Abstract
Neutrophil extracellular traps (NETs), in addition to their function as a host defense mechanism, play a relevant role in thrombus formation and metastatic dissemination of cancer cells. Here we screened different cancer cell lines endogenously expressing a variety of integrins for their ability to bind to NETs. To this end, we used NETs isolated from neutrophil-like cells as a substrate for adhesion assays of HT1080, U-87 MG, H1975, DU 145, PC-3 and A-431 cells. Levels of α5, αIIb, αv, β1, β3 and β5 chains were determined by western blot analysis in all cell lines and levels of whole integrins on the plasma membrane were assessed by fluorescence-activated cell sorting (FACS) analysis. We found that high levels of α5β1, αvβ3 and αvβ5 enhance cell adhesion to NETs, whereas low expression of α5β1 prevents cell attachment to NETs. Excess of cyclic RGD peptide inhibited cell adhesion to NETs by competing with fibronectin within NETs. The maximal reduction of such adhesion was similar to that obtained by DNase 1 treatment causing DNA degradation. Our findings indicate that NETs from neutrophil-like cells may be used as a substrate for large screening of the adhesion properties of cancer cells expressing a variety of RGD-binding integrins.
Highlights
Neutrophil extracellular traps (NETs) are web-like structures released from activated neutrophils exposed to different pathogens including bacteria, viruses or fungi [1]
Conditioned medium from treated cells was subjected to centrifugation to obtain cell-free NET-enriched suspensions that were subsequently employed as an adhesion substrate
Our study showed that isolated NETs, obtained from stimulation of neutrophil-like cells, express the same major markers of NETs released from circulating human neutrophils and maintained similar structural features
Summary
Neutrophil extracellular traps (NETs) are web-like structures released from activated neutrophils exposed to different pathogens including bacteria, viruses or fungi [1] They are composed of decondensed chromatin associated with histones and other granular proteins such as myeloperoxidase (MPO) and neutrophil elastase (NE) [2]. In response to inflammatory stimuli, neutrophils migrate from the blood stream to the site of infection where, in addition to the well-known mechanisms of phagocytosis and degranulation, they can extrude NETs with a multistep process termed netosis [5,6,7] This process is characterized by morphological changes of polymorphonucleocytes (PMN), including chromatin decondensation and loss of the classical lobulated nuclear morphology, disruption of nuclear membrane and subsequent mixing of nuclear and cytoplasmic content followed by extrusion into the extracellular environment through plasma membrane disintegration [8,9]. Adhesion of human K562 cells, differentially expressing α5β1 and αvβ integrins, to NETs was inhibited by both degradation of DNA and blocking antibodies against those integrins
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