Name: modeling spontaneous metastatic renal cell carcinoma (mrcc) in mice following nephrectomy
Uploaded: 2014-04-29T15:00:00
Description: Watch this Scientific Journal Video about Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy at JoVE.com

Vi er taknemmelige for laboratoriet af Dr. Robert S. Kerbel (University of Toronto, Sunnybrook Research Institute, Toronto, Canada) for teknisk hjælp og ekspertise inden for udvikling af denne procedure. Vi vil også gerne takke Dr. Sandra Sexton og Roswell Park Cancer Institute Department of Laboratory Animal Resources. Dette arbejde blev støttet af en pris fra Roswell Park Alliance Foundation (til JMLE).

1. Ortotopisk Nyre Tumor Implantation <ol><li> Celledyrkning <ol><li> Før orthotopisk implantation vokse muse RENCA LUC-celler som et monolag til 75% konfluens. </li><li> Efter trypsinisering og resuspension i 5% FBS, der indeholder medier, centrifuger cellerne ved 1.000 rpm, 4 ° C i 5 minutter, gentagelse 3 gange for at vaske i PBS. Derefter resuspenderes cellerne i serumfrie medier til en koncentration på 5 x 10 4 RENCA LUC / 5 pi serum-frie medier. Bemærk: Afhængig a...

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Formålet med denne protokol er at vurdere klinisk relevant spontan metastatisk sygdom ved hjælp af en syngen tumor musemodel til at beskrive implantation / resektion teknikker. I øjeblikket har de fleste af prækliniske studier, som evaluerede nye eksperimentelle behandlingsformer omfatter ikke undersøgelse af metastatisk sygdom, og kun få rekapitulere stadier af primær tumor vækst, kirurgisk resektion, og eventuel spontan metastatisk spredning. Til dato gensplejsede musemodeller (GEMMs) genereret at fremkalde sp...

Den største årsag til dødelighed hos patienter med renalcellecarcinom (RCC) er systemisk metastatisk sygdom, der typisk forekommer efter kirurgisk fjernelse af en primær tumor vokser i nyrerne. Men meget få prækliniske tumormodeller evaluering eksperimentelle lægemidler i mus har metastatisk sygdom, og endnu færre trofast rekapitulere de kliniske stadier af lokaliseret vækst, kirurgi og spontan mikro-metastatisk initiering og progression 1-3. Dette hul i test er blevet stadig vigtigere i evalueringen af nye behandlingsformer, som undertiden slående anti-tumor effekt set i dyremodeller har ikke altid oversat til tilsvarende vellykket behandling af patienter på 4. Sådanne forskelle i resultaterne kan stamme fra differentierede efficacies narkotika mellem lokaliserede ektopiske eller ortotopisk primær tumor modeller og sen-fase metastatisk sygdom 5-7. I tilfælde af RCC, har kun få undersøgelser anvendte etableret enNimal protokoller, der omfatter spontane tilbagevendende sygdom, der efterligner patienter, der typisk har haft tumor-bærende nyrer helt eller delvist fjernet 2,3. Årsagerne til denne mangel i musemodel test varierer. Først er der den høje dyr omkostninger og iboende variabilitet af tumorcelle udvælgelse og metastatisk potentiale. For eksempel humane nyrecellelinier tendens til sjældent metastaserer, og skal udvælges over flere runder af orthotopisk primære implantation og metastatisk valg at udlede varianter, der konsekvent udbrede og danner fjerne læsioner (se beskrivelse af en sådan human cellelinie afledning 8-10) . Omvendt museceller i immunkompetente modeller tendens til at opføre sig aggressivt og lavt celleantal skal injiceres med Matrigel at reducere øjeblikkelig spredes systemisk 3. For det andet, de tekniske vanskeligheder med at udføre korrekt implantation kirurgisk resektion (nephrectomy), og sporing (og kvantificere) spontan metastatic vækst kan være udfordrende og flere kritiske variabler skal tages i betragtning ansætte denne teknik (se Diskussion for detaljer). Formålet med denne protokol er at beskrive de væsentlige skridt (samt potentielle faldgruber) af ortotopisk implantation, resektion (nefrektomi), og overvågning af spontan metastatisk RCC sygdom og tilbyde en guideline for standardiseret (og mere udbredt) bruger blandt videnskabelige laboratorier at vurdere effekten af ​​eksperimentelle lægemidler.

