title: Mammary carcinoma cell derived cyclooxygenase 2 suppresses tumor immune surveillance by enhancing intratumoral immune checkpoint activity.
abstract: BACKGROUND
  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 (Th) cells and CD8+ cytotoxic immune cells (CTL) consistent with increased immune surveillance. The ratio of Th markers Tbet (Th1) to GATA3 (Th2) 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 Th1 over immune suppressive Th2 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.
authors.forename: Nune
authors.initials: N
authors.lastname: Markosyan
authors.forename: Edward P
authors.initials: EP
authors.lastname: Chen
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Evans
authors.forename: Victoire
authors.initials: V
authors.lastname: Ndong
authors.forename: Robert H
authors.initials: RH
authors.lastname: Vonderheide
authors.forename: Emer M
authors.initials: EM
authors.lastname: Smyth
mesh_terms: D000818:Animals; D060890:B7-H1 Antigen / Q000235:genetics / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D002277:Carcinoma / Q000276:immunology* / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000276:immunology; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D007157:Immunologic Surveillance; D008213:Lymphocyte Activation / Q000276:immunology; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D018345:Mice, Knockout; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000276:immunology; D010641:Phenotype; D019139:Tumor Escape / Q000276:immunology
chemical_list: D060890:B7-H1 Antigen; D051546:Cyclooxygenase 2