Name: a murine orthotopic bladder tumor model and tumor detection system
Uploaded: 2017-01-12T15:00:00
Description: Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

This work was funded by a grant from the National Medical Research Council of Singapore (NMRC/CIRG/1335/2012) awarded to Professor Kesavan Esuvaranathan.

Alt dyr arbejde levet op til de institutionelle Animal Care og brug Udvalg (IACUC) retningslinjer for brug af dyr og håndtering (Protokol nummer 084/12) på National University of Singapore. 1. Voksende MB49-PSA celler in vitro og Måling PSA Sekretion <ol><li> Oprethold murine blære cancerceller MB49-PSA 15 i fuldstændig Dulbeccos modificerede Eagle medium (DMEM) suppleret med 10% føtalt bovint serum (FBS), 2 mmol / l L-glutamin og 0,05 mg / mL penicillin-...

<ol>
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De mest kritiske trin i protokollen er 1) med held opretholde tumorigeniciteten af ​​cellelinien; 2) at sikre målelig PSA sekretion før tumorcelleimplantation i mus; 3) generering af et enkelt-cellesuspension til implantation for at reducere variationen i tumorstørrelse; og 4) bibringe celler ved en langsom hastighed for at forhindre vesico-ureteral reflux, hvilket resulterer i tumorcelleimplantation i nyren. Efter forlænget passage in vitro, kan MB49 / MB49-PSA celler miste...

Målet med denne fremgangsmåde er at generere murine ortotopisk blæretumorer og overvåge de implanterede tumorer så præcist som muligt, således at mus uden tumorimplantation ikke menes at være blevet hærdet ved slutpunktsanalyse. Samlet set viste metode vil reducere behovet for et stort antal mus til eksperimentel analyse og sikre større nøjagtighed ved bestemmelse terapeutiske resultater. Udviklingen af ​​et ortotopisk model for kræft er vigtig, da implanterer tumorceller subkutant ikke rekapitulere miljøet af den kliniske sygdom eller muliggøre udviklingen af ​​terapeutiske strategier. Arkitekturen af ​​blæren tillader inddrypning af blæren cancerterapier direkte ind i blæren med minimale systemiske virkninger. Således dyremodeller der rekapitule- dette miljø, såsom en orthotopisk model, er vigtigt at evaluere nye behandlingsformer. Konklusionerne fra enhver forsøgsopstilling er dependent ved begrænsningerne af modellen. Adskillige teknikker er blevet udviklet til fremstilling af ortotopisk blæretumorer hos mus. Disse er afhængige af at beskadige glycosaminoglycan lag af blæren, så tumorceller, der skal implanteres. De anvendte teknikker omfatter elektrokirurgi, hvilket resulterer i et enkelt skader i blærevæggen, hvilket fører til tumorudvikling på samme sted i blæren 1,2. Men succesraten for tumorimplantation hjælp elektrokauterisation er operatørafhængigt mellem 10 - 90%, og det indeholder den fare, blærevæggen vil blive punkteret, hvilket fører til tumorer udvikler i bughulen. Kemisk cautery udføres ved hjælp af sølvnitrat, hvilke skader blærevæggen 3. Tilsvarende har syre blevet anvendt til at ødelægge blærevæggen 4. Trypsin er også blevet anvendt til at beskadige blæren samt 5. Disse fremgangsmåder kan resultere i udviklingen af ​​mere end én tumor i blæren.Desuden er der fare for ødelæggelse blæren hvis kemikalierne efterlades i kontakt med blærevæggen for længe. Metoden er udviklet af Ninalga et al. anvender den positivt ladede poly-L-lysin (PLL) 6 molekyler til pels blærevæggen; dette muliggør de negativt ladede tumorceller til at klæbe til glycosaminoglycan lag af blæren. Denne metode resulterer generelt i mere end én tumor udvikler sig i blæren, men tumorimplantation er ved 80 - 100% 4,7. Teknisk set er det også den nemmeste procedure at udføre. For at sikre, at de tumorer, der udvikler er ret selv i størrelse, er det vigtigt, at tumorcellerne ikke er grupperet i store klumper før implantation. For at evaluere terapeutisk effektivitet, er det bedst at udføre denne undersøgelse på mus med nogenlunde samme størrelse tumorer. Således er en god detektering system, der kan kvantificere tumorstørrelse hurtigt efter implantation er vigtig. Adskillige strategier har bin anvendes til at evaluere tumorer. Disse omfatter magnetisk resonans (MRI) 8-10, fluorescens 11, bioluminescens 12,13, ultralyd 14, og enzymkoblet immunosorbentassay (ELISA) 15,16. Mens MRI og ultralyd ikke kræver modifikationer af tumorcellerne, er der behov for følsomt udstyr og kontrastmidler til MRI. De fluorescence-, luminescence-, og ELISA-assays kræver ændring af tumorcellerne til at udtrykke markørproteiner, der kan opdages af disse metoder. For luminescens, er et substrat kræves til påvisning af luciferaseaktiviteten; der er således en ekstra skridt og øgede omkostninger. Både luminescens og fluorescens kræver specialiseret udstyr. At producere fluorescens, grønt fluorescerende protein (GFP) ringslutning, som katalyseres af molekylært oxygen, er påkrævet. Således kan GFP udtryk være variabel inden for en tumor masse afhængigt af adgang til ilt, hvilket gør dette en temmelig upålidelig markør <sup&gt; 17. Modifikation af den murine blærecancer-cellelinie MB49 til at udskille human prostataspecifikt antigen (PSA) 15,16 som en surrogatmarkør er en anden strategi. Disse markører giver også en alternativ måde at bekræftelse tumor tilstedeværelse ved afslutning af forsøget, hvilket gør dem et alternativ til immunhistokemi. Denne undersøgelse rapporterer PLL fremgangsmåde til ortotopisk tumorimplantation og præsenterer en sammenligning af tumor detektionssystemer, nemlig ELISA, fluorescens, og ultralydsbilleddannelse.

