Tumor Egfl6 is a new driver of neutrophil extracellular traps (NETs) in ovarian cancer 2310080

Abstract Introduction Ovarian cancer (OvCa) is characterized by immunosuppressive tumor microenvironment (TME). We displayed the tumor-secreted factor EGF-like domain multiple-6 (EGFL6) drives an immunosuppressive myeloid phenotype, inducing CXCL2, IL-8, and other factors associated with neutrophil extracellular trap (NET) formation. Our preliminary data showed in Egfl6-expressing ID8p53-/- tumors NET infiltration increased compared to parental tumors. Here, we evaluated the role of EGFL6 as a regulator of NETosis in the OvCa TME and whether pharmacological NETosis blockade can reverse Egfl6-induced immunosuppression. Methods In vitro assays were used to evaluate the direct role of recombinant Egfl6 (rEgfl6) on NETosis. OvCa patient tissues were analyzed by immunostaining and qPCR to assess EGFL6 expression, immune cell abundance, and NET markers, such as Citrullinated histone 3 (Cit H3) and neutrophil elastase (NE). To test whether NETosis inhibition reprograms the EGFL6-induced immunosuppression, Egfl6+/- ID8p53-/- tumor-bearing mice were treated with GSK484, a selective PAD4 inhibitor. Immune profile analyzed within murine ascites and tumors via flow cytometry. Results rEgfl6 directly enhanced NETosis in vitro. OvCa patient tissues showed high EGFL6 and NE expression, and NK cells were frequently localized near CitH3+ NETs. In vivo, GSK484 treatment increased cytotoxic CD107+ NK cells and reduced expression of inhibitory receptors including NKG2A and KLRG1. In myeloid cells, GSK484 upregulated antigen-presenting markers (MHCII, CD86) and reduced infiltration of Tregs in Egfl6+ TME. Conclusion Our findings identify EGFL6 as a previously unrecognized tumor-derived driver of NETosis, contributes to immunosuppression in OvCa. NETs directly impair NK cell functions in Egfl6+ tumors, while NETosis inhibition with GSK484 partially restores anti-tumor immunity by enhancing NK cell cytotoxic and reprogramming myeloid cells. These data support targeting NETosis as a therapeutic approach to enhance current therapies in OvCa. Funding Source NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)