Mammary Carcinoma Cell Derived Cyclooxygenase 2 Suppresses Tumor Immune Surveillance by Enhancing Intratumoral Immune Checkpoint Activity

INTRODUCTION: Systemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreases the risk of breast cancer and its recurrence. However, the biology of COX-2 in the multicellular tumor microenvironment is poorly defined. METHODS: Mammary tumor onset and multiplicity were examined in ErbB2 transgenic mice that were deficient in mammary epithelial cell COX-2 (COX-2(MEC)KO) compared to wild type (WT) mice. Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, for proliferation, apoptosis, angiogenesis and immune microenvironment. Lentiviral shRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breast cancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenic recipient mice, with or without depletion of CD8(+ )immune cells. RESULTS: Mammary tumor onset was delayed, and multiplicity halved, in COX-2(MEC)KO mice compared to WT. COX-2(MEC)KO tumors showed decreased expression of Ki67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2, endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumor vascularization. COX-2(MEC)KO tumors contained more CD4(+ )T helper (T(h)) cells and CD8(+ )cytotoxic immune cells (CTL) consistent with increased immune surveillance. The ratio of T(h )markers Tbet (T(h)1) to GATA3 (T(h)2) was higher, and levels of Retnla, a M2 macrophage marker, lower, in COX-2(MEC)KO tumor infiltrating leukocytes compared to WT, suggesting a prevalence of pro-immune T(h)1 over immune suppressive T(h)2 lymphocytes, and reduced macrophage polarization to the immune suppressive M2 phenotype. Enhanced immune surveillance in COX-2(MEC)KO tumors was coincident with increased intratumoral CXCL9, a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitory receptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was also decreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumors in immune competent mice was markedly suppressed. However, robust growth of COX-2KD tumor cells was evident when recipients were depleted of CD8(+ )cells. CONCLUSIONS: The data strongly support that, in addition to its angiogenic function, tumor cell COX-2 suppresses intratumoral cytotoxic CD8(+ )immune cell function, possibly through upregulation of immune checkpoints, thereby contributing to tumor immune escape. COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy.