Background: Low density neutrophils (LDN) which are co-purified with mononuclear cells by Ficoll-Hypaque density gradient preparations, have been reported to be increased in circulating blood in patients with autoimmune diseases, sepsis and cancer. Recent studies have suggested the LDN contributes to lupus pathogenesis as well as the development of organ damage in sepsis.

Method: In patients who underwent curative surgery for gastrointestinal malignancy, peripheral blood was obtained at the timing of laparotomy (preoperative) and just before wound closure (postoperative) and the ratios of LDN were evaluated with flowcytometry. The LDN and mononuclear cells (MNC) in postoperative samples were separated using CD66b microbeads and MACS column and the proliferation of T cells were evaluated by in vitro culture on anti-CD3 coated plate using CFSE dilution method. The LDN were cultured for 2 hours and neutrophil extracellular traps (NETs) were observed with the addition of SYTOX green, dye for extracellular double strand (ds)-DNA. In adhesion experiments, human colon cancer, DLD-1, were stained by PKH-26 and added on the cultured LDN for 5 min. After gentle washing, the remaining cells were counted under fluorescein microscope.

Results: 1. Ratios of CD66b(+) LDG in CD45(+) leukocytes were markedly increased in postoperative as compared with preoperative blood samples. 2. Division of CD3(+) T cells were significantly suppressed by the addition autologous CD66b(+) LDN. 3. Short term culture of the LDN produced massive thread-like structures stained with SYTOX green. Immunostaining with specific mAbs revealed that histone and myeloperoxidase (MPO) were colocalized on the fibrous structure, indicating NETs 3. DLD-1, selectively attached to the NETs, which was totally abrogated by the pretreatment with 100u/ml DNAse.

Conclusion: During abdominal surgery, immunosuppressive LDN are markedly increased in circulating blood. The LDN extensively produces NETs which support the metastasis formation by trapping the circulating tumor cells in target organ. Functional blockade of the LDN might effectively reduce the recurrence rate after curative surgery.

Citation Format: Yuko Kumagai, Rihito Kanamaru, Hideyuki Ohzawa, Hisanaga Horie, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Low-density neutrophils (LDN) in circulating blood of postoperative patients may participate in the development of distant recurrence through the production of neutrophil extracellular traps (NETs) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B77.