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.