Name: neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment
Uploaded: 2018-08-03T14:30:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

We thank Ms. J. Shinohara and I. Nieda for technical and clerical work.&#160;Also, we thank Drs. Shiro Matsumoto, Hidenori Haruta, Kentaro Kurashina, and Kazuya Takahashi for their cooperation for sample acquisition in operating room.&#160;This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science (17K10606).

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
<ol>
	<li>Sample acquisition
	<ol>
		<li>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.</li>
		<li>Lavage the abdominal cavity extensively for at least 1 min. 
		NOTE: It is recommended that the infused fluid is slowly stirred with the surgeon&#39;s hands so that the samples are uniform.</li>
		<li>Recover 200 mL of lavage fluid with four 50 mL syringes. 
		NOTE: Sometimes a rubber connector is used to take up the fluids.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Transfer the peritoneal lavage fluid to a 50 mL tube. 
		NOTE: In this step, pass the fluids through a 100 &#181;m nylon filter to remove impurities.</li>
		<li>Centrifuge the peritoneal fluid at 270 x g for 7 min at RT.</li>
		<li>Resuspend the pellets in 5 mL of PBS with 0.02% EDTA.</li>
		<li>Carefully overlay the 5 mL of cell suspension on a 3 mL density gradient solution.</li>
		<li>Centrifuge at 1,700 x g for 15 min at RT without any breaks.</li>
		<li>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.</li>
		<li>Centrifuge the peritoneal fluid at 400 x g for 7 min at RT and discard the supernatant.</li>
		<li>Add another 10 mL of PBS with 0.02% EDTA, and centrifuge at 270 x g for 7 min at RT. Discard the supernatant.</li>
		<li>Dissolve the cell pellet (1 x 107) in 60 &#181;L of buffer for the magnetic cell separation kit.</li>
		<li>Add 20 &#181;L of Fc block to the pellets and incubate for 10 min at 4 &#176;C.</li>
		<li>Add 20 &#181;L of the anti-CD66b conjugated with microbeads and incubate for an additional 10 min at 4 &#176;C.</li>
		<li>Wash and re-suspend the pellets in 500 &#181;L of MACS buffer.</li>
		<li>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.</li>
		<li>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 &#62;95% of the CD66(+) fraction are positive for CD11b, CD15, and CD16, but negative for CD14.</li>
	</ol>
	</li>
	<li>Generation of NETs
	<ol>
		<li>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.</li>
		<li>Culture the LDN on a 6-well poly-L-lysine coated plate (6 cm in diameter) for 2 h at 37 &#730;C in 5% CO2.</li>
		<li>Add the green fluorescent counterstain (a membrane-impermeable dye to stain the nucleus and chromosomes) at the final concentration of 5 &#181;M.</li>
		<li>Immediately observe the morphology of NETs (the extracellular DNA components expelled from the LDN under fluorescence microscopy).</li>
	</ol>
	</li>
</ol>
2. Staining of Tumor Cells with Red Fluorescent Cell Linker Dye
<ol>
	<li>Prepare human cancer cell lines MKN45, NUGC, and OCUM-1.</li>
	<li>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.</li>
	<li>Add 1 mL of solution for dye staining to the pellets, and pipette gently.</li>
	<li>Dissolve 4 &#181;L of Red Fluorescent Cell Linker dye in 1 mL of solution for staining and mix with the cell suspension from step 2.2.</li>
	<li>Incubate the pellets for 4 min at RT.</li>
	<li>Add 4 mL of DMEM (10% FBS) to stop the staining reaction.</li>
	<li>Centrifuge 270 x g for 7 min and discard the supernatant.</li>
	<li>Repeat steps 2.6 and 2.7 twice.</li>
	<li>Check with a fluorescence microscope (excitation = 551 nm, emitting = 567 nm) that the tumor cells are stained red.</li>
</ol>
3. Analysis of Tumor Cell Adhesion to NETs
<ol>
	<li>Re-suspend the red fluorescent stained tumor cells (1 x 106) in 1 mL of RPMI 1640 supplemented with 0.1% BSA.</li>
	<li>Add the tumor cells to the LDN culture that produced the NETs as described in step 1.3.</li>
	<li>Incubate for 5 min at 37 &#730;C, which enables the tumor cells to contact the NETs. 
	NOTE: Long incubation induces tumor cell adhesion to LDN or to the plates.</li>
	<li>Remove the medium and gently wash the wells by adding 2 mL of prewarmed media (0.1% BSA + RPMI 1640) and swirling the dish.</li>
	<li>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.</li>
	<li>Add the green fluorescent dye for staining the nucleus and chromosomes at a final concentration of 5 &#181;g/mL for visualization of the NETs.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
4. Time Lapse Video Analysis of the Trapped Tumor Cells
<ol>
	<li>Culture the peritoneal LDN in a 35 mm round dish coated with poly-L-lysine as described in step 1.3.</li>
	<li>Add the green fluorescent dye for staining the nucleus and chromosomes to visualize NETs as described in step 1.3.</li>
	<li>After 1 min of incubation, remove the media and add 2 mL of 0.1% BSA + RPMI 1640.</li>
	<li>Remove the media and add unstained 1 x 106 MKN45 cells suspended in 0.1% BSA + RPMI 1640. Incubate for 5 min at RT.</li>
	<li>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 &#181;g/mL streptomycin.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Superimpose the images at each timepoint and construct time-lapse videos using Image Viewer software.</li>
</ol>

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The authors declare that they have no competing financial interests.

