The authors have declared that no competing interests exist.
Conceived and designed the experiments: KR. Performed the experiments: SA AA KK PM IA AM ES. Analyzed the data: KK CP PM IK PS ES IM AG. Contributed reagents/materials/analysis tools: IK IM AG. Wrote the paper: SA AA KK CP IA AM IK PS IM AG KR.
‡ These authors co-supervised this work.
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.
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
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
These data support further the role of neutrophils and NETs in cancer biology. We also suggest their involvement on cancer cell growth.
Cancer is a leading cause of morbidity and mortality worldwide, with metastasis and cancer-associated thrombosis comprising the main causes of death in patients with malignancy. It is well known that the relationship between cancer and the immune system is not limited only to surveillance against rising monoclonal disorders, but also against established malignant tumors. The interplay between the immune system and cancer determines many aspects of the clinical presentation and the progression of the disease [
Recently, a key mechanism of neutrophils, Neutrophil Extracellular Traps (NETs), has redefined their role in tumour biology [
Moreover, Tissue factor (TF) the main
In this study we investigated the presence of NETs in solid tumours and metastatic lymph nodes of colon adenocarcinoma patients and their
For this study, formalin-fixed paraffin-embedded surgical tissue specimens from 10 patients who had undergone colectomy for adenocarcinoma, including the draining lymph nodes, were examined. Patient characteristics are demonstrated in
In addition, 10 healthy donors were enrolled for blood samples in order to isolate polymorphonuclear neutrophils (PMNs).
Written consent was granted by all individuals involved in this study. The study protocol design was in accordance with the Declaration of Helsinki and was approved by the Ethics Review Board of the University Hospital of Alexandroupolis.
Sections were deparaffinized and immunocytochemical staining was performed using a LSAB/HRP kit (DAKO) as previously described [
For immunofluorescence, sections were stained as previously described [
Peripheral blood neutrophils were isolated from heparinized blood from healthy donors as previously described [
Caco-2 [Caco2] (ATCC® HTB-37™) (ATCC, Manassas, USA) cells were cultured in 5% CO2, at 37°C, in L-glutamine Eagle's Minimum Essential Medium (EMEM; Gibco BRL, New York, USA). AML cells were cultured in 5% CO2, at 37°C, in Myeloid Long-Term Culture Medium (MyeloCult™ H5100; Stem Cell Technologies, Vancouver, Canada). Both Caco-2 and AML cells were stimulated with 500 ng NET structures isolated from neutrophils treated with the aforementioned agents, or whole PMN pretreated with these agents, for 4 days. For NET scaffold inhibition, isolated NET structures were pre-incubated with DNase1 (10 U/ml; Fermentas, Vilnius, Lithuania) or heparin (heparin sodium, 100 μg/ml; LEO Pharma A/S, Ballerup, Denmark) for 60 min. For TF inhibition an IgG1 mouse anti-human TF mAb (10 μg/ml; Sekisui Diagnostics) was used.
Four images randomly taken from different regions of each well with 100x magnification per experiment were analysed. Average surface area covered by cells was calculated with Fiji/ImageJ [
To generate NETs, 1.5 × 106 neutrophils were seeded in six-well culture plates (Corning Incorporated, New York, USA) in low-serum RPMI medium (Gibco BRL) as previously described [
For the analysis of cell proliferation, Caco-2 and AML cells were stained with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma-Aldrich) [
For the analysis of apoptosis/necrosis, Caco-2 and AML cells were stained with FITC-annexin V (BD Biosciences) and propidium iodide (PI; Sigma-Aldrich). AML cells were also stained with CD34 APC (clone 8G12; BD Biosciences).
Proliferation and apoptosis analysis were performed after 4 days of co-culture with NETs in a FACScalibur flow cytometer (BD Biosciences). All data were analyzed with Flowjo V10.
Statistical analyses were performed using one-way analysis of variance (ANOVA) with Scheffé test for post hoc comparisons. P values less than 0.05 were considered significant. All statistical analyses were performed with OriginPro 8.