<table border="0" cellpadding="0" cellspacing="0" width="893">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:404px;">MB49-PSA cells</td>
			<td style="width:188px;">N/A</td>
			<td style="width:188px;">N/A</td>
			<td style="width:113px;">ref (Wu QH, 2004)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640 media</td>
			<td>HyClone</td>
			<td>SH30027.01&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) media</td>
			<td>Biowest</td>
			<td>L0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS) South American, Premium</td>
			<td>Biowest</td>
			<td>S181B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal Bovine Serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30088.03&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine</td>
			<td>Biowest</td>
			<td>X0550</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin-Streptomycin</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hygromycin B</td>
			<td>Invitrogen</td>
			<td>10687-010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">free PSA (Human) ELISA kit</td>
			<td>Abnova</td>
			<td>KA0209</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TRIzol Reagent for RNA extraction&#160;</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>4374967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Mouse Actb</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Mm00607939_s1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TaqMan Gene Expression Assay &#8211; Human KLK3</td>
			<td>Applied Biosystems</td>
			<td>4331182</td>
			<td>Hs00426859_g1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">C57BL/6J female mice</td>
			<td>In Vivos</td>
			<td></td>
			<td>4-6 wk old</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anesthesia (75mg/kg Ketamine and 1mg/kg Medetomidine)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reversal drug (1mg/kg Atipamezole)</td>
			<td>Local pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ear punch</td>
			<td>Electron Microscopy Sciences</td>
			<td>72893-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hartmann&#39;s solution or Compound sodium lactate</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ophthalmic ointment - Duratears sterile ocular lubricant ointment</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat pack - HotHands handwarmers</td>
			<td>Heatmax Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Introcan Certo IV catheter</td>
			<td>B Braun</td>
			<td>4251300</td>
			<td>24G x 3/4&#8243;</td>
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			<td>Local pharmacy</td>
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			<td height="17" style="height:17px;">&#160;Poly-L-lysine solution, 0.01%,</td>
			<td>Sigma</td>
			<td>P4707</td>
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			<td height="17" style="height:17px;">cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
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			<td height="17" style="height:17px;">Quantichrom Creatinine Assay Kit</td>
			<td>BioAssay Systems</td>
			<td>DICT-50&#160;</td>
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			<td height="17" style="height:17px;">Fluorescent dye - VivoTrack 680</td>
			<td>Perkin Elmer</td>
			<td>NEV12000&#160;</td>
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			<td height="17" style="height:17px;">RNAlater-ICE Frozen Tissue Transition Solution</td>
			<td>Ambion</td>
			<td>4427575</td>
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			<td height="17" style="height:17px;">Name</td>
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			<td>Catalog number</td>
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		<tr height="17">
			<td height="17" style="height:17px;">7500 Realtime PCR System</td>
			<td>Applied Biosystems</td>
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			<td height="17" style="height:17px;">7500 Software v2.3</td>
			<td>Applied Biosystems</td>
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			<td height="17" style="height:17px;">Metabolic Cage</td>
			<td>Tecniplast&#160;</td>
			<td>Vertical type rack for 12 cages</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSCanto I system&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BD FACSDiva software v7</td>
			<td>BD Biosciences</td>
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			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">IVIS SpectrumCT in vivo imaging system&#160;</td>
			<td>Caliper Life Sciences&#160;</td>
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			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Living Image Software v3.1</td>
			<td>Caliper Life Sciences&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vevo 2100 imaging system&#160;</td>
			<td>VisualSonics&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine orthotopic bladder tumor model and tumor detection system

