Introduction: Neutrophils constitute 50-70% of the white blood cells in the human body. It is now known that, asin the case of macrophages, neutrophils with N1 and N2 phenotypes can be distinguished. An important role in modulating the neutrophil phenotype is played by the interferon receptor, which is activated by interferon type 1.IFN-based therapy has been shown to promote polarization of tumor-associated neutrophils (TANs) toward N1phenotype. While interferon receptor silencing promotes neutrophil development towards a N2 phenotype. N2TANs promote, directly or indirectly, tumor growth, as well as cancer cell spread by secreting ECM remodellingenzymes and proangiogenic factors that promote metastasis and angiogenesis. However, it is currently unclearhow neutrophils communicate with the tumor and whether EVs are involved in this communication. Therefore,our objective was to characterise EVs secreted by pro-oncogenic and anti-tumor neutrophils and to comparetheir ability to modulate angiogenesis. Methods: We isolated EVs using SEC. We assessed the size,concentration, morphology, and presence of typical EV markers using ZetaView, TEM, and Western Blot.Furthermore, we determined the proteome composition of neutrophils and EVs secreted by them using the SILACtechnique. We verified the influence of EVs on the angiogenesis process using the aortic ring assay (permission#51/2021, Katowice, Poland) and the assessment of endothelial cell migration and proliferation. Results:Morphology analysis using TEM and ZetaView showed the size of EVs in the range of 80-150 nm. Furthermore, weconfirmed the presence of proteins such as CD81, CD63, TSG101, CD9, Ly-6G in the obtained EVs samples.Proteomic analysis revealed more than 5-fold higher levels of PTK2B, HSPH1, SLC39A8, VPS13C proteins in N2TAN than in N1 TAN. A similar pattern of change was observed in isolated EVs. N2 TAN-secreted EVs also causedincreased cell angiogenesis in the aortic ring assay, increased endothelial cell migration, and proliferation.Summary/Conclusion: Knockdown of the interferon receptor in neutrophils causes changes in the levels ofproteins present in EVs. The result is an increase in angiogenesis, migration and proliferation of endothelial cellsstimulated by EVs secreted by N2 TAN. Funding: The research was financed by the National Science Centre, grantno. 2023/48/C/NZ5/00001.
Smolarz et al. (Sun,) studied this question.