Abstract A71: HER2/neu oncogene induced tumors in mammary epithelial cell COX-2 deficient mice have disease delaying modifications of tumor microenvironment.

Cyclooxygenase (COX) 2 expression is associated with more than 40% of breast cancers and its inhibition leads to decreased disease risk and recurrence. COX-2 promotes tumor cell proliferation and survival but also contributes to the interplay between tumor and stromal cells that drives disease progression. We examined the specific role of mammary epithelial cell (MEC) COX-2 in a HER2/neu oncogene transgenic mouse model of breast cancer. Deletion of MEC COX-2 (COX-2MECKO) delayed mammary tumor onset compared to wild type (WT) littermates (tumor onset at age 23 and 29 weeks, respectively, n=12-17, p=0.006), and lowered tumor multiplicity (4.1 versus 7.6, respectively, n=14-18, p=0.05). Similarly, in WT mice, growth of HER2/neu expressing mammary cancer cells as orthotopic tumors was suppressed by knock-down of tumor cell COX-2 with shRNA. Compared to WT, COX-2MECKO tumors showed reduced Ki67, a marker of proliferation, and CD31, an endothelial cell marker, indicating decreased tumor growth and vascularization. Reduced gene expression of the angiogenic factor VEGF-A and its receptor VEGFR-2, as well as VEGFR-3, the receptor for the lymphangiogenic factor VEGF-C, were all lower in COX-2MECKO tumors, consistent with the reported pro-angiogenic function of COX-2 in tumors. Flow cytometric analysis of COX-2MECKO tumors indicated higher percentages of tumor-associated CD3+CD8+ cytotoxic T lymphocytes, CD3-CD8+ natural killer cells, as well as CD3+CD4+ T helper (Th) lymphocytes, compared to WT. Leukocytes were isolated from COX-2MECKO and WT tumors, using microbeads coated with antibodies against the pan-leukocyte marker CD45, and analyzed for phenotypic marker gene expression. A higher ratio of Tbet (Th1 marker)/GATA3 (Th2 marker), and reduced Retnla (a M2 macrophage marker), was evident in leukocytes from COX-2MECKO tumors compared to WT tumors, indicating prevalence of pro-immune Th1 over immune suppressive Th2 lymphocytes, as well as reduced polarization of macrophages to the pro-tumorigenic M2 phenotype. Taken together, these data reveal a less tolerant immune microenvironment in COX-2MECKO tumors, with enhanced cytotoxic and pro-immune type 1, and suppressed pro-tumorigenic and immune suppressive type 2 tumor-associated macrophages and lymphocytes. This restoration of immune surveillance in COX-2MECKO tumors may be explained, at least in part, by decreased gene expression of the T lymphocyte co-inhibitory receptors CTLA4 and PD1, as well as the PD1 ligand PD-L1. Indeed, PD-L1 protein, measured by flow cytometry, was decreased in IFNγ-treated COX-2 knock-down mammary cancer cells in vitro, and was restored by treatment with prostaglandin (PG) E2, the dominant product of COX-2 in tumors. The data strongly support that, in addition to its pro-angiogenic role, tumor cell COX-2 is paracrine mediator that promotes a permissive tumor immune microenvironment and contributes to tumor immune escape. COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy. Citation Format: Nune Markosyan, Chen Edward, Victoire Ndong, Emer Smyth. HER2/neu oncogene induced tumors in mammary epithelial cell COX-2 deficient mice have disease delaying modifications of tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A71.