Denne protokol beskriver genereringen af ​​blæretumorer i C57BL / 6J mus under anvendelse af murine blærekræft cellelinje MB49, der er blevet modificeret til at udskille human prostataspecifikt antigen (PSA), og proceduren til bekræftelse af tumorimplantation. Kort fortalt er musene bedøvet ved anvendelse injicerbare lægemidler og er lavet til at lægge i rygleje. Urin er forladt fra blæren og 50 pi af poly-L-lysin (PLL) langsomt indpodet med en hastighed på 10 gl / 20 s under anvendelse af en 24 G IV-kateter. Det overlades i blæren i 20 minutter ved tilstoppet kateteret. Kateteret fjernes, og PLL er forladt af let tryk på blæren. Dette efterfølges af inddrypning af den murine blærecancer-cellelinje (1 x 10 5 celler / 50 pi) med en hastighed på 10 pi / 20 s. Kateteret tilproppet at forhindre for tidlig evakuering. Efter 1 time musene genoplivet med en vending lægemiddel, og blæren er forladt. Den langsomme instillation sats er vigtig,da det reducerer vesico-ureteral reflux, hvilket kan forårsage tumorer at forekomme i de øvre urinveje og i nyrerne. Cellelinien skal være godt resuspenderes at reducere sammenklumpning af celler, da dette kan føre til ujævn tumorstørrelser efter implantation. Denne teknik inducerer tumorer med høj effektivitet. Tumorvækst monitoreres ved urin PSA sekretion. PSA markør overvågning er mere pålidelig end ultralyd eller fluorescens billeddannelse til påvisning af tilstedeværelsen af ​​tumorer i blæren. Tumorer i mus generelt nå en maksimal størrelse, der negativt påvirker sundhed af omkring 3 - 4 uger, hvis venstre ubehandlet. Ved at overvåge tumorvækst, er det muligt at differentiere mus, der blev hærdet fra dem, der blev ikke held implanteret tumorer. Med kun end-point analyse, kan sidstnævnte være fejlagtigt antages at være blevet helbredt ved terapi.

PSA-sekretion fra MB49-celler viste sig at variere med vækstmediet. MB49-PSA dyrkes i DMEM-medium, fordi dette resulterer i øget PSA sekretion (figur 1A). For at bestemme følsomheden af ​​PSA ELISA og real-time PCR, blev forskellige antal MB49-PSA-secernerende celler blandet med MB49 parentale celler. PSA ELISA registrerer et minimum på 1 x 10 5 PSA-secernerende celler / 1 x 10 6 celler (figur 1b), mens realtids-PCR analyse ...

Watch this Scientific Journal Video about A Murine Orthotopic Bladder Tumor Model and Tumor Detection System at JoVE.com

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

Denne protokol beskriver frembringelsen af ​​murine ortotopisk blæretumorer i C57BL / 6J-mus og overvågning af tumorvækst.

En Muse ortotopisk Blære Tumor Model og Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Research

JoVE Journal

Cancer Research

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