Prostate cancer peripheral blood NK cells show enhanced CD9, CD49a, CXCR4, CXCL8, MMP-9 production, and secrete monocyte-recruiting and polarizing factors

Abstract

ABSTRACTBackground Natural killer (NK) cells are effector lymphocytes of the innate immunity. Two major NK cell subsets are mostly present in the peripheral blood (pNKs): the cytotoxic CD56dimCD16+ NK cell subset (90-95% of pNKs), and the low cytotoxic, highly cytokine-producing CD56brightCD16-/low NK cell subset (5-10% of pNKs). It has been demonstrated that NK cells in peripheral blood of patients with several tumors are altered. We have shown that in NSCLC and colon cancer, tumor associated circulating NK (pTA-NK) and tumor infiltrating NK (TI-NK) are skewed towards the CD56brightCD16-/low phenotype. We have detected the production of pro-inflammatory and pro-angiogenic cytokines and chemokines. Other groups are reporting similar observations. There is still a lack of knowledge concerning the phenotype of pNK cells in prostate cancer (PCa). Here, we phenotypically and functionally characterized peripheral blood NK (pNK) from PCa patients (PCa pTA-NKs) and investigated their production of soluble factors, with endothelial cells and macrophage stimulatory action.Methods NK cell subset distribution was investigated in the peripheral blood of PCa patients, by multicolor flow cytometry (FC) for surface antigens expression. Protein arrays were performed to characterize the secretome on FACS-sorted pNK cells. Secreted products from FACS-sorted PCa TA-NKs were used to characterize their production of pro-inflammatory molecules. Secreted products from FACS-sorted PCa pTA-NKs were also used to stimulate endothelial cells and monocytes and macrophages, determining their ability to recruit and polarize them. Alterations of endothelial cells and monocytes, following exposure to secreted products from FACS-sorted PCa pTA-NKs, was assessed by RT-PCR. To confirm these observations, secreted products from 3 different PCa (PC-3, DU-145, LNCaP) cell lines were used to assess their effects on human NK cell polarization, by multicolor flow cytometry.Results Circulating NK cells from prostate cancer patients have been studied before, mostly for their impaired lytic functions. However, here we are the first to report that circulating pNK cells from PCa patients acquire a CD56brightCD9+CD49a+CXCR4+ phenotype with pro-inflammatory properties. We observed a similar polarization of heathy-donor derived pNK cells exposed to secreted products of three different PCa cell lines. Increased production of CXCL8, CXCR4, MMP-9, pro-inflammatory and reduced production of TNFα, IFNγ and Granzyme-B was detected. PCa TA-NKs released factors able to support angiogenesis in vitro and increased the expression of CXCL8, ICAM-1 and VCAM-1 mRNA in endothelial cells, confirming a pro-inflammatory signature. Secretome analysis revealed the ability of PCa pTA-NKs to release pro-angiogenic cytokines/chemokines involved in monocyte recruitment and M2-like polarization. In experimental setting, secreted products from PCa pTA-NKs can recruit THP-1 monocyte and polarize THP-1-differentiated macrophage towards CD206/Arginase1/IL-10/CXCL8-expressing M2-like/TAMs.Conclusions Our results show that PCa pTA-NKs are effector cells able to produce pro-inflammatory angiogenesis factors able to stimulate endothelial cells, attract monocytes and polarize macrophage to an M2-like type. Our data provides a rationale for the possible use of pNK profiling in clinical studies on PCaGraphical Abstract: Representative cartoon illustrating the pro-angiogenic features of PCa pTA-NKs.A) direct effects of PCa pTA-NKs in supporting angiogenesis by interacting with endothelial cells. B) Proposed model for PCa pTA-NK pro-angiogenic activities via macrophage recruitment and polarization.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONSADCCAntibody Dependent Cellular CytotoxicityADKAdenocarcinomaANGAngiogeninANGIOP1Angiopoietin 1ANOVAAnalysis of VarianceCCLChemokine Ligand (C-C motif)cDNAcomplementary DNACMConditioned MediaCRPCCastration Resistant Prostate CancerCXCLChemokine Ligand (C-X-C motif)DNAM-1DNAX accessory molecule 1dNK cellsdecidual Natural Killer cellsEBMEndothelial cell Basal MediumEGMEndothelial cell Growth MediumFACSFluorescence-Activated Cell SortingFBSFetal Bovine SerumFCFlow CytometryFSCForward ScatterGAPDHGlyceraldehyde-3-Phosphate DehydrogenaseGM-CSFGranulocyte-Macrophage Colony-Stimulating FactorHBVHepatitis B VirusHCHealthy ControlHCVHepatitis C VirusHIVHuman Immunodeficiency VirusHUVECHuman Umbilical Vein Endothelial CellICAMInter Cellular Adhesion Molecule-1IFN-γ = Interferon γIHCImmuno Histo ChemistryIL-Interleukin-I-TACInterferon-inducible T-cell Alpha ChemoattractantmAbsmonoclonal AntibodiesMCP-1/CCL2Monocyte Chemoattractant Protein-1MMPsMatrix Metallo ProteinasesMNCsMono Nuclear CellsNKNatural KillerNKG2DNatural Killer receptor Group 2 DNSCLCNon-Small Cell Lung CancerPAIPlasminogen Activator InhibitorPBPeripheral Blood cellsPBMCsperipheral Blood Mononuclear CellsPCaProstate CancerPD-L1Programmed Death receptor Ligand 1PGE2Prostaglandin E2PlGFPlacental Growth FactorPMAPhorbol Merystate AcetateP/SPenicillin / StreptomycinPTA-NKsProstate Tumor Associated Natural Killer cellsRANTESRegulated upon Activation, Normal T Cell Expressed and Presumably SecretedSSCSide ScatterSTATSignal Transducer and Activator of TranscriptionTAMsTumor Associated MacrophagesTGFβTransforming Growth Factor-βTIMPTissue Inhibitor of Metallo-ProteinaseTMETumor MicroenvironmentTNF αTumor Necrosis Factor-αTregT Regulatory CellsuPARurokinase-type Plasminogen Activator ReceptorVCAMVascular Cellular Adhesion Molecule-1VEGFVascular Endothelial Growth FactorVEGFRVascular Endothelial Growth Factor ReceptorView Full Text