Previous studies have reported that circulating tumor cells can be trapped by NET substrates in vivo10,11. 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 stimulation15. These observations suggest a possible role for mechanical involvement of NETs in the metastatic process. However, the detailed molecular mechanisms that mediate adhesive interactions between the NETs and tumor cells is still unclear. Recent studies have implicated the involvement of integrin in this process through the use of NET stock isolated from activated neutrophils that serve as the adhesion substrate12,13. However, purified and concentrated NETs components are supposed to have largely different molecular structures from original NETs. In comparison, our method uses intact NETs without physical manipulation, which is a strong advantage when examining the detailed mechanisms of adhesive interactions with NETs. With this simple technique, researchers can detect the specific attachment of many tumor cells to NETs quickly (within 5 min).
The most important feature of the protocol is the use of a poly-L-lysine coated plate to fix the NET structures on the bottom of the plate. When intact plastic plates were used for the LDN culture, only a few NETs were detected at the bottom. Furthermore, many green-positive clusters were observed as freely floating in the culture media, when they should have been detached from the plate (Figure 1D). We also examined NET formation on plates coated with collagen, fibronectin, and laminin, which enabled the detection of web-like structures in the bottom layer. However, the NETs appeared to attach to the substrates less tightly than to poly-L-lysine, and they were easily detached by the washing procedure. Therefore, these other options are insufficient for adhesion assays. In fact, if we use plates without poly-L-lysine coatings, NETs and any attached tumor cells can become easily removed from the plate by simply adding media to the wells.
The critical step in the adhesion assay is the incubation period of the co-culture with the tumor cells. Five minutes is sufficient for tumor cells to bind to the NETs. Under the experimental conditions, when adding the solution containing tumor cells to the 6-well plate, all tumor cells make physical contact with the NET structures formed at the bottom of the plate. However, co-culturing for a longer period (e.g., longer than 10 minutes) results in the binding of tumor cells to LDN or poly-L-lysine substrates. Therefore, the incubation time should be short to detect specific binding to the NETs. The maneuvering of gentle washing is another essential point. We made sure to slowly add the warmed media from the side-wall of each well, gently swing the plate, and remove the supernatant with an aspirator. Since NETs stick to the plate weakly, this process should be performed in an especially cautious manner.
This technique highlights the fact that a different molecular mechanism is needed for tumor cells binding to NETs within minutes, than that shown in an adhesion assay with a ~30 - 60 minute incubation to NET stocks12,13. Blocking experiments using this method can elucidate the detailed molecular mechanisms in which real NETs trap various materials. However, a limitation of this technique is that the washing process can differ when the adhesive interactions occur between NETs and tumor cells in vivo. In the clinical setting, the density of tumor cells is much lower than that used in this protocol, which may present another limitation.
Time-lapse video analysis is highly useful for monitoring the outcomes of various cellular events over a period of days. Using the whole-cell view system, we show that NETs are gradually degraded, presumably by DNAse contained in serum, whereas MKN45 trapped by NETs start to proliferate vigorously thereafter. This video only showed the division of tumor cells attached to NETs, with no comparison to MKN45 attached to the plate. Contaminated neutrophils and the added nuclear staining dye may have also affected the growth of the tumor cells. Therefore, we could not draw a definite conclusion regarding the net effects of NETs on tumor cell growth. However, this analysis does indicate that tumor cells are not killed by the NETs and can in fact survive following entrapment by NETs.

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 diseases1,2, sepsis3, and cancer4,5. 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 neutrophils2,7. 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 metastases10,11. 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 &#946;1 and &#946;3 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 &#34;cell-free NET stock&#34; 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.

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

neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment

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.

