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
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 gas...

<ol>
	<li>Hacbarth, E., Kajdacsy-Balla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+neutrophils+in+patients+with+systemic+lupus+erythematosus,+rheumatoid+arthritis,+and+acute+rheumatic+fever.">Low density neutrophils in patients with systemic lupus erythematosus, rheumatoid arthritis, and acute rheumatic fever.</a> Arthritis and Rheumatology. 29 (11), 1334-1342 (1986).</li><li>Denny, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinct+subset+of+proinflammatory+neutrophils+isolated+from+patients+with+systemic+lupus+erythematosus+induces+vascular+damage+and+synthesizes+type+I+IFNs.">A distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type I IFNs.</a> The Journal of Immunology. 184 (6), 3284-3297 (2010).</li><li>Morisaki, T., Goya, T., Ishimitsu, T., Torisu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increase+of+low+density+subpopulations+and+CD10+(CALLA)+negative+neutrophils+in+severely+infected+patients.">The increase of low density subpopulations and CD10 (CALLA) negative neutrophils in severely infected patients.</a> Surgery Today. 22 (4), 322-327 (1992).</li><li>Schmielau, J., Finn, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+granulocytes+and+granulocyte-derived+hydrogen+peroxide+are+the+underlying+mechanism+of+suppression+of+t-cell+function+in+advanced+cancer+patients.">Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients.</a> Cancer Research. 61 (12), 4756-4760 (2001).</li><li>Brandau, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+in+the+peripheral+blood+of+cancer+patients+contain+a+subset+of+immature+neutrophils+with+impaired+migratory+properties.">Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties.</a> Journal of Leukocyte Biology. 89 (2), 311-317 (2011).</li><li>Carmona-Rivera, C., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+granulocytes:+a+distinct+class+of+neutrophils+in+systemic+autoimmunity.">Low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity.</a> Seminars in Immunopathology. 35 (4), 455-463 (2013).</li><li>Villanueva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netting+neutrophils+induce+endothelial+damage,+infiltrate+tissues,+and+expose+immunostimulatory+molecules+in+systemic+lupus+erythematosus.">Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.</a> The Journal of Immunology. 187 (1), 538-552 (2011).</li><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Demers, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancers+predispose+neutrophils+to+release+extracellular+DNA+traps+that+contribute+to+cancer-associated+thrombosis.">Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (32), 13076-13081 (2012).</li><li>Cools-Lartigue, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+sequester+circulating+tumor+cells+and+promote+metastasis.">Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis.</a> Journal of Clinical Investigation. , (2013).</li><li>Tohme, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Extracellular+Traps+Promote+the+Development+and+Progression+of+Liver+Metastases+after+Surgical+Stress.">Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress.</a> Cancer Research. 76 (6), 1367-1380 (2016).</li><li>Monti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-dependent+cell+adhesion+to+neutrophil+extracellular+traps+through+engagement+of+fibronectin+in+neutrophil-like+cells.">Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.</a> Public Library of Science One. 12 (2), e0171362 (2017).</li><li>Najmeh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+sequester+circulating+tumor+cells+via+beta1-integrin+mediated+interactions.">Neutrophil extracellular traps sequester circulating tumor cells via beta1-integrin mediated interactions.</a> International Journal of Cancer. 140 (10), 2321-2330 (2017).</li><li>Najmeh, S., Cools-Lartigue, J., Giannias, B., Spicer, J., Ferri, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+Human+Neutrophil+Extracellular+Traps+(NETs)+Isolation+and+Handling.">Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.</a> Journal of Visualized Experiments. (98), (2015).</li><li>Kanamaru, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+neutrophils+(LDN)+in+postoperative+abdominal+cavity+assist+the+peritoneal+recurrence+through+the+production+of+neutrophil+extracellular+traps+(NETs).">Low density neutrophils (LDN) in postoperative abdominal cavity assist the peritoneal recurrence through the production of neutrophil extracellular traps (NETs).</a> Scientific Reports. 8 (1), 632 (2018).</li><li>Eades-Perner, A. M., Thompson, J., van der Putten, H., Zimmermann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+transgenic+for+the+human+CGM6+gene+express+its+product,+the+granulocyte+marker+CD66b,+exclusively+in+granulocytes.">Mice transgenic for the human CGM6 gene express its product, the granulocyte marker CD66b, exclusively in granulocytes.</a> Blood. 91 (2), 663-672 (1998).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cells+induce+metastasis-supporting+neutrophil+extracellular+DNA+traps.">Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps.</a> Science Translational Medicine. 8 (361), 361ra138 (2016).</li></ol>

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 s...

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&#8482; 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&#8217;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&#8217;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, &#8805;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 extracellula...

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 ...

