Name: an in vivo murine sciatic nerve model of perineural invasion
Uploaded: 2018-04-23T13:00:00
Description: Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

Forfatterne anerkender de tekniske tjenester, som molekylære cytologi facilitet og Dyrefacilitet af Memorial Sloan Kettering Cancer Center. Dette arbejde blev støttet af NIH tilskud CA157686 (til R.J. Wong) og P30 CA008748 (Memorial Sloan Kettering Cancer Center støtte grant).

Alle procedurer med animalsk emner blev godkendt af det institutionelle Animal Care og brug udvalg på Memorial Sloan Kettering Cancer Center.
1. forberedelse af kræftceller
<ol><li>Høst sub sammenflydende Panc02-H7 celler med 0,25% trypsin i 5 min ved 37 ° C. Indsamle cellerne i en 15 mL centrifugeglas. Bemærk: Cellerne dyrkes i T-225 kolbe, som indeholder omkring 12 x 106 celler pr. kolbe på 80% confluency og 4 mL trypsin / kolbe bruges.</li>
	<li>Der centrifugeres celle...

<ol>
	<li>Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H., Albo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+in+cancer.">Perineural invasion in cancer.</a> Cancer. 115 (15), 3379-3391 (2009).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11 (10), 695-707 (2011).</li><li>Deborde, S., Wong, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Schwann+cells+facilitate+cancer+progression+in+nerves.">How Schwann cells facilitate cancer progression in nerves.</a> Cell Mol Life Sci. 341 (177-186), 1236361-1236416 (2017).</li><li>Abiatari, I., 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=Consensus+transcriptome+signature+of+perineural+invasion+in+pancreatic+carcinoma.">Consensus transcriptome signature of perineural invasion in pancreatic carcinoma.</a> Mol Cancer Ther. 8 (6), 1494-1504 (2009).</li><li>Ayala, G. 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=In+vitro+dorsal+root+ganglia+and+human+prostate+cell+line+interaction:+redefining+perineural+invasion+in+prostate+cancer.">In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.</a> Prostate. 49 (3), 213-223 (2001).</li><li>Gil, Z., Cavel, O., 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102 (2), 107-118 (2010).</li><li>Deborde, S. T., 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=Schwann+cells+induce+cancer+cell+dispersion+and+invasion.">Schwann cells induce cancer cell dispersion and invasion.</a> J Clin Invest. 126 (4), 1538-1554 (2016).</li><li>Pour, P. M., Egami, H., Takiyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+growth+and+metastases+of+induced+pancreatic+cancer+in+relation+to+the+prognosis+and+its+clinical+implications.">Patterns of growth and metastases of induced pancreatic cancer in relation to the prognosis and its clinical implications.</a> Gastroenterology. 100 (2), 529-536 (1991).</li><li>Eibl, G., Reber, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+xenograft+nude+mouse+model+for+perineural+invasion+and+recurrence+in+pancreatic+cancer.">A xenograft nude mouse model for perineural invasion and recurrence in pancreatic cancer.</a> Pancreas. 31 (3), 258-262 (2005).</li><li>Stopczynski, R. 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=Neuroplastic+changes+occur+early+in+the+development+of+pancreatic+ductal+adenocarcinoma.">Neuroplastic changes occur early in the development of pancreatic ductal adenocarcinoma.</a> Cancer Res. 74 (6), 1718-1727 (2014).</li><li>Gil, Z., 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=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13 (21), 6479-6485 (2007).</li><li>Cardiff, R. D., Miller, C. H., Munn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+hematoxylin+and+eosin+staining+of+mouse+tissue+sections.">Manual hematoxylin and eosin staining of mouse tissue sections.</a> Cold Spring Harb Protoc. 2014 (6), 655-658 (2014).</li><li>Cremer, H., 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=Inactivation+of+the+N-CAM+gene+in+mice+results+in+size+reduction+of+the+olfactory+bulb+and+deficits+in+spatial+learning.">Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning.</a> Nature. 367 (6462), 455-459 (1994).</li><li>He, 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=The+chemokine+(CCL2-CCR2)+signaling+axis+mediates+perineural+invasion.">The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.</a> Mol Cancer Res. 13 (2), 380-390 (2015).</li><li>He, 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=GFR&#945;1+released+by+nerves+enhances+cancer+cell+perineural+invasion+through+GDNF-RET+signaling.">GFR&#945;1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling.</a> P Natl Acad Sci USA. , 02944 (2014).</li></ol>

I denne protokol beskriver vi en i vivo murine model af perineural invasion, der giver mulighed for kvantificering af iskiasnerven invasion af kræft i bugspytkirtlen celler. Denne model giver mulighed for undersøgelse af molekylære mekanismer af nerve invasion. Vellykkede eksperimenter ved hjælp af denne teknik kræver en forsigtig tilgang til tre kritiske trin i processen: 1) indsprøjtningen af kræftceller (trin 2.7, 2.8), 2) udvinding af invaderede nerver (trin 3,4), og 3) behandling af høstede nerver (...

