VEGF is a major pro-angiogenic factor, and VEGF signaling plays a central role in both physiological and pathological angiogenesis. It has been shown that human neutrophils carry an intracellular pool of VEGF and release it during the process of degranulation, upon stimulation with PMA, fMet-Leu-Phe (fMLP), and TNF-α [112]. Interestingly, neutrophils were found to be the main source of the circulating VEGF in the majority of patients with metastatic breast cancer or anal carcinoma [120]. Another study has reported that circulating neutrophils release higher amounts of VEGF in patients with oral cavity cancer, compared to healthy donor counterparts [121]. Furthermore, it has been shown that neutrophil depletion is linked to the inhibition of angiogenesis because of the reduced release of pre-formed neutrophil VEGF stores [122]. Importantly, elevated infiltration of tumor tissue by CD11b⁺Gr⁺ neutrophils, characterized by the increased expression of VEGF, was demonstrated to contribute to tumor angiogenesis in a mouse model of fibrosarcoma and melanoma [32].

Surprisingly, VEGF was also shown to indirectly affect neutrophil recruitment. It was reported that IL-8 expression by endothelial cells after VEGF stimulation induced neutrophil migration in vitro [123]. Moreover, VEGF production by neutrophils might promote the further recruitment of neutrophils, in a positive feedback loop, to establish pro-angiogenic signaling inside the tumor and support tumor angiogenesis.

Additionally, neutrophils have been shown to produce and/or contribute to the release of FGF-2. The direct FGF-2 expression of tumor-infiltrating neutrophils was demonstrated in the mouse model of hepatic metastases from gastrointestinal tumors [116]. In this study, neutrophils from hepatic metastases were shown to abundantly express FGF-2 mRNA and promote angiogenesis. Furthermore, neutrophil activity stimulates the release and bioavailability of sequestered FGF-2 in the extracellular matrix [124,125].

Oncostatin M is a cytokine produced by T cells, monocytes, and neutrophils. In early studies, oncostatin M has been shown to inhibit cancer cell proliferation and tumor growth [126,127]. However, neutrophil-secreted oncostatin M has been suggested to contribute to angiogenesis and invasiveness of breast cancer [115]. When co-cultured with breast cancer cells in vitro, human blood neutrophils have been shown to express oncostatin M [115]. Furthermore, both the cell–cell contact and GM-CSF production of breast cancer cells were demonstrated to be necessary for the oncostatin M release from neutrophils. Importantly, GM-CSF-dependent production of oncostatin M by neutrophils has been shown to promote the invasiveness and VEGF expression of MDA-MB-231 and T47D human breast cancer cells [115].

Neutrophil-derived IL-17 has also been suggested to induce tumor angiogenesis [128]. Numasaki et al. demonstrated that IL-17 enhances the angiogenic activity and in vivo growth of non-small cell lung cancer (NSCLC) by promoting the expression of angiogenic chemokines (CXCL1, CXCL5, CXCL6, and CXCL8) [128]. Interestingly, tumor infiltrating T cells and neutrophils were identified as the major source of IL-17 in NSCLC tissue [128].

MMPs are reported to be highly expressed in various cancer types and are commonly associated with the tumor progression [129,130,131]. MMP-9 is a gelatinase that mediates the remodeling of the extracellular membrane, and it has been demonstrated to play an important role in tumor angiogenesis by triggering the angiogenic switch [132]. Importantly, tumor-associated neutrophils were reported to have significantly more abundant MMP-9, readily available for rapid release, than tumor-associated macrophages [133]. Neutrophil-produced MMP-9 mediates degradation of the basal membrane and leads to the liberation and activation of ECs. The degradation of the basal membrane, by MMP-9, indirectly leads to the release of pro-angiogenic factors VEGF and FGF-2, stored in extracellular matrix [97,116,125,134]. VEGF, sequestered in the tumor extracellular matrix, after being released by MMP-9, increases further neutrophilic expression of MMP-9 in a positive feedback fashion [135]. VEGF-A was also demonstrated as a chemoattractant for MMP-9-expressing neutrophils [136]. Moreover, increased levels of VEGF-A in hypoxic regions recruit a distinct subpopulation of pro-angiogenic neutrophils that are characterized by the expression profile of CD49d⁺VEGFR1^hiCXCR4^hi [137]. Neutrophil MMP-9 also increases VEGF-A bioavailability and bioactivity and contributes to the inflammation-induced lymphangiogenesis [138]. Li et al. recently identified high levels of IL-17⁺ neutrophils as the major source of MMP-9 in the invasive margin of gastric cancer [139]. In this study, IL-17 producing neutrophils were shown to promote the pro-angiogenic activity of cancer cells, both in vivo and in vitro. Moreover, such neutrophils stimulated cancer cells to produce neutrophil chemoattractants, which, in turn, supported further neutrophil recruitment [139]. The pro-angiogenic role of MMP-9-secreting neutrophils in tumor vascularization was also validated in other types of cancer. In primary tumors formed by highly disseminating variants of human fibrosarcoma and prostate carcinoma, an elevated accumulation of MMP-9-positive neutrophils was demonstrated. The inhibition of neutrophil recruitment into such tumors, by IL-8 neutralization, diminished tumor angiogenesis [140].

