Monti, Marcello (2018) Neutrophil extracellular traps (NETs) in cancer-associated thrombosis and tumor progression: role of integrins α5β1, αvβ3 and αIIbβ3. [Tesi di dottorato]

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Upon infection of pathogens, neutrophils are the first white blood cells activated to entrap and kill invading microorganisms through the processes of phagocytosis or degranulation. A newly discovered mechanism used by neutrophils to eliminate bacteria, viruses and fungi is the release of neutrophil extracellular traps (NETs), web-like structures composed of nucleic acids, histones and selected cytoplasmic proteins, like myeloperoxidase and neutrophil elastase. In addition to their main defensive function, NETs were reported to promote thrombotic events and metastatic dissemination of cancer cells, but the mechanisms underlying these processes have not been elucidated yet. In this PhD thesis project, we tested the role of α5β1, αvβ3 and αIIbβ3 integrins in the adhesion of different human cancer cell lines using cell-free isolated NETs as substrate. Neutrophil-like cells, obtained after differentiating HL-60 cell line with DMSO, were stimulated with calcium ionophore A23187 and used as a stable source of NETs. Two human leukemia cell lines, differentially expressing α5β1 and αvβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or αvβ3, 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 αvβ3 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. Co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. Then, we screened a panel of human cancer cell lines endogenously expressing different protein levels of α5β1, αvβ3 and αIIbβ3 integrins in the adhesion to NETs: the concomitant expression of α5β1, αvβ3 and αIIbβ3 integrins was associated to an enhanced adhesion to NETs and the addition of an excess of cyclic RGD peptide inhibited cell adhesion at a different extent in each cell line. Interestingly, the maximal reduction of integrin-dependent adhesion to NETs was similar to that obtained after DNAse 1 treatment, confirming that both DNA and fibronectin determined cell attachment to NETs. Since low or undetectable levels of α5β1 integrin prevents cell adhesion to NETs, this integrin is need to anchor cells in the web-like structure of NETs, allowing a close interaction between cells and DNA/histone complexes. This PhD thesis demonstrated that α5β1, αvβ3 and αIIbβ3 integrins modulate cell adhesion to NETs by binding to their common substrate fibronectin, found to be a protein component of NETs. In addition to mechanical trapping, integrin-mediated cell adhesion to NETs should be taken into account as a mechanism promoting cell-cell interactions at the interface with NETs. Therefore, by using integrins-specific antibodies or the RGD cyclic peptide is possible impairing the homing of different integrins-expressing cell types such as platelets, endothelial and cancer cells to specific sites of NETs accumulation, thus reducing NETs-dependent cancer-associated thrombosis and tumor progression.

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