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

Systemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreasesthe risk of breast cancer and its recurrence. However, the biology of COX-2 in themulticellular tumor microenvironment is poorly defined.

Mammary tumor onset and multiplicity were examined in ErbB2 transgenic mice thatwere deficient in mammary epithelial cell COX-2 (COX-2^MECKO) comparedto wild type (WT) mice.

Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, forproliferation, apoptosis, angiogenesis and immune microenvironment. LentiviralshRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breastcancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenicrecipient mice, with or without depletion of CD8⁺ immune cells.

Mammary tumor onset was delayed, and multiplicity halved, in COX-2^MECKOmice compared to WT. COX-2^MECKO tumors showed decreased expression ofKi67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2,endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumorvascularization. COX-2^MECKO tumors contained more CD4⁺ Thelper (T_h) cells and CD8⁺ cytotoxic immune cells (CTL)consistent with increased immune surveillance. The ratio of T_h markersTbet (T_h1) to GATA3 (T_h2) was higher, and levels of Retnla,a M2 macrophage marker, lower, in COX-2^MECKO tumor infiltratingleukocytes compared to WT, suggesting a prevalence of pro-immune T_h1over immune suppressive T_h2 lymphocytes, and reduced macrophagepolarization to the immune suppressive M2 phenotype. Enhanced immune surveillancein COX-2^MECKO tumors was coincident with increased intratumoral CXCL9,a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitoryreceptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was alsodecreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumorsin immune competent mice was markedly suppressed. However, robust growth ofCOX-2KD tumor cells was evident when recipients were depleted of CD8⁺ cells.

The data strongly support that, in addition to its angiogenic function, tumor cellCOX-2 suppresses intratumoral cytotoxic CD8⁺ immune cell function,possibly through upregulation of immune checkpoints, thereby contributing to tumorimmune escape. COX-2 inhibition may be clinically useful to augment breast cancerimmunotherapy.