Prostate cancer peripheral blood NK cells show enhanced CD9, CD49a, CXCR4, CXCL8, MMP-9 production, and secrete monocyte-recruiting and polarizing factors
Authors: Baci Denisa , Gallazzi Matteo , Lorenzo Mortara , Bosi Annalisa , Buono Giuseppe , Naselli Angelo , Guarneri Andrea , Dehò Federico , Capogrosso Paolo , Albini Adriana , Noonan Douglas M. , Bruno Antonino 
Published: July 2020

ABSTRACTBackgroundNatural 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-/lowNK 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-/lowphenotype. 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.MethodsNK 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.ResultsCirculating 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 angiogenesisin vitroand increased the expression ofCXCL8, ICAM-1andVCAM-1mRNA 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 towardsCD206/Arginase1/IL-10/CXCL8-expressing M2-like/TAMs.ConclusionsOur 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 ABSTRACTGraphical 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.