Abstract 2972: Neutrophil extracellular traps sequester circulating tumor cells in vitro and in a murine model of metastasis

Abstract

Abstract Introduction: Emerging evidence suggests that neutrophil mediated factors may be implicated in cancer progression, however the mechanisms for this are unclear. Neutrophil extracellular traps (NET's) constitute a mechanism by which pathogens are trapped and killed in extracellular neutrophil derived DNA webs containing antimicrobial proteins. The role of NETs in cancer progression is unknown. We hypothesized that circulating tumor cells could become trapped within NET's, potentially favoring the development of metastatic disease. Materials and Methods: Static adhesion assays were performed using H59 lung cancer cells added to neutrophil monolayers. NETs were induced with phorbol myristate acetate (PMA). NET formation was inhibited with DNAse. Adhesion under dynamic conditions was quantified under flow at 1 dyne/cm2. Lung cancer cells (A549 or H59) were perfused over neutrophil monolayers stimulated with PMA, PMA + DNAse, or media alone, and tumor cell adhesion was quantified. NET formation after stimulation was verified by staining extracellular DNA with Sytox green and assessed for the presence of tumor cell-NET association. To study in vivo interactions between NETs and cancer cells, control and bacterial lipopolysaccharide (LPS) stimulated mice (4 hours prior) were prepared for spinning disc confocal intravital microscopy and H59 cells were injected intra-arterially. Neutrophils were identified by injection of anti-GR1 Alexa 647 conjugated mAb and NETs were stained by injection of anti-histone H2A.X Alexa 555 conjugated mAb.Results: Static H59 cells adhesion to neutrophils stimulated with PMA increased 8-fold over media alone, an effect that was completely attenuated by DNAse. Under flow conditions, PMA increased A549 and H59 cell adherence to neutrophil monolayers 10-fold over media alone, DNAse reduced cancer cell adhesion in PMA stimulated neutrophils by a factor of 3. Staining with Sytox green demonstrated a high degree of co-localization of NETs with clusters of malignant cells in neutrophils stimulated with PMA alone, but not in the presence of DNAse or media alone. Mice stimulated with LPS demonstrated heavy histone H2A.X staining suggestive of NET formation and co-localization with H59 cells. LPS induced NET formation correlated with a previously published 50% increase in H59 cell adhesion to hepatic sinusois. Conclusions: Neutrophil extracellular trap formation is associated with increased adherence and capture of cancer cells in both static and dynamic conditions. This is the first time this in vitro finding has been described and correlates with the first demonstration of in vivo NET/cancer cell interactions. These results suggest a novel mechanism by which neutrophils may promote cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2972. doi:1538-7445.AM2012-2972