Since it has been recently suggested that NETs are implicated in cancer progression and metastasis in murine lung and mammary tumour models [
To this end, we used confocal microscopy to examine tumour specimens of colectomy for adenocarcinoma from ten patients. We observed significant deposition of NETs in tumour sections, as extracellular colocalization of neutrophil elastase with citrullinated H3 (
(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.
Furthermore, NETs (
Together, these data indicate that both neutrophils and NETs are present in the vicinity of cancer cells.
Considering the presence of inflammation in cancer and that inflammation generates a concentration gradient of cytokines and chemokines [
We observed a gradient of neutrophil accumulation in proportion to the distance from the center of the tumour mass (
—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.
These findings indicate that the intensity of neutrophil infiltration and NET generation is proportional to the proximity to the tumour.Since TF has a key role in tumour biology and thromboinflammation and recent publications demonstrate that, in thrombotic disorders, NETs express functional TF [
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.
These data suggest that neutrophils and NETs are important sources of TF in tumour microenvironment.
Based on the above findings we investigated the possible role of NETs in solid tumour progression in
Both PMA and sepsis serum-induced NETs inhibited
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.
We next investigated whether NETs inhibit Caco-2 growth via apoptosis. Thus, Caco-2 cells were treated with the aforementioned agents and demonstrated increased levels of apoptotic and late apoptotic cells, as assessed by Annexin V/PI flow cytometry (
To further clarify whether this inhibitory role of NETs in cancer cell growth is specific only to colon adenocarcinoma or occurs with other types of malignancy as well, we investigated their effects in human hematopoietic cancer. Thus, we conducted
Similarly to Caco-2 cells, PMA or sepsis-induced NETs or respectively pre-stimulated neutrophils significantly suppressed the growth 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.
These data taken together with the previous findings suggest that NETs, irrespectively of the stimulus, have also a potentially universal inhibitory role in primary human cancer cell cultures, as was observed in cells of acute myeloid leukemia.
In this study, we demonstrated for the first time the presence of NETs, decorated with TF, in colon adenocarcinoma sections of both the primary tumour mass and the respective metastatic lymph nodes. We also provide evidence for a potential role of NETs to limit
In line with previous reports indicating the presence of NETs in cancer biopsies [
We observed that NETs both in the primary tumour mass and in the metastatic lymph nodes are decorated with TF, the main
The biological significance of NETs present, however, remains unclear. On one hand, they may represent a reaction of the tumour environment against the growing cancer. On the other hand, they may play an adverse role in tumour growth offering a scaffold with an array of biologically active molecules attached on it, which may promote malignant cell survival, growth and, ultimately, local tumour expansion [
In conclusion, we show here for the first time the presence of NETs in colon cancer tissue and the respective metastatic lymph nodes. The pattern of neutrophilic distribution close to the area of tumour and neutrophil specific immunohistochemical method could provide a useful tool for the determination of proper surgical margin. The TF immunostaining is more prominent on NETs than in cancer cells pointing neutrophils as a significant source of TF in cancer microenvironment. Moreover, the subsequent thromboinflammatory and angiogenetic activity of TF may be involved in thrombosis and metastasis. Although NETs in our
H&E staining of colon adenocarcinoma patient specimens and respective metastatic lymph node specimens. Arrows demonstrate neutrophils. One representative out of ten independent experiments is shown. Original magnification 400x.
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(A) May Grünwald-Giemsa staining of Caco-2 cells co-cultured with PMNs pretreated with PMA or sepsis serum. One representative out of four independent experiments is shown. Original Magnification 100x. (B) May Grünwald-Giemsa staining of AML cells co-cultured with either PMA or sepsis pretreated PMNs, or 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. (C) and (D) Annexin V/PI flow cytometry of AML cells co-cultured with PMA or sepsis serum-induced NETs in the presence or absence of NET scaffold inhibitors. (C) demonstrates representative scatter plots. (D) Data from four independent experiments presented as mean±SD. n.s.—not significant compared to control, *p < 0.05.
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