Abstract 2754: Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model
Authors: Somasundaram Veena , Basudhar Debashree , McGinity Christopher , Cheng Robert Y. , Ridnour Lisa A. , Lockett Stephen J. , Wink David A. 
Published: September 2020

Abstract
Progression of aggressive, triple negative breast cancers (TNBC) is driven by inflammation and the presence of tumor infiltrating lymphocytes (TIL) improves survival. Recent studies show that TNBCs show high levels of TILs but these cells are exhausted as evident from decreased IFNγ, Ki67 and granzyme B. High expression of the inflammation-associated protein, inducible nitric oxide synthase (NOS2) and co-expression with high cyclooxygenase 2 (COX2) means poor prognosis in ER- breast cancer. Interestingly, NOS2 and COX2 are upregulated in different cells in the tumor microenvironment (TME). Thus, we hypothesized that the tandem use of NOS2 and COX2 inhibitors would curtail tumor progression by targeting different cell types within the TME simultaneously. As COX2 has also been reported to regulate tumor infiltration of myeloid cells, this could regulate both the lymphoid and myeloid arms of the immune system for better response. We investigated the effects of NOS2 and COX2 on tumorigenesis and pulmonary metastasis in a murine model of TNBC by injecting 4T1 cells into the fourth mammary fat pad of wild type (WT) and NOS2 knockout (NOS2-/-) BALB/c mice followed by treatment with COX inhibitor, indomethacin. Changes in infiltrating immune cells was analyzed using flow cytometry and confocal microscopy. We measured the levels of serum cytokines using bead-based, LegendPlex assay. We found that NOS2-/- mice developed fewer pulmonary metastatic lesions than WT mice while indomethacin directly reduced primary tumor growth in both WT and NOS2-/- mice. Indomethacin treated NOS2-/- mice showed better median survival (60 days compared 51 in WT mice). Two NOS2-/-, indomethacin treated mice showed complete remission and long term (5-months) tumor growth delay after 4T1 re-challenge, indicating immune activation in mice lacking NOS2 and COX2 function. Flow cytometry of primary tumors showed higher infiltration of activated CD4, CD8 cells, macrophages and reduction of Tregs in treated mice. Microscopy revealed a physical ‘walling -off' of TILs by CD11b+ cells in untreated tumors that was abrogated upon indomethacin treatment. As many TNBC patients succumb to pulmonary metastasis and because we found reduced metastasis in NOS2-/- mice, we studied the immune cells in metastatic lungs. Lung niche of NOS2-/- mice had more activated T-cells, bone marrow-derived macrophages and neutrophils that are not conducive to establishment of metastasis. Serum of NOS2-/- mice had elevated IL-6 that drove increased IL17A subsequently contributing to reduced metastasis and this effect was improved upon indomethacin treatment. IL6 is also a potential inhibitor of Tregs thus further improving immune surveillance. COX inhibition can treat aggressive TNBC directly by reducing cancer cell proliferation and indirectly by immune activation. Tandem NOS2 inhibition can render the pulmonary microenvironment unfavorable for metastatic spread and induce a favorable systemic cytokine storm that further improves outcome.
Citation Format: Veena Somasundaram, Debashree Basudhar, Christopher McGinity, Robert Y. Cheng, Lisa A. Ridnour, Stephen J. Lockett, David A. Wink. Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2754.