Pancreatectomy Induces Cancer-Promoting Neutrophil Extracellular Traps

Abstract

Background: Neutrophil extracellular traps (NETs) occur when neutrophil chromatin is decondensed and extruded into the extracellular space in a web-like structure. Originally described as an anti-microbial function, this process has been implicated in the pathogenesis of pancreatic disease. In addition, NETs are upregulated during physiologic wound-healing and coagulation. This study evaluated how the inflammatory response to pancreatic surgery influences NET formation.

Methods: For this study, 126 patients undergoing pancreatectomy gave consent before participation. Plasma was collected at several time points (preoperatively and through the postoperative outpatient visit). Plasma levels of NET markers, including cell-free DNA (cfDNA), citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-6, and granulocyte colony-stimulating factor (G-CSF) were measured using enzyme-linked immunosorbent assay (ELISA). Patient clinical data were retrospectively collected from a prospectively maintained database.

Results: After pancreatic resection, NET markers (cfDNA and CitH3) were elevated, peaking on postoperative days 3 and 4. This increase in NETs was due to an inherent change in neutrophil biology. Postoperatively, NET-inducing cytokines (IL-8, IL-6, and G-CSF) were increased, peaking early in the postoperative course. The patients undergoing the robotic approach had a reduction in NETs during the postoperative period compared with those who underwent the open approach. The patients who experienced a pancreatic leak had an increase in NET markers during the postoperative period.

Conclusions: Pancreatectomy induces cancer-promoting NET formation. The minimally invasive robotic approach may induce fewer NETs, although the current analysis was limited by selection bias. Pancreatic leak resulted in increased NETs. Further study into the potential for NET inhibition during the perioperative period is warranted.

Keywords: Neutrophil extracellular traps; Neutrophils; Pancreatectomy; Pancreatic leak; Robotic approach.

Figure 1.. Pancreatectomy induces cancer promoting neutrophil extracellular traps.

Circulating NET markers, cfDNA and CitH3, measured in plasma pre-, intra-, and post-operation of all patients undergoing pancreatic resection (n=126) and those with PDAC (A, n=64), demonstrating an increase in NET markers following surgery. ANC values of patients following pancreatic resection does not significantly change following surgery (B, n=103). Neutrophils extracted from patient 97 (PC-97) for an ex vivo NETosis assay measuring cf-DNA release pre- and post-operation (C). Patients undergoing pancreatectomy have a non-significant increase in the average cf-DNA release in ex vivo NETosis assay, persisting through POD3/4 (n = 13), suggesting an increased propensity to form NETs (D). Representative images of patient 97 (PC-97) neutrophils following the ex vivo NET assay either stimulated or not (E). Representative bioluminescence image of MPO activity and ROS levels in neutrophils isolated from 10 patients undergoing pancreatic resection at various time points (F). Corresponding bioluminescence quantification of area under the curve of (G) basal and (H) PMA activated neutrophil fold change (normalized to pre-operative time point). Luminol measures MPO activity while lucigenin assesses the ROS superoxide anion. L-012 quantifies MPO and ROS levels more generally. (n = 10 patients) (Pre Op); blood drawn at pre op, post op, post op day 1 and post op day 3/4. Multiple nonparametric t test *p < 0.05, ** p < 0.01, ***p < 0.001.

Figure 2.. Pancreatectomy induces release of inflammatory cytokines known to induce NET formation.

Plasma IL-8, IL-6, and G-CSF measured pre-operatively through the first follow up visit (POOV) in patients who underwent pancreatectomy (n=126). All three cytokines were elevated following pancreatic resection, peaking on POD1. * = p-value<0.05

Figure 3.. Pancreatectomy surgical approach influences NET formation post operatively.

Levels of circulating NET markers (cf-DNA and CitH3) in patient plasma stratified into groups undergoing robotic vs. open pancreatectomies. The total cohort (A) and PDAC only (B) patients are shown (, n=108 robot, n=18 open). The minimally invasive procedure had several timepoints with significantly reduced induction of NETs following pancreatic resection compared to the open. * = p-value<0.05

Figure. 4.. Elevated circulating NET markers are associated with pancreatic leak.

Circulating NET markers (cfDNA and citH3) in those patients that did have a pancreatic leak (n=8) and those that did not (n= 118) (A-B). There was a rise in circulating NET markers in those that did have a leak starting at POD1 and maintaining until the first post-op visit (POOV). In the PDAC only cohort, when comparing those that did have a leak (n=3) to those that did not (n=61), we saw a rise in NET markers that was not back to baseline by POOV (C). * = p-value<0.05