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 distinct class of neutrophils⁶. It should be noted that LDN in circulating blood are more likely to produce neutrophil extracellular traps (NETs) than normal density neutrophils^2,7. NETs are web-like structures composed of nucleic acids, histones, proteases, and granular and cytosolic proteins, and they can efficiently entrap and destroy pathogens⁸.

Recently, NETs have been shown to capture not only microbes, but also platelets and circulating tumor cells which can assist in thrombus formation⁹ and tumor metastases^10,11. However, the molecular mechanisms behind the adhesive interactions between NETs and platelets or tumor cells are still unclear. More recently, an in vitro adhesion assay revealed that myeloid leukemia cells (K562¹²) and lung carcinoma cells (A549¹³) attach to NETs via β1 and β3 integrins. The authors used NET stock isolated from neutrophils and activated by phorbol 12-myristate 13-acetate (PMA) as the adhesion substrate¹⁴. Although this assay allows detection of real interactions with NET components in the absence of neutrophils, it is arguable whether the "cell-free NET stock" isolated by high-speed centrifugation retains the molecular structure identical to NETs produced in vivo. Recently, we found that peritoneal lavage fluid after abdominal surgery contained many mature LDN, which generated massive NETs and attached to tumor cells causing peritoneal metastases¹⁵. In this study, we successfully examined the adhesion of tumor cells to intact NETs without any physical manipulation. Here, we show details of a technique to detect adhesive interactions between NETs and free tumor cells.

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 within one minute. This is routinely performed as a postoperative peritoneal washing without significant effects on patients.
   2. Lavage the abdominal cavity extensively for at least 1 min.
      NOTE: It is recommended that the infused fluid is slowly stirred with the surgeon's hands so that the samples are uniform.
   3. Recover 200 mL of lavage fluid with four 50 mL syringes.
      NOTE: Sometimes a rubber connector is used to take up the fluids.
2. Perform purification of peritoneal LDN using granulocyte specific mAb, CD66b¹⁶.
   NOTE: Because the intermediate layer after density gradient centrifugation contains many mononuclear cells, positive selection of polymorph neutrophils using CD66b mAb was performed.
   1. Transfer the peritoneal lavage fluid to a 50 mL tube.
      NOTE: In this step, pass the fluids through a 100 µm nylon filter to remove impurities.
   2. Centrifuge the peritoneal fluid at 270 x g for 7 min at RT.
   3. Resuspend the pellets in 5 mL of PBS with 0.02% EDTA.
   4. Carefully overlay the 5 mL of cell suspension on a 3 mL density gradient solution.
   5. Centrifuge at 1,700 x g for 15 min at RT without any breaks.
   6. Harvest the ~2 - 3 mL of solution containing the intermediate layers (Figure 1A) using a pipette and mix it with 10 mL of PBS with 0.02% EDTA.
   7. Centrifuge the peritoneal fluid at 400 x g for 7 min at RT and discard the supernatant.
   8. Add another 10 mL of PBS with 0.02% EDTA, and centrifuge at 270 x g for 7 min at RT. Discard the supernatant.
   9. Dissolve the cell pellet (1 x 10⁷) in 60 µL of buffer for the magnetic cell separation kit.
   10. Add 20 µL of Fc block to the pellets and incubate for 10 min at 4 °C.
   11. Add 20 µL of the anti-CD66b conjugated with microbeads and incubate for an additional 10 min at 4 °C.
   12. Wash and re-suspend the pellets in 500 µL of MACS buffer.
   13. Apply the cell suspension to a magnetic column in the magnetic field of a suitable magnetic separator and collect the flow-through containing CD66b(-) cells by washing the column with 15 mL of buffer.
   14. Remove the column from the magnetic separator and immediately flush out the magnetically labeled CD66b(+) cells by firmly pushing the plunger into the column.
       NOTE: The CD66b(+) cell population consists mostly of neutrophils as determined by FACS analysis indicating that >95% of the CD66(+) fraction are positive for CD11b, CD15, and CD16, but negative for CD14.
3. Generation of NETs
   1. After centrifugation at 270 x g for 7 min at RT, re-suspend the isolated LDN (5 x 10⁶) in 1 mL of RPMI1640 with 10% FCS.
   2. Culture the LDN on a 6-well poly-L-lysine coated plate (6 cm in diameter) for 2 h at 37 ˚C in 5% CO₂.
   3. Add the green fluorescent counterstain (a membrane-impermeable dye to stain the nucleus and chromosomes) at the final concentration of 5 µM.
   4. Immediately observe the morphology of NETs (the extracellular DNA components expelled from the LDN under fluorescence microscopy).

