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

Vi takker Ms. J. Shinohara og I. Nieda for teknisk og kontorarbejde. Vi takker også, Drs. Shiro Matsumoto, Hidenori Haruta, Kentaro Kurashina og Kazuya Takahashi for deres samarbejde for prøven erhvervelse på operationsstuen. Dette arbejde blev støttet af en licensbetaling for videnskabelig forskning fra ministeriet for uddannelse, videnskab, sport, og kultur af Japan og Japan samfund til fremme af videnskab (17K 10606).

LDN er fremstillet af patienter tilmeldt i denne undersøgelse og blev godkendt af den institutionelle Review Board af Jichi medicinske universitet.
1. isolering af LDN fra bughulen Lavages og netto påvisning
<ol><li>Prøven erhvervelse<ol>
<li>Indgyde 1.000 mL normalt sterilt saltvand direkte ind i bughulen før sår lukning i patienter, der har gennemgået abdominal kirurgi på grund af gastrointestinale malignitet. Bemærk: Prøver blev indhentet fra patienter, der undergik en gastrek...

<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>

Tidligere undersøgelser har rapporteret, at cirkulerende tumorceller kan blive fanget af netto substrater i vivo10,11. Metastatisk brystkræftceller har vist sig at stimulere neutrofile og fremkalde dannelse af redskaber, som hjælper i tumorcellevækst i target organ17. Desuden fandt vi, at kortsigtede kulturer af LDN fra postoperative lavage væske produceret massive redskaber, der effektivt kunne lokke tumorceller uden yderlig...

Polymorph nukleare neutrofiler i cirkulerende blod er typisk adskilt fra mononukleære celler gennem metoden tæthed gradient forberedelse. Nogle neutrofiler, kendt som low-density neutrofile (LDN), med CD11b(+), CD15(+), CD16(+) og CD14(-) fænotyper, er imidlertid Co renset med mononukleære celler. Det relative antal LDN øger i forskellige patologiske betingelser herunder autoimmune sygdomme1,2, sepsis3og kræft4,5. Tidligere undersøgelser har vist, at LDN er en fænotype og funktionelt adskilte klasse af neutrofiler6. Det skal bemærkes, at LDN i cirkulerende blod er mere tilbøjelige til at producere neutrofile ekstracellulære fælder (NETs) end normal tæthed neutrofiler2,7. NETs er web-lignende strukturer sammensat af nukleinsyrer, histoner, proteaser og kornede og cytosole proteiner, og de kan effektivt have fat og ødelægger patogener8.
For nylig, net har vist sig at fange ikke kun mikrober, men også blodplader og cirkulerende tumorceller, som kan hjælpe med blodprop dannelse9 og tumor metastaser10,11. Men de molekylære mekanismer bag de selvklæbende interaktioner mellem net og blodplader eller tumorceller er stadig uklart. En in vitro- vedhæftning analyse afslørede for nylig, at myeloid leukæmiceller (K56212) og lunge cancer celler (A54913) tillægger net via B1 og β3 integriner. Forfatterne anvendte NET stock isoleret fra neutrofile og aktiveres af phorbol 12-myristate 13-acetat (PMA) som vedhæftning substrat14. Selv om denne analyse giver påvisning af virkelige interaktioner med netto komponenter i fravær af neutrofiler, kan det diskuteres hvorvidt de "celle-gratis NET stock" isoleret ved højhastigheds centrifugering bevarer den molekylære struktur identisk med net produceret i vivo. For nylig, fandt vi at peritoneal lavage væske efter abdominal kirurgi indeholdt mange modne LDN, som genereres massive redskaber og knyttet til tumorceller forårsager peritoneal metastaser15. I denne undersøgelse undersøgte vi med held vedhæftning af tumorceller til intakt redskaber uden fysisk manipulation. Her, viser vi oplysninger om en teknik til at registrere klæbende interaktioner mellem net og gratis tumorceller.

<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

Aktiveret neutrofiler release neutrofile ekstracellulære fælder (net), som kan fange og ødelægge mikrober. Nylige undersøgelser tyder på at NETs er involveret i forskellige sygdomsprocesser, såsom autoimmune sygdom, trombose og tumor metastaser. Her viser vi en detaljeret in vitro- teknik til at detektere netto aktivitet under diffusering af gratis tumorceller, der vokser efter tilknytning til NETs. Først, vi indsamlet lavdensitet neutrofile (LDN) fra postoperative peritoneal lavage væske fra patienter, der gennemgik laparotomies. Kortsigtede dyrkning af LDN resulterede i massiv netto dannelse, der blev visualiseret med grøn fluorescerende nukleare og kromosom kontrastfarve. Efter Co inkubation af menneskelige gastrisk kræft cellelinjer MKN45, dokumenter-1 og NUGC-4 med garnene, blev mange tumorceller fanget af nettene. Efterfølgende, den vedhæftede fil blev fuldstændig ophævet ved nedbrydning af net med DNase I. Time-lapse video afslørede, at tumorceller fanget af garnene ikke gjorde dø, men i stedet voksede kraftigt i en kontinuerlig kultur. Disse metoder kan anvendes til påvisning af selvklæbende interaktioner mellem net og forskellige typer af celler og materialer.

I 2-timers kultur, CD66b(+) LDN stammer fra peritoneal lavage væske viste streng strukturer farves med grønt-fluorescerende farvestof for nukleare og kromosom (figur 1B), mens CD66b(-) mononukleære celler ikke (figur 1 c). Men når LDN kulturer blev forbehandlet med 100 U/mL DNase, den karakteristiske struktur var ødelagt (fig. 1 d), der angiver, at de var sammensat af ekstracellulære DNA bortvi...

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

Vi præsenterer her, en metode hvor menneskelige low-density neutrofile (LDN), inddrives fra postoperative peritoneal lavage væske, producere massive neutrofile ekstracellulære fælder (net) og effektivt fælde gratis tumorceller, der senere vokser.

Neutrofile ekstracellulære fælder genereret af Low Density neutrofiler fremstillet af Peritoneal Lavage væske mægle tumorcellevækst og vedhæftet fil

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

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