Nerver danne en bestemt tumor mikromiljø, der stimulerer kræft vækst og migration1,2,3. Perineural invasion (PNI) er den proces, gennem hvilken kræft celler invadere i og omkring nerverne. Det kan betragtes som en unik rute af metastaser da kræft invasion strækker sig fra steder, hvor oprindelse langs nerverne. PNI er fundet i flere kræft typer herunder i bugspytkirtlen, prostata, hoved & hals, spyt, livmoderhalskræft, og tyktarms-og endetarmskræft med en incidens spænder fra 22% til 100%1,2. PNI er forbundet med smerte og korrelerer med dårlig prognose og værre overlevelse satser1,2.
Udvikling af modeller af perineural invasion er vigtigt at belyse de cellulære og molekylære mekanismer af denne proces, og at teste kandidat terapeutiske agenter for at formindske PNI. In vitro metoder til at studere samspillet mellem kræft og nerver omfatter kræftceller med nerve explants4, dorsalrods ganglier5,6,7, eller med specifikke celler fælles kultur fra nerve celler mikromiljø såsom Schwann7. In vivo tilgange, er imidlertid mere fysiologisk relevante, omfatter anvendelse af kræft musemodeller som kræft er blevet induceret eller transplanteret og har fordel af regnskab for hele nerve mikromiljø. I orthotopic modeller af pancreas eller prostata cancer, PNI har været rapporteret8,9,10 og forekomsten af PNI kan registreres, men på grund af den lille størrelse af nerverne i disse organer, er det svært at se den hele nerve og dermed at opgøre omfanget af PNI. Den model vi beskrive her er en i vivo model af PNI i hvilke kræft celler injiceres i iskiasnerven af mus gennem en enkel kirurgisk procedure11. Heterotopisk transplantation invaderer inden for nerve mod rygmarven. Længden af nerve invasionen fra webstedet af injektion på rygmarven kan måles, og omfanget af kræft i nerve. Vigtigere, kan invaderede nerven også indsamles for en bred vifte af assays herunder mikroskopisk, og molekylære analyser. En bred vifte af kræftceller kan afprøves, og værten mus, der er blevet genetisk modificeret eller behandlet med særlige forbindelser kan bruges som godt. Denne kraftfulde assay giver mulighed for kræftceller og vært mikromiljø ændres til undersøgelse af mekanismerne af PNI.

<table border="0" cellpadding="0" cellspacing="0" style="width:973px;" width="973">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Mouse</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;">Number and age variable depending on experimental needs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td>
			<td>Any</td>
			<td></td>
			<td>Steps 1.1, 1.2, 1.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tube, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Step 1.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge tube 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td>Step 1.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electric razor</td>
			<td>WAHL</td>
			<td>9962</td>
			<td>Step 2.1. Can be substituted with commercial hair removal agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, 250 mL</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td>Step 2.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hypoallergenic surgical tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td>Step 2.3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Betadine Swapsticks</td>
			<td>PDI</td>
			<td>SKU 41350</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile surgical tools (scissors and forceps)</td>
			<td></td>
			<td></td>
			<td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 &#956;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Steps 2.7, 2.8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Micro spatula</td>
			<td>Fisher Scientific</td>
			<td>S50823</td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacine, 1 g</td>
			<td>Enzo Life Sciences</td>
			<td>BML-NA139-0001</td>
			<td>Step 2.9. Reconstitute to 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td>Step 2.9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Step 4.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek Cryomold Molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Step 4.1</td>
		</tr>
	</tbody>
</table>

an in vivo murine sciatic nerve model of perineural invasion

Kræftceller invadere nerverne gennem en proces, der kaldes perineural invasion (PNI), i hvilke kræft celler formere sig og vandrer i nerve mikromiljø. Denne form for invasion er udstillet ved en række kræft typer og meget ofte er fundet i bugspytkirtelkræft. Den mikroskopiske størrelse af nervefibre i mus bugspytkirtlen gør studiet af PNI vanskeligt i orthotopic murine modeller. Her, beskriver vi en heterotopisk i vivo model af PNI, hvor vi indsprøjte syngeneic pancreas cancer cellelinie Panc02-H7 i murine iskiasnerven. I denne model, er ischiadicus nerver af bedøvede mus udsat og injiceres med kræftceller. Kræftcellerne invadere i nerver proksimalt mod rygmarven fra punktet, injektion. Invaderede ischiadicus nerver er derefter udvundet og forarbejdet med OCT for frosne skæring. H & E og immunofluorescens farvning af disse sektioner giver mulighed for kvantificering af både graden af invasion og ændringer i protein udtryk. Denne model kan anvendes til en lang række undersøgelser af PNI givet sin alsidighed. Ved hjælp af mus med forskellige genetiske modifikationer og/eller forskellige typer af kræftceller giver mulighed for undersøgelse af de cellulære og molekylære mekanismer af PNI og forskellige kræft typer. Virkningerne af terapeutiske agenter på nerve invasion kan endvidere studeres ved at anvende behandling til disse mus.

I denne metode beskrives kirurgisk implantation af kræft i bugspytkirtlen celler i murine iskiasnerven at oprette en i vivo model af kvantificerbare nerve invasion. Figur 1 illustrerer den anatomiske placering af iskiasnerven og injektionsstedet. Figur 2 viser to ischiadicus nerver af en nøgen mus. PBS injiceres nerve (til venstre) kan sammenlignes med en nerve sprøjtes med MiaPaCa-2 kræftceller (til højre). Nerven...

Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

En In Vivo Murine iskiasnerven Model af Perineural Invasion

Vi beskriver en i vivo murine model af perineural invasion ved at indsprøjte syngeneic bugspytkirtelkræftceller i iskiasnerven. Modellen giver mulighed for kvantificering af omfanget af nerve invasion, og understøtter undersøgelse af de cellulære og molekylære mekanismer af perineural invasion.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

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Cancer Research

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

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Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

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Rumlig og tidsmæssig kontrol af murine melanom initiering fra mutant melanocyte stamceller

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