Angiogenesis, the formation of new blood vessels from already existing vasculature, is tightly regulated by pro- and anti-angiogenic stimuli and occurs under both physiological and pathological conditions. Tumor angiogenesis is central for tumor development, and an “angiogenic switch” could be initiated by multiple immune cells, such as neutrophils. Tumor-associated neutrophils promote tumor angiogenesis by the release of both conventional and non-conventional pro-angiogenic factors. Therefore, neutrophil-mediated tumor angiogenesis should be taken into consideration in the design of novel anti-cancer therapy. This review recapitulates the complex role of neutrophils in tumor angiogenesis and summarizes neutrophil-derived pro-angiogenic factors and mechanisms regulating angiogenic activity of tumor-associated neutrophils. Moreover, it provides up-to-date information about neutrophil-targeting therapy, complementary to anti-angiogenic treatment.

Angiogenesis plays an important role during tumor growth and metastasis. We could previously show that Type I interferon (IFN)‐deficient tumor‐associated neutrophils (TANs) show strong pro‐angiogenic activity, and stimulate tumor angiogenesis and growth. However, the exact mechanism responsible for their pro‐angiogenic shift is not clear. Here, we set out to delineate the molecular mechanism and factors regulating pro‐angiogenic properties of neutrophils in the context of Type I IFN availability. We demonstrate that neutrophils from IFN‐deficient (Ifnar1^−/−) mice efficiently release pro‐angiogenic factors, such as VEGF, MMP9 or BV8, and thus significantly support the vascular normalization of tumors by increasing the maturation of perivascular cells. Mechanistically, we could show here that the expression of pro‐angiogenic factors in neutrophils is controlled by the transcription factor forkhead box protein O3a (FOXO3a), which activity depends on its post‐translational modifications, such as deacetylation or phosphorylation. In TANs isolated from Ifnar1^−/− mice, we observe significantly elevated SIRT1, resulting in SIRT1‐mediated deacetylation of FOXO3a, its nuclear retention and activation. Activated FOXO3a supports in turn the transcription of pro‐angiogenic genes in TANs. In the absence of SIRT1, or after its inhibition in neutrophils, elevated kinase MEK/ERK and PI3K/AKT activity is observed, leading to FOXO3a phosphorylation, cytoplasmic transfer and inactivation. In summary, we have found that FOXO3a is a key transcription factor controlling the angiogenic switch of neutrophils. Post‐translational FOXO3a modifications regulate its transcriptional activity and, as a result, the expression of pro‐angiogenic factors supporting development of vascular network in growing tumors. Therefore, targeting FOXO3a activity could provide a novel strategy of antiangiogenic targeted therapy for cancer.

Head and Neck Cancers (HNCs) have highly immunosuppressive properties. Small extracellular vesicles (sEVs), including exosomes, nanosized mediators of intercellular communication in the blood, carry immunosuppressive proteins and effectively inhibit anti-tumor immune responses in HNCs. This study evaluates immunosuppressive markers on sEVs from 40 HNC patients at different disease stages and 3- and 6-month follow-up after surgery and/or chemoradiotherapy. As controls, sEVs from normal donors (NDs) are examined. Immunoregulatory surface markers on sEVs were detected as relative fluorescence intensity (RFI) using on-bead flow cytometry, and their expression levels were monitored in the early and late stages of HNC and during follow-up. In parallel, the sEV-mediated apoptosis of CD8⁺ Jurkat cells was assessed. Together with TGF-β1 and PD-L1 abundance, total sEV proteins are elevated with disease progression. In contrast, total sEV protein, including TGF-β1, PD-1 and PD-L1, decrease upon therapy response during follow-up. Overall survival analysis implies that high sEV PD-1/PD-L1 content is an unfavorable prognostic marker in HNC. Consistently, the sEV-mediated induction of apoptosis in CD8⁺ T cells correlates with the disease activity and therapy response. These findings indicate that a combination of immunoregulatory marker profiles should be preferred over a single marker to monitor disease progression and therapy response in HNC.