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 extracellular DNA expelled from neutrophils. When LDN were cultured on plastic plates without coating, many NET clusters floating in media were observed, with few NETs on the bottom (Figure 1E).
Next, to examine adhesion of the tumor cell lines to NETs, MKN45 cells labelled with red fluorescent dye were added to the NETs and co-incubated for 5 minutes. After gentle washing, many MKN45 cells were found attached (Figure 2A and 2C). In contrast, few MKN45 cells attached when LDN were pretreated with DNase I (Figure 2B and 2C). The merged figure clearly showed that a majority of the attached MKN45 cells were co-localized with NETs, seen as structures formed on the bottom of the plate (Figure 2D). Similar adhesion patterns were observed with OCUM-1 and NUGC-4 (Figure 2E and 2F).
Since NETs have been shown to be cytotoxic to bacteria, we then examined whether tumor cells trapped by NETs die or survive in a continuous co-culture using time-lapse video. Interestingly, while NETs gradually disappeared within several hours, the MKN45 cells trapped by NETs started to proliferate vigorously afterwards (Video 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58201/58201fig1.jpg" /> 
Figure 1: NET formation after the culture of peritoneal LDN. (A) After density gradient centrifugation of postoperative lavage fluid, cells recovered from the intermediate layer are shown. (B) Then, CD66b(+) LDN were separated from CD66b(-) mononuclear cells and cultured on poly-L-lysine coated 6-well plates, followed by nuclear staining dye addition and immediate observation with fluorescence microscopy. (C) The same number of CD66b(-) mononuclear cells were cultured under the same conditions. (D) The LDN monolayer was incubated with DNase I for 5 minutes before nuclear staining. (E) CD66b(+) LDN were cultured on plastic plates without coating. White bars represent 100 &#181;m. This figure was modified from a previous publication15. <a href="https://www.jove.com/files/ftp_upload/58201/58201fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58201/58201fig2.jpg" /> 
Figure 2: Adhesion of tumor cells to NETs. (A) Peritoneal LDN were cultured on poly-L-lysine coated 6-well plates for 2 h, which produced massive NETs. Red fluorescein-labelled MKN45 were then added. After a 5-minute incubation, unattached cells were removed by gentle washing, and remaining tumor cells were observed under a fluorescence microscope. (B) Before the addition of MKN45, the LDN monolayer was pretreated with DNase I for 5 min. (C) In experiments (A) and (B), the number of attached MKN45 cells were counted in 3 random fields under the fluorescence microscope and mean &#177; SD was expressed. (D) After gentle washing, green fluorescent dye for nuclear and chromosome was added to the co-culture. In representative fields, photos of attached MKN45 and NET structures were taken using appropriate optical wavelength filters, and the two figures were merged. The same experiments performed in (D) were also performed for OCUM-1 (E) and NUGC-4 (F). White bars represent 100 &#181;m. This figure was modified from a previous publication15. <a href="https://www.jove.com/files/ftp_upload/58201/58201fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/58201/58201video1.jpg" /> 
Video 1: Post-adhesion behavior of the trapped tumor cells. LDN were cultured on poly-L-lysine coated 6-well plates for 2 hours, and NET formation was confirmed by green fluorescent dye for nuclear and chromosome. Then, unstained MKN45 cells were added and co-incubated for 5 minutes, and unattached cells were removed by gentle washing and continued to be co-cultured in 10% FCS + DMEM. Using a whole-view cell observation system, areas where tumor cells were trapped by NETs were searched for. Photographs of the field were then taken every 6 minutes under normal light and fluorescence, which enabled clear figures of tumor cells and NETs, respectively. Finally, the images were superimposed with Image Viewer software, and a time lapse video was created at a 3600x higher speed than real-time. <a href="https://www.jove.com/files/ftp_upload/58201/NET+MKN.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

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

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.

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

neutrophil-extracellular-traps-generated-low-density-neutrophils

Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.7K Views.  Jichi Medical University. 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.

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Immunology and Infection

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Author Spotlight: Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

Efficient Rat Bone Marrow Neutrophil Extracellular Trap Isolation

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

Author Spotlight: Optimized Method for Isolating Rat Neutrophils and NETs from Bone Marrow

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood neutrophils.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone ...

Biology

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

Time-lapse Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Analyze Post-adhesion Interaction between Cancer Cells and NETs

Source: Kanamaru, R. et al. <a href="https://www.jove.com/video/58201" target="_blank">Neutrophil Extracellular Traps Generated by Low-density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment.</a> J. Vis. Exp. (2018)
In this video, we analyze the post-adhesion behavior of cancer cells trapped in neutrophil extracellular traps (NETs) by co-culturing low-density neutrophils and gastric cancer cells. The time-lapse video analysis helps monitor various cellular events and study the role of NETs in pathological conditions.

Source: Kanamaru, R. et al. Neutrophil Extracellular Traps Generated by Low-density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell ...

JoVE Encyclopedia of Experiments

Gastrointestinal Cancer

Assays and techniques

Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive Interaction Between Cancer Cells and NETs

Source: Kanamaru, R. et al. <a href="https://www.jove.com/video/58201" target="_blank">Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment.</a> J. Vis. Exp. (2018)
This video demonstrates the fluorescent staining of neutrophil extracellular traps (NETs), the extracellular DNA components expelled by neutrophils. This technique enables the visualization of adhesive interaction between cancer cells and NETs.

Source: Kanamaru, R. et al. Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell ...