Neutrophils isolated from the CT-26 tumor-bearing mice, when co-cultured both with naïve and tumor-bearing NK cells, displayed a decreased production of IFNγ; treatment with PD-L1 neutralizing antibody was effective in limiting tumor-bearing neutrophil inhibitory effects on tumor-bearing NK cells, but did not exert the same inhibitory effect on naïve NK cells ^[3][52]. Additionally, administration of PD-1 neutralizing antibodies was able to antagonize the inhibitory effect of neutrophils on NK cells ^[3][52].

The NKp46 NCR is considered a key molecule for NK-related killing capacity; Valayer et al., showed that NKp46 decreasing on NK cells is related to activated neutrophil-derived serine proteases and in particular, cathepsin G (CG) is responsible for NKp46 extracellular cleavage ^[4][53] (Figure 12Ci), thus causing defective activation of NK cells in in vitro experiments using human-derived cells. The use of a specific CG inhibitor, α1ACT, abrogated the capability of neutrophil-derived conditioned media to decrease NKp46 on the NK cell surface ^[4][53].

Neutrophils can activate or suppress the NK cells cytotoxic functions and therefore can indirectly interfere with the angiogenic process. Romero et al., demonstrated that neutrophil-derived ROS can modulate NKp46 expression on NK targeted cells ^[5][54] (Figure 12Cii). Indeed, neutrophil-derived ROS downmodulated NKp46 in CD56^dim NK cells ^[5][54] (Figure 12Cii), while the opposite effect was exerted in CD56^bright NK cells, in which NKp46 was increased, probably due to high anti-oxidative intrinsic capability ^[5][54] (Figure 12Cii), and this modulation is revered by catalase ^[5][54]. Thus, neutrophil-derived ROS, by NKp46 modulation of NK cells, can enhance or reduce their cytotoxic effect against endothelial cells (ECs), as reported by Dondero and colleagues, that showed that NKp46 is involved in ECs killing in multiple myeloma (MM). Similar to ROS and CG, elastase and lactoferrin act on NK cells, increasing their cytotoxicity ^[6][55] (Figure 2Ciii). Arginase I (ARG1), released by TANs, also participate in supporting NK cell pro-angiogenic features, suppressing NK cell capability to produce anti-tumor factors such as IFNγ ^[7][56] (Figure 2Di).

IL12 is also produced by neutrophils and is crucial for optimal IFNγ and perforin production by both murine and human NK cells ^[8][57]. IL12 signaling, through STAT4 activation, induces IFNγ production in NK cells, as confirmed in a mouse model lacking Stat4, in which NK cells display lower IFNγ production and thus decreased cytolytic function ^[9][58] (Figure 12Dii). The same mechanism occurs in humans, as shown by using the human NKL cell line in which STAT4 activation by IL12 is directly related to perforin expression in in vitro experiments ^[10][59]. Moreover, IFNγ can exert its effect on TANs. As observed in C57BL/6 mice implanted with the MCA205 murine fibrosarcoma cell line, NK cell depletion and IFNγ deficiency allowed for an increased tumor growth compared to the control mice ^[11][60]. This effect is mediated by TANs, thus the absence of NK cells increased the pro-angiogenic features of TANs ^[11][60] (Figure 12E).

Neutrophil and NK cell interactions have been reported to be crucial in supporting the metastatic process. Spiegel et al., using a murine model (BALB/c mice) injected with 4T1 mammary carcinoma cells, showed that neutrophils can suppress intraluminal NK-mediated tumor cell elimination and enhance extravasation of disseminated carcinoma cells ^[12][61].

2. Contribution to Tumor Angiogenesis

Neutrophils represent one of the first infiltrating cell type within TME and can shape TME promoting tumor growth and metastasis formation by angiogenesis stimulation ^[13][14][8,63]. In a skin model of wound healing in CD18-deficient mice that lack neutrophils, it has been shown that neovascularization is compromised compared to wild-type animals ^[15][64], suggesting that neutrophil infiltration ameliorates/improves angiogenesis. Angiogenesis stimulation can be mainly mediated by the release of soluble factors including the main pro-angiogenic master regulator VEGF ^[16][17][65,66]. Vessel-associated neutrophils are a key source of VEGF upon stimulation with CXCL1 ^[18][67] and G-CSF ^[19][68] are one of the main producers of MMP9, which contributes to angiogenic switch in Rip-tag pancreatic cancer in the in vivo mouse model, where neutrophil depletion at the early stage inhibits angiogenesis progression ^[20][69]. In addition, through gene expression analysis, Schruefer and colleagues identified novel pro-angiogenic factors produced by human neutrophils such as ephrin A2 and B2, thrombospondin, TGFβ receptor 2 and 3 (TGFβR2 and TGFβR3), tissue inhibitor of metalloproteinase 2, and restin ^[15][64]. Conversely, neutrophils can also produce anti-angiogenic ^[21][70] factors as they can release enzymatic activities that in vitro generate active angiostatin fragments that in turn inhibit basic fibroblast growth factor (bFGF/FGF2) and VEGF-induced EC proliferation ^[21][70], thus negatively interfering with the angiogenic process. On ECs, VEGF can increase ICAM-1 and VCAM-1 expression, which in turn, can respectively mediate neutrophil and NK cell recruitment by interaction with CD18 expressed by both neutrophils and NK and CD49d on the NK cell surface ^[17][22][23][66,71,72]. NK cells can play a dual role related to angiogenesis by acting as the inhibitor and promoter of this process. In response to IL12 ^[22][71], cytotoxic NK cells start producing IFNγ ^[23][72], which exerts an anti-angiogenic effect through IFNγ inducible protein-10 (IP-10) accumulation ^[24][73] (Figure 23A).