Differential Neutrophil Extracellular Traps Release (NETosis) In Cancer-Related Neutrophils

Introduction: Neutrophils play a major role in tumor biology. At least three distinct populations of circulating neutrophils have been described in the context of cancer, including the mature high-density neutrophils (HDN) as well as mature and immature low-density neutrophils (LDN), with pro- and anti-tumor characteristics. Among other functions, cancer-related neutrophils were shown to release upon activation Neutrophil-extracellular traps (NETs), mesh-like structures of decondensed chromatin fibers, in a process named NETosis. In this work, we aimed to characterize the cellular mechanisms leading to NETosis in murine and human cancer-related neutrophils and the differential ability of neutrophil subpopulations in releasing NETs.

Methods: Mice were injected with AB12 (mesothelioma) or LLC (carcinoma) cell lines. Circulating neutrophils were isolated from tumor bearing mice and from peripheral blood of Lung Cancer patients using a density gradient centrifugation. Following stimulation with Ionomycin or PMA, cells were fixed and stained for Myeloperoxidase, citrullinated-Histones3 and DAPI. NET formation was determined as the percentage of cells identified positive for MPO and CitH3 signal. Data were acquired using a Nikon Confocal microscope and analyzed by ImageJ.

Results: We find that HDN and LDN show comparable NETotic potential following activation. As suggested by previous studies, we find that circulating neutrophils isolated from tumor bearing mice display higher ability to release NETs than healthy controls. Surprisingly, HDN isolated from mice bearing GCSF-enriched tumors do not display higher NETotic ability compared to neutrophils isolated from control mice. However, the addition of G-CSF to the media strongly induced the release of NETs. Moreover, we find that neutrophils’ expression of TNFα is important for the release of NETs by LDNs, but not by HDN. Human HDN present significantly higher release of NETs compared to murine neutrophils (~15% vs 3% respectively). Similarly to murine neutrophils, G-CSF stimulation enhanced NETosis in human HDN.

Conclusions: Our findings demonstrate that NETosis is preponderant in cancer-related neutrophils, both in mice and humans. Our data suggest that the constitutive stimulation by G-CSF is required to maintain the enhanced susceptibility of neutrophils to form NETs independently of the cancer-induced predisposition.