2. Staining of Tumor Cells with Red Fluorescent Cell Linker Dye

1. Prepare human cancer cell lines MKN45, NUGC, and OCUM-1.
2. Wash 1 x 10⁷ cells with PBS + 0.02% EDTA in a 15 mL tube and centrifuge at 270 x g for 7 min at RT.
3. Add 1 mL of solution for dye staining to the pellets, and pipette gently.
4. Dissolve 4 µL of Red Fluorescent Cell Linker dye in 1 mL of solution for staining and mix with the cell suspension from step 2.2.
5. Incubate the pellets for 4 min at RT.
6. Add 4 mL of DMEM (10% FBS) to stop the staining reaction.
7. Centrifuge 270 x g for 7 min and discard the supernatant.
8. Repeat steps 2.6 and 2.7 twice.
9. Check with a fluorescence microscope (excitation = 551 nm, emitting = 567 nm) that the tumor cells are stained red.

3. Analysis of Tumor Cell Adhesion to NETs

1. Re-suspend the red fluorescent stained tumor cells (1 x 10⁶) in 1 mL of RPMI 1640 supplemented with 0.1% BSA.
2. Add the tumor cells to the LDN culture that produced the NETs as described in step 1.3.
3. Incubate for 5 min at 37 ˚C, which enables the tumor cells to contact the NETs.
   NOTE: Long incubation induces tumor cell adhesion to LDN or to the plates.
4. Remove the medium and gently wash the wells by adding 2 mL of prewarmed media (0.1% BSA + RPMI 1640) and swirling the dish.
5. Repeat the washing procedure in step 3.4 twice.
   NOTE: Since NETs weakly attach to the plate, washing should be done as gently as possible to avoid removal of the NETs themselves.
6. Add the green fluorescent dye for staining the nucleus and chromosomes at a final concentration of 5 µg/mL for visualization of the NETs.
7. Observe the NETs and attached tumor cells using the appropriate filters (green, excitation = 504 nm, emitting = 523 nm; red, excitation = 551 nm, emitting = 567 nm).
8. Merge the figures to show the tumor cells that are trapped by the NETs.
   NOTE: In some experiments, DNA degradation enzyme was added to the LDN culture (final concentration of 100 U/mL) 5 min before co-incubation for 5 min.

4. Time Lapse Video Analysis of the Trapped Tumor Cells

1. Culture the peritoneal LDN in a 35 mm round dish coated with poly-L-lysine as described in step 1.3.
2. Add the green fluorescent dye for staining the nucleus and chromosomes to visualize NETs as described in step 1.3.
3. After 1 min of incubation, remove the media and add 2 mL of 0.1% BSA + RPMI 1640.
4. Remove the media and add unstained 1 x 10⁶ MKN45 cells suspended in 0.1% BSA + RPMI 1640. Incubate for 5 min at RT.
5. Remove any unattached tumor cells by gently washing as described in step 3.4 and add DMEM with 10% FCS supplemented with 100 u/mL Penicillin and 100 µg/mL streptomycin.
6. Mount the culture dish in a whole-view cell observation system and select the appropriate field in which tumor cells are trapped by the NETs.
7. Continue to co-culture for an additional 2 days and take continuous photos of the field every 6 min under normal light and fluorescence, which detects MKN45 cells and NETs, respectively.
8. Superimpose the images at each timepoint and construct time-lapse videos using Image Viewer software.

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