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			<td height="21" style="height:21px;width:397px;">DMEM-high glucose with Pyr. And L-Glutamine</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">FBS</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.25% Trypsin EDTA</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1x DPBS without Calcium &#38; Magnesium</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Matrigel</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td>must be kept on ice</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Artifical tears-lubricant opthalmic ointment</td>
			<td style="width:147px;">Akorn Animal Health</td>
			<td style="width:119px;">17478-162-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Pocket pro pet trimmer</td>
			<td style="width:147px;">Braintree scientific</td>
			<td style="width:119px;">CLP9931B</td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Alcohol swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">326895</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Betadine solution swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">67618-152-01</td>
			<td></td>
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			<td height="21" style="height:21px;">MICRO DISSECTING sissors straight,blunt - 25mm blades - 4.5&#34;&#160;</td>
			<td style="width:147px;">Southpointe surgical</td>
			<td style="width:119px;">RS-5982</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Iris Forceps, serrated, curved, 10cm long</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>need two of these</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">10&#181;l Hamilton syringe</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7635-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">30G, 45 degree, RN needle</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7803-07</td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Sterile cotton tipped appicator</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10805-144</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">High temperature cautery kit</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">5-0 coated Vicryl, conventional cutting needle&#160;</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:119px;">J834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clip applier for 7mm clips</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clips, 7mm, non-sterile</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Removing forceps, 12cm lone</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.9% Sodium Chloride</td>
			<td style="width:147px;">Baxter Healthcare</td>
			<td style="width:119px;">2B1322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Buprenorphine 0.01mg/mL</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">25G 5/8&#34; needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1mL syringe w/out needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">D-Luciferin</td>
			<td style="width:147px;">Gold Bio technology</td>
			<td style="width:119px;">LUCK-1G</td>
			<td></td>
		</tr>
	</tbody>
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modeling spontaneous metastatic renal cell carcinoma (mrcc) in mice following nephrectomy

En af de vigtigste udfordringer for forbedret afprøvning af nye eksperimentelle lægemidler i renalcellecarcinom (RCC) er udviklingen af ​​modeller, der trofast rekapitulere metastatisk sygdomsprogression tidlige og den sene fase. Typiske tumor implantation modeller udnytte ektopisk eller orthotopisk primær tumor implantation, men få indbefatter systemisk spontan metastatisk sygdom, der efterligner det kliniske miljø. Denne protokol beskriver de vigtigste skridt til at udvikle RCC sygdomsprogression stadier tilsvarende patienter. For det første anvender en meget metastatisk musetumor cellelinie i en syngen model til at vise ortotopisk tumorcelle implantation. Metoder omfatter overfladisk og intern implantation i sub-kapsulær rum med celler kombineret med matrigel at forhindre lækage og tidlig spredning. Derefter beskrives procedurerne for excision af tumor-bærende nyre (nefrektomi), med kritisk præ-og post-kirurgisk mus pleje. Endelig skitserer de nødvendige skridt til at overvåge og vurderemikro-og makro-metastatisk sygdomsprogression, herunder selvlysende billedbehandling samt giver en detaljeret visuel obduktion guide til at score systemisk sygdom distribution. Målet med denne protokol beskrivelse er at lette den udbredte brug af klinisk relevante metastatisk RCC modeller til at forbedre den prædiktive værdi af fremtidig terapeutisk test.

Figur 1A viser en skematisk skitserer de procedurer, der er beskrevet i denne protokol resumé. Flere vigtige faktorer skal overvejes for hvert trin. For eksempel er det i trin 1 viser to metoder til sub-kapsulær tumorcelle implantation i nyrerne. Tumorceller kan implanteres i sub-kapsulær rum med en lille hvid-bubble bekræfter lokaliseret placering af celler med lækage forhindres ved omhyggelig fjernelse af nålen og pensling overskydende væskeudslip (Figur 1B-i). For at forhindre...

Watch this Scientific Journal Video about Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy at JoVE.com

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Modeller af spontan metastatisk renalcellekarcinom (RCC) sygdomsprogression kan anvendes til at evaluere behandlinger i klinisk relevant indstilling. Denne protokol demonstrerer forskellige procedurer for ortotopisk nyre tumor celle implantation, ordentlig nefrektomi, og endelig skitserer en obduktion vejledning for visuel og selvlysende scoring af metastatisk byrde og lokalisering.

Modeling Spontan metastaserende renalcellecarcinom (MRCC) i mus efter nefrektomi

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

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Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

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