Gradient Infiltration of Neutrophil Extracellular Traps in Colon Cancer and Evidence for Their Involvement in Tumour Growth

Abstract

Background: The role of neutrophils in tumour biology is largely unresolved. Recently, independent studies indicated either neutrophil extracellular traps (NETs) or Tissue Factor (TF) involvement in cancer biology and associated thrombosis. However, their individual or combined role in colonic adenocarcinoma is still unexplored.

Methods: Colectomy tissue specimens and variable number of draining lymph nodes were obtained from ten patients with adenocarcinoma of the colon. NETs deposition and neutrophil presence as well as TF expression were examined by immunostaining. The effect of NETs on cancer cell growth was studied in in vitro co-cultures of Caco-2 cell line and acute myeloid leukemia primary cells. Proliferation and apoptosis/necrosis of cancer cells were analyzed by flow cytometry.

Results: TF-bearing NETs and neutrophil localization were prominent in tumour sections and the respective metastatic lymph nodes. Interestingly, neutrophil infiltration and NETs concentration were gradually reduced from the tumour mass to the distal margin. The in vitro-generated NETs impeded growth of cancer cell cultures by inducing apoptosis and/or inhibiting proliferation.

Conclusions: These data support further the role of neutrophils and NETs in cancer biology. We also suggest their involvement on cancer cell growth.

Fig 1. Presence of NETs and neutrophils in colon adenocarcinoma tumour and metastatic lymph nodes.

(A) NETs visualized in colon adenocarcinoma specimens as extracellular structures decorated with neutrophil elastase and citrullinated H3 or neutrophil elastase and MPO, (confocal microscopy). (B) Abundance of neutrophil presence in colon adenocarcinoma demonstrated by neutrophil elastase immunohistochemical staining (light microscopy). Magnified section demonstrates neutrophils stained with neutrophil elastase. NETs (C) and neutrophil (D) presence in metastatic lymph nodes in patients with colon adenocarcinoma. (C) NE/cit-H3 and NE/MPO immunofluorescence confocal microscopy and (D) neutrophil elastase immunohistochemical staining. (A), (C) i-ii Green: NE, Red: cit-H3, Blue: DAPI/DNA, iii Green: MPO, Red: NE, Blue: DAPI/DNA (A), (B), (C), (D) One representative out of ten independent experiments is shown. (A), (C) Original magnification 600x, Scale bar– 5μm. Original magnification (B) 400x, (D) 200x.

Fig 2. Neutrophil and NETs presence is diminished accordingly to the distance from the center of tumour.

—Representative experiment where NETs and neutrophils were diminished at 3 cm from tumour mass. (A) Macroscopic photography of colectomy biopsy specimen for demonstration of successive section sampling. (B) Neutrophil elastase immunohistochemical staining in successive biopsy specimens from the center of tumour, up to 3–4 cm from the tumor mass in healthy tissue in patients with colon adenocarcinoma. (C) Neutrophil elastase and citrullinated H3 immunofluorescence staining in successive biopsy specimens from the center of tumour, up to 3–4 cm from the tumor mass in healthy tissue in patients with colon adenocarcinoma. Green: NE, Red: cit-H3, Blue: DAPI/DNA. (B),(C) One representative out of ten independent experiments is shown. Original magnification (B) 200x, (C) 600x, Scale bar– 5μm.

Fig 3. TF localization on NETs present in colonic adenocarcinoma and metastatic lymph nodes.

TF and neutrophil elastase immunofluorescence staining in (A) colonic adenocarcinoma specimens and (B) respective metastatic lymph nodes. Green: TF, Red: NE, Blue: DAPI/DNA. One representative out of four independent experiments is shown. Original magnification 600x, Scale bar– 5μm.

Fig 4. NETs inhibit growth and induce apoptosis in Caco-2 cultures.

A) May Grünwald-Giemsa staining of Caco-2 cells co-cultured with PMA or sepsis serum-induced NETs in the presence or absence of NET scaffold inhibitors. One representative out of four independent experiments is shown. Original Magnification 100x. (B) Percentage of surface area covered by cells. (C), (D) Annexin V/PI flow cytometry of Caco-2 cells co-cultured with PMA or sepsis serum-induced NETs in the presence or absence of NET scaffold inhibitors. (C) Representative scatter plots. (B), (D) Data from four independent experiments presented as mean±SD. n.s.—not significant compared to control, *p < 0.05.

Fig 5. NETs reduce proliferation of AML cells.

(A), (B) CFSE flow cytometry of AML cells co-cultured with either PMA or sepsis serum-induced NETs in the presence or absence of NET scaffold inhibitors. (A) Representative scatter plots. (B) Data from four independent experiments presented as mean±SD. n.s.—not significant compared to control, *p < 0.05.