Hereditary hemorrhagic telangiectasia (HHT) is characterized by mucocutaneous telangiectases and visceral vascular malformations. Individuals suffering from HHT have a significantly increased risk of bacterial infections, but the mechanisms involved in this are not clear. White blood cell subpopulations were estimated with flow cytometry in 79 patients with HHT and 45 healthy individuals, and association with clinicopathological status was assessed. A prominent decrease in absolute numbers of T cells in HHT was revealed (0.7 (0.5–1.1) vs. 1.3 (0.8–1.6), 10⁶/mL, p < 0.05), and in multivariate regression analysis, hemoglobin level was associated with lymphopenia (OR = 0.625, 95% CI: 0.417–0.937, p < 0.05). Although no changes in absolute numbers of neutrophils and monocytes were observed, we revealed a significant impairment of neutrophil antibacterial functions in HHT (n = 9), compared to healthy individuals (n = 7), in vitro. The release of neutrophil extracellular traps (NETs) against Pseudomonas aeruginosa MOI10 was significantly suppressed in HHT (mean area per cell, mm²: 76 (70–92) vs. 121 (97–128), p < 0.05), due to impaired filamentous actin organization (% of positive cells: 69 (59–77) vs. 92 (88–94), p < 0.05). To conclude, this study reveals the categories of patients with HHT that are prone to immunosuppression and require careful monitoring, and suggests a potential therapeutic strategy based on the functional activation of neutrophils.

The role of neutrophils during cancer formation and elimination is diverse. Here, for the first time, we investigate neutrophil helper cells (N_BH), their influence on B cell activity in the regional lymph nodes (RLN) of head-and-neck cancer patients and the effect of this neutrophil/B cell interaction on patient prognosis. Circulating and RLN neutrophils of patients with stage I–IV head-and-neck squamous cell carcinoma were investigated with flow cytometry and qPCR. In addition, neutrophil/B cell co-localization in RLNs was evaluated using immunohistochemistry. B cell proliferation was assessed and correlated with the distance to neutrophils. Patient survival was evaluated. Neutrophils with the helper cell phenotype were identified in the RLN of HNC patients. B cells in close proximity to such N_BH showed significantly higher proliferation rates, together with elevated activation-induced cytidine deaminase (AID) expression. Notably, patient survival was significantly higher in individuals with high N_BH frequencies in the B follicles of RLNs. Neutrophils in RLN can support T cell-independent activation of the adaptive immune system through B cell stimulation, capturing helper cell phenotype character. The presence of such helper neutrophils in the RLNs of HNC patients positively correlates with patient prognosis.

Neutrophils are known to play an important regulatory role during tumor progression in several types of cancer. However, the mechanisms responsible for their tumorigenic bias and extended lifespan in cancer are not clear to date. This study uncovers a previously unknown mechanism by which tumor-derived small extracellular vesicles (sEVs), via their serpin cargo, reprogram neutrophils to adopt a tumor-supporting phenotype. We demonstrated here an elevated content of plasma sEVs during head and neck cancer progression, and their significant cargo enrichment with inhibitors of neutrophil serine proteases: serpins A1 and A3. Mechanistically, neutrophils educated with serpin-rich tumor-derived sEVs displayed typical pro-tumoral characteristics, including prolonged lifespan and activated CD62L ^low CD11b ^high PDL1 ^high phenotype. Functionally, such neutrophils demonstrated a strong ability to promote the epithelial-to-mesenchymal transition of tumor cells. Moreover, such neutrophils induced remarkable suppression of cytotoxic CD8 T cells, significantly reducing their tumor cell-killing capacity. Importantly, serpin cargo was essential for this activity, as serpin-depleted sEVs failed to reprogram neutrophils. These findings again highlight the clinical significance of sEVs and suggest their serpin content as important mediators of pro-tumoral functionality. Targeting the biogenesis or uptake of such immunosuppressive sEVs, or modifying their cargo, could potentially serve as a potent adjuvant anti-cancer therapy.