy for lung cancer is limited by radiation toxicity to lung tissue that results from a chronic inflammatory response, leading to pneumonitis and fibrosis. We have reported in preclinical mouse models that treatment with soy isoflavones mitigates inflammatory cytokines and fibrosis, but the cellular mediators of radioprotection remain unclear. Macrophages possess the plasticity to respond to environmental stressors in tissues that functionally may range from proinflammatory to immunosuppressive phenotypes. We hypothesize that soy isoflavones mediate radioprotection of normal lung tissue by modifying macrophage phenotype and function in irradiated lungs. In this study, we investigate the role of lung macrophage subsets in radioprotection of lung tissue by soy. BALB/c mice received a single 10 Gy dose of thoracic irradiation with soy isoflavones given orally at 1 mg per day, prior-to and continuously after radiation for up to 18 weeks. Bronchoalveolar lavage (BAL) fluid and lungs were harvested at early and late time points post-radiation. Differential cell counts on BAL fluid cytospins were performed and ratios of enlarged, foamy macrophages and smaller macrophages were calculated. At 4 weeks, lungs were dissociated into single cell suspensions and cells were stained with anti-CD45, anti-F4/80, and anti-CD11c fluorescent antibodies to analyze interstitial (F4/80+CD11c-) and alveolar (F4/80+CD11c+) lung tissue macrophages by flow cytometry. In situ M1 macrophages were detected by immunohistochemical staining in lung tissue sections for the pan-macrophage marker F4/80 and the M1 macrophage activation marker NOS2. At 18 weeks after radiation, there was a significant increase in the percentage of enlarged, foamy macrophages in BAL fluid to 79.0±6.7% compared with 14.6±5.3% in control (p = 0.0003). Soy significantly inhibited this radiation-induced increase to 33.1±8.3% compared with radiation alone (p = 0.0091). In situ staining of F4/80 and NOS2 in lung tissue sections revealed an increase of activated M1 macrophages caused by radiation in contrast to relatively low NOS2 levels in lungs of mice treated with radiation and soy or control. Soy significantly reduced the percentage of F4/80+CD11c- interstitial macrophage (p = 0.0313) in lung tissue post-radiation. F4/80+CD11c+ alveolar macrophages in lungs are significantly decreased (p = 0.0074) after radiation, however soy did not have a significant effect (p = 0.7160). These data indicate that radiation-induced proinflammatory M1 macrophage activation is inhibited by soy. Further studies are ongoing to clarify the role of interstitial and alveolar macrophages in radiation-induced lung inflammation and its regulation by soy. These findings suggest that soy modulation of the macrophage subset functions in response to radiation may play a critical role in soy-mediated radioprotective effects in lungs. Citation Format: Lisa M. Abernathy, Matthew D. Fountain, John M. David, Christopher K. Yunker, Michael C. Joiner, Gilda G. Hillman. Radioprotection of lung tissue involves modulation of radiation-induced macrophage activation by soy isoflavones. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 277. doi:10.1158/1538-7445.AM2015-277