Neutrophil-derived Bv8 and S100 proteins (S100A8 and S100A9) increase tumor proliferation and angiogenesis by supporting neutrophil mobilization, EC activation, and proliferation [46,141,142,143]. Tumor-associated neutrophils have been shown to produce Bv8 and S100A8 in a mouse melanoma model [105]. Bv8 has been suggested as a modulator of myeloid-cell-dependent tumor angiogenesis, and neutrophils have been reported to produce Bv8 robustly, after stimulation with G-CSF or GM-CSF [142,144]. Recently, Itatani et al. demonstrated that tumor-infiltrating neutrophils strongly expressed Bv8 in a mouse model of colorectal cancer. Importantly, in the same study, significantly higher plasma levels of Bv8 were observed in CRC patients, compared to healthy donors. Moreover, high plasma levels of Bv8 positively correlated with a bad prognosis and decreased overall survival of such patients [145]. In another study, treatment with anti-Bv8 antibodies reduced the number of angiogenic islets in transgenic mouse model of multistage pancreatic β-cell tumorigenesis [111]. Moreover, such anti-Bv8 treatment inhibited the recruitment of CD11b⁺Gr⁺ neutrophils to the neoplastic lesions [111,118,146]. Apart from direct stimulation of tumor angiogenesis, neutrophil-derived Bv8 also plays a role in tumor resistance to anti-VEGF therapy (see chapter 7) [145]. Increased levels of plasma G-CSF, after anti-VEGF therapy of mice bearing VEGF-resistant colorectal tumors, were shown to promote neutrophil infiltration into tumor stroma and stimulate angiogenesis via elevated neutrophil Bv8 expression [145]. This study demonstrates that blocking neutrophil-derived Bv8 increases the efficacy of anti-VEGF antibody therapy in colorectal cancer [145].

STAT3 is transcription factor that is involved in cell proliferation, survival, and angiogenesis [147]. STAT3 has been reported to be constitutively activated in both tumor and immune cells in the tumor microenvironment [147,148,149]. Neutrophil STAT3 activity (triggered via G-CSF/G-CSFR pathway) is important for their production, survival, and mobilization from the bone marrow [150,151,152]. STAT3 signaling in mice has been shown to have a regulatory role in neutrophil pro-angiogenic activity by controlling the expression of VEGFA, FGF-2, and MMP-9 genes [153]. Importantly, it has also been demonstrated that neutrophils isolated from tumors have activated STAT3 and induce angiogenesis in vitro [153]. STAT3 signaling in neutrophils was suggested to activate neutrophil pro-angiogenic functions, even before they entered the TME [96]. Stimulation by G-CSF and IL-6 was shown to increase Stat3 expression, which then led to the upregulation of MMP-9 and Bv8 genes in bone marrow neutrophils [96]. Mechanistically, G-CSF-mediated STAT3 activation induces binding of phospho-STAT3 to the Bv8 promotor and elevates the expression of Bv8 [154]. The inhibition of Stat3, by type I IFN signaling, impairs VEGF and MMP-9 production by neutrophils and suppresses tumor angiogenesis [32].