Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Summary

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Abstract

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Introduction

Polymorph nuclear neutrophils in circulating blood are typically separated from mononuclear cells through the density gradient preparation method. However, some neutrophils known as low-density neutrophils (LDN), with CD11b(+), CD15(+), CD16(+), and CD14(-) phenotypes, are co-purified with mononuclear cells. The relative number of LDN significantly increases in various pathological conditions including autoimmune diseases^1,2, sepsis³, and cancer^4,5. Previous studies have shown that LDN are a phenotypically and functionally disti....

Protocol

LDN were obtained from patients enrolled in this study and were approved by the Institutional Review Board of Jichi Medical University.

1. Isolation of LDN from Abdominal Cavity Lavages and NET Detection

1. Sample acquisition
   1. Infuse 1,000 mL of normal sterile saline directly into the abdominal cavity before wound closure in patients who have undergone abdominal surgery due to gastrointestinal malignancy.
      NOTE: Samples were obtained from patients who underwent a gastrectomy, colectomy, or esophagectomy without bias based on age or sex. Saline was transferred into a container and poured into the whole abdomen with....

Results

In the 2-hour culture, CD66b(+) LDN derived from peritoneal lavage fluid showed string structures stained with green-fluorescent dye for nuclear and chromosome (Figure 1B), while CD66b(-) mononuclear cells did not (Figure 1C). However, when the LDN cultures were pretreated with 100 U/mL DNase I, the characteristic structure was destroyed (Figure 1D), indicating that they were composed of extracellula.......

Discussion

Previous studies have reported that circulating tumor cells can be trapped by NET substrates in vivo^10,11. Metastatic breast cancer cells have been shown to stimulate neutrophils and induce formation of NETs, which assists in tumor cell growth in the target organ¹⁷. In addition, we found that short-term cultures of LDN from postoperative lavage fluid produced massive NETs that could efficiently entrap tumor cells without further s.......

Materials

Name	Company	Catalog Number	Comments
Ficoll-Paque PLUS	GE Healthcare, SWEDEN	17-1440-02
StraightFrom™ Whole Blood CD66b MicroBeads	Miltenyi Biotec, Bergisch Gladbach, Germany	130-104-913
Fc block	Miltenyi Biotec, Bergisch Gladbach, Germany	130-059-901
MACS Rinsing Solution	Miltenyi Biotec, Bergisch Gladbach, Germany	130-091-222
MACS BSA Stock Solution	Miltenyi Biotec, Bergisch Gladbach, Germany	130-091-376
LS Columns	Miltenyi Biotec, Bergisch Gladbach, Germany	130-041-306
MACS Magnetic Separator	Miltenyi Biotec, Bergisch Gladbach, Germany	130-042-501
SYTOX green nucleic acid stain 5mM solution in DMSO	Thermo Fisher Scientific, Waltham, MA, USA	S7020
PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling	Sigma-Aldrich, St Louis, MO, USA	P9691
Diluent C	Sigma-Aldrich, St Louis, MO, USA	CGLDIL
RPMI1640 Medium	Sigma-Aldrich, St Louis, MO, USA	R8758
Dulbecco’s Modified Eagle Medium-high glucose (DMEM)	Sigma-Aldrich, St Louis, MO, USA	D5796
Dulbecco’s Phosphate Buffered Saline (DPBS)	Sigma-Aldrich, St Louis, MO, USA	D8537
0.5mol/l-EDTA Solution (pH 8.0)	nacalai tesque, Japan	06894-14
Fetal Bovine Serum, qualified, USDA-approved regions	gibco by life technologies, Mexico	10437-028
Bovine Serum Albumin lyophilized powder, ≥96% (agarose gel electrophoresis)	Sigma-Aldrich, St Louis, MO, USA	A2153
Penicillin Streptomycin	Life Technologies Japan	15140-122
Plasmocin Prophylactic	InvivoGen, San Diego, CA-USA	ant-mpp
DNase I	Worthington, Lakewood NJ)	LS002138
Poly-L-Lysine-Coated MICROPLATE 6Well	IWAKI, Japan	4810-040
Poly-L-Lysine-Coated MICROPLATE 24Well	IWAKI, Japan	4820-040
fluorescein stereomicroscope	BX8000, Keyence, Osaka Japan	BZ-X710
Whole view cell observation system	Nikon, Kanagawa, Japan	BioStudio (BS-M10)
MKN45 human gastric cancer cell line	Riken, Tukuba Japan	N/A
NUGC-4 human gastric cancer cell line	Riken, Tukuba Japan	N/A
OCUM-1 human gastric cancer cell line	Osaka City University, Japan	N/A	Gift from Dr. M.Yashiro