Analysis of the proteomic profile of pro- and anti-tumor neutrophils and the sEVs they secrete

Introduction: Neutrophils constitute the most numerous group of cells in the human immunesystem (50–70% of WBCs). Similar to macrophages, they exist in two phenotypes: pro- andanti-tumor. A significant factor modulating their phenotype is the type I interferon receptor(IFNAR1): its presence promotes anti-tumor properties, while its silencing (Ifnar1−/−) favorsa pro-tumor phenotype. Pro-tumor neutrophils promote tumor growth and metastasis bysecreting factors that modulate the tumor microenvironment (VEGF-A, TGF-β, MMP-9, IL-8)and forming neutrophil traps (NETs). One mechanism of communication between neutrophilsand tumors may be small extracellular vesicles (sEVs). They are secreted by neutrophils andtransport molecules such as proteins, lipids and nucleic acids. The composition of sEVs isdetermined to some extent by the phenotypic state of the parent cell. Therefore, they caninfluence distant cells without direct cell-to-cell interactions.Methods: Conditionally immortalized progenitor cells (ER-Hoxb8) were used in this study;wild-type (WT) and interferon receptor I knockout (Ifnar1−/−). Before proteomic analysis, cellswere labeled using the SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture)method. To differentiate the cells into neutrophils, β-estradiol was removed from the medium5 days before sEVs isolation. Two days before isolation, the medium was replaced withmedium containing exosome-depleted FBS. sEVs isolation was performed using mini-SEC.The sEVs fraction was characterized using NAT, TEM, and typical exosomal markers (CD63,CD81, TSG101). The obtained cell lysates and sEVs were subjected to proteomic analysisusing nano-LC-MS/MS. Raw data were processed using Proteome Discoverer, and theresulting protein identifications were analyzed using the STRING bioinformatics platform. Results: Proteomic analysis identified 3089 proteins in neutrophils and 365 proteins in secretedsEVs. In Ifnar1−/− neutrophils, 338 upregulated proteins were identified compared to WTneutrophils. In sEVs derived from Ifnar1−/− neutrophils, 107 proteins were found to beoverexpressed compared to sEVs derived from WT neutrophils. Comparative analysis ofproteins overrepresented in neutrophils and sEVs revealed 23 proteins in common. All of themexhibit pro-tumor properties by stimulating cell migration, proliferation, and differentiation.Furthermore, they activate pathways such as MAPK/ERK, PI3K/AKT, and NF-κβ, therebyinfluencing the activity of proangiogenic factors and metalloproteinases.Conclusion: WT and Ifnar1−/− neutrophils have distinct proteomic profiles, which are alsoevident in the sEVs they secrete. Some of the proteins overrepresented in Ifnar1−/− neutrophilsare identical to those identified in the sEVs they secrete. Therefore, sEVs may be excellentmodulators of the tumor microenvironment, stimulating oncogenesis without direct cell-to-cellcontact.