Neutrophils in lung cancer patients: Activation potential and neutrophil extracellular trap formation

Abstract

Background: Patients with cancer have an increased risk of developing venous thromboembolism. Neutrophils and neutrophil extracellular traps (NETs) reportedly influence the risk of cancer-associated thrombosis. Subpopulations of high and low-density neutrophils (HDN/LDN) are of specific interest, as they might have different functions in cancer patients.

Objectives: We aimed to investigate differences between HDNs and LDNs of patients with lung cancer and healthy controls, and their ability of activation and NET formation.

Methods: Within the framework of the Vienna Cancer and Thrombosis Study, a prospective observational cohort study, HDNs and LDNs from 20 patients with lung cancer and 20 controls were isolated by density gradient centrifugation. The ability of neutrophil subpopulations for activation and NET formation was investigated by flow cytometry.

Results: Compared to controls, patients with cancer had higher numbers of total leukocytes, HDNs, and LDNs. LDNs of patients were more frequently in an activated state (CD62L↓/CD16↑) at baseline (median [IQR] 5.9% [3.4-8.8] vs 2.5% [1.6-6.7]). HDNs and LDNs from patients showed a significantly increased response to stimulation with ionomycin (CD11b HDN: 98.5 [95.4-99.4] vs 41.7 [13.4-91.6]; LDN: 82.9 [63-94] vs 39.6 [17.3-72.1]). In addition, HDNs from patients showed a higher capability of NET formation after ionomycin stimulation compared to HDNs from healthy controls (18509.5 [12242.5-29470.3] vs 10001 [6618.8-18384.3]).

Conclusion: Protumorigenic LDNs were elevated, and neutrophil subpopulations showed an increased activation profile and ability for NET formation in patients with cancer. These mechanisms might be involved in tumor promotion and contribute to the prothrombotic phenotype of neutrophils in cancer.

Keywords: cancer-associated thrombosis; high-density neutrophil; low-density neutrophil; lung cancer; neutrophil extracellular traps.

Figure 1

HDN/LDN counts and subsets in patients with lung cancer compared to healthy controls. (A) HDN count and (B) LDN count of healthy controls and patients with lung cancer. Flow cytometric characterization of neutrophil subsets in (C) HDNs and (D) LDNs. The proportions of CD62L↑/CD16↑ (mature), CD62L↓/CD16↑ (activated) and CD62L↑/CD16↓ (newly released) neutrophils to CD66b positive cells are indicated. Light colour indicates healthy control samples; darker colour indicates samples from patients with cancer. Statistical analysis was performed using Mann-Whitney-U-test. HDN, high-density neutrophil; LDN, low-density neutrophil; ∗P ≤ .05; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ 0.0001.

Figure 2

Differences of HDNs between patients with lung cancer and healthy controls. HDNs isolated from 20 healthy controls and 20 patients with lung cancer at baseline (immediately fixed after isolation), untreated and after stimulation with IO and PMA. (A) MFI of CD66b and (B) CD11b as well as (C) % of CD66b high and (D) % of CD11b high of healthy controls and patients with lung cancer after treatment for 30 minutes at 37°C. (E) MFI of H3Cit after stimulation for 3 hours at 37°C. Statistical analysis was performed using Mann-Whitney-U-test. HDN, high-density neutrophil; MFI, mean fluorescent intensity; IO, ionomycin, PMA, phorbol 12-myristate 13-acetate; H3Cit, citrullinated histone H3; ∗P ≤ .05; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001; n.s., not significant.

Figure 3

Differences of LDNs between patients with lung cancer and healthy controls. LDNs isolated from 20 healthy controls and 20 patients with lung cancer at baseline (immediately fixed after isolation), untreated and after stimulation with IO and PMA. (A) MFI of CD66b and (B) CD11b as well as (C) % of CD66b high and (D) % of CD11b high of healthy controls and patients with APS after treatment for 30 minutes at 37°C. (E) MFI of H3Cit after stimulation for 3 hours at 37°C. Statistical analysis was performed using Mann-Whitney-U-test. LDN, low-density neutrophil; MFI, mean fluorescent intensity; IO, ionomycin, PMA, phorbol 12-myristate 13-acetate; H3Cit, citrullinated histone H3; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; n.s., not significant.