This method can help answer key questions about the role of neutrophil subpopulations in tumor immunology especially about the mechanical involvement of neutrophil extracellular traps, or NETs, in tumor metastasis. The main advantage of this technique is that it allows visualization of the adhesive interactions of tumor cells to intact NETs. The implications of this technique extend towards the therapy of cancer.As NETs derived from specific neutrofil subsets may promote tumor metastasis. Demonstrating the procedure will be Junko Shoniohara from our laboratory. To acquire the neutrophils first infuse one liter of normal sterile saline immediately before wound closure directly into the abdominal cavity of a patient who has just undergone abdominal surgery due to gastrointestinal malignancy and lavage the abdominal cavity extensively for at least one minute.Then stir the saline within the cavity and recover 200 milliliters of lavaged fluid with four 50-milliliter syringes. In the lab transfer the peritoneal lavage fluid through a 100 micrometer nylon filter into individual 50 milliliter tubes for centrifugation. Re-suspend the pellets in 5 milliliters of PBS supplemented with 0.02%EDTA and carefully overlay each cell suspension onto three milliliters of density gradient solution.After density gradient separation harvest two to three milliliters of the intermediate layer from each tube and wash the peritoneal fluid samples in 10 milliliters of fresh PBS plus EDTA per tube. Pool the pellets in 10 milliliters of fresh PBS plus EDTA for an additional wash and dilute the cells to a one times 10 to the seventh cells per 60 microliters of magnetic activated cell sorting, or MACS, buffer concentration. Block any nonspecific binding with 20 microliters of Fc block for 10 minutes at four degrees Celsius followed by the addition of 20 microliters of anti-CD66b magnetic beads.After 10 minutes at four degrees Celsius wash and re-suspend the cells in 500 microliters of MACS buffer and add the cells to an appropriately sized magnetic column within the magnetic field of a suitable magnetic separator. When all of the CD66b negative cells have run through the column add 15 millilitres of fresh MACS buffer to the column reservoir and transfer the column from the magnetic separator into a new conical tube. Then immediately plunge the column to flush the magnetically labeled CD66b positive into the tube.After centrifugation re-suspend the isolated low density neutrophils at a five times 10 to the sixth cells per milliliter of RPMI1640 medium supplemented with FBS concentration and seed 1 milliliter of neutrophils per well on the six well poly-L-lysine coated plate. After two hours at 37 degrees Celsius and 5%carbon dioxide stain the cells in each well with an appropriate fluorescent membrane impermeable nuclear and chromosomal dye according to the manufacturer's instructions and immediately observe the morphology of the NETs by fluorescence microscopy. To assess tumor cell adhesion to the NETs re-suspend gastric tumor cells stained with the contrasting fluorescent dye at a one times 10 to the sixth per milliliter of RPMI1640 supplemented with 0.1%bovine serum albumin concentration and add one milliliter of tumor cells to each well of the low density neutrophil cultures.After five minutes at 37 degrees Celsius remove the supernatant from each well and gently wash the cells two times with 2 milliliters of pre warmed RPMI1640 medium supplemented with BSA per wash. A five minute incubation is long enough for detecting NET specific binding. Add the warmed medium to the side of each well slowly gently swirl the plate and remove the supernatant with an aspirator.The NETs and the attached tumor cells can then be observed by fluorescence microscopy. For a time lapse video analysis of the trapped tumor cells culture and stain the peritoneal low density neutrophils in a 35 millimeter dish, poly-L-lysine coated dish, as demonstrated followed by a brief wash in two milliliters of 0.1%BSA plus RPMI1640. Replace the medium with one times 10 to the sixth unstained gastric tumor cells suspended in two milliliters of RPMI supplemented with BSA for a five minute incubation at room temperature.At the end of the incubation rinse the cells in two milliliters of fresh RPMI supplemented with BSA for five minutes at room temperature followed by gentle swirling to remove any unattached tumor cells. Then replace the supernatant with DMEM supplemented with FBS and antibiotics and mount the dish in a whole view cell observation system for time lapse analysis of tumor cell trapping by the NETs. After two hours of culture CD66b positive low density neutrophils derived from peritoneal lavage fluid exhibit string structures by green fluorescent nuclear and chromosomal staining while CD66b negative mononuclear cells do not.When the low density neutrophil cultures are pre-treated with 100 units per milliliter of DNAse I, however, the characteristics structures are destroyed indicating that the structures are composed of extracellular DNA expelled from the neutrophils. When the low density neutrophils are cultured on uncoated plastic plates many NET clusters can be observed floating within the medium with few NETs attached to the bottom of the dish. After five minutes of incubation with a fluorescently labeled gastric tumor cell line many tumor cells attach to the neutrophils within the NETs a phenomenon that is not observed after pre-treatment with DNAse I.Similar adhesion patterns are also seen for other tumor cells.OKUM-1 and NUGC-4. Interestingly while the NETs gradually disappear within several hours of extended tumor cell co-culture the tumor cells trapped by the NETs begin to proliferate vigorously after NET release. During this procedure it's important to remember that because the NETs stick to the plate weakly the washing and dish transfer processes should be performed in an especially cautious manner.After its development this technique paves the way for researchers in various fields of biomedical science to explore the causal relationship of NETs with various diseases such as thrombosis, systemic inflammatory response syndrome, and autoimmune diseases.

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Research

JoVE Journal

Cancer Research

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 &#181;m zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 &#181;m of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

The present protocol develops an image-based technique for rapid, non-destructive, and label-free regional cell density and viability measurement within 3D tumor aggregates. Findings revealed a cell-density gradient, with higher cell densities in core regions than outer layers in developing aggregates and predominantly peripheral cell death in HER2+ aggregates treated with Trastuzumab.

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These ...

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Far-Red Fluorescent Senescence-Associated &amp;#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...