Name: an orthotopic bladder cancer model for gene delivery studies
Uploaded: 2013-12-01T22:00:00
Description: Watch this Scientific Journal Video about An Orthotopic Bladder Cancer Model for Gene Delivery Studies at JoVE.com

Detta arbete stöddes av NIH R21 CA143505 till Christina Voelkel-Johnson.

Alla som deltar i djurförsök har granskats och godkänts av Institutional Animal Care och användning kommittén vid medicinska universitetet i South Carolina. Protokollet godkändes enligt USDA kategori D för smärta. 1. Cell Implantation <ol><li> Två dagar innan du utför proceduren, plåt 1 x 10 6 MB49 celler till T-25 kolvar. Använd hög glukos DMEM kompletterat med 10% FBS (och antibiotika, om så önskas). En kolv är tillräcklig för varje grupp av upp till fem mös...

<ol>
	<li>Siegel, R., Naishadham, D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2012.">Cancer statistics, 2012.</a> CA Cancer. J. Clin. 62 (1), 10-29 (2012).</li><li>Seager, C. M., Puzio-Kuter, A. 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=Intravesical+delivery+of+rapamycