Abstract
Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5β1 and ανβ3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5β1 and ανβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or ανβ3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5β1 or ανβ3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.
Highlights
Neutrophil extracellular traps (NETs) are web-like structures composed of nucleic acids, histones and selected cytoplasmic proteins that are released by activated neutrophils to entrap and kill different pathogens [1, 2]
Our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin, which was found to co-localize with citrullinated histone H3 (Cit H3) inside the web-like structure of NETs and to interact directly or indirectly with histone H3
A23187-stimulated neutrophil-like cells was performed using anti-fibronectin antibody followed by SDS-PAGE of the immunoprecipitated proteins and immunoblotting with the indicated antibodies
Summary
Neutrophil extracellular traps (NETs) are web-like structures composed of nucleic acids, histones and selected cytoplasmic proteins that are released by activated neutrophils to entrap and kill different pathogens [1, 2]. In addition to their function as a host defense mechanism, a growing body of evidence indicates that NETs promote thrombosis by providing a scaffold for platelet and red blood cell adhesion [3, 4] as well as metastatic dissemination of cancer cells by entrapment of circulating tumor cells [5]. A recent study in an animal model reproducing surgical stress of hepatic resection for metastatic colorectal cancer reported that NETs formation from mouse neutrophils was associated with High Mobility Group Box 1 (HMGB1) release and activation of Toll-like receptor 9 (TLR9)-dependent pathways in cancer cells promoting adhesion, proliferation, migration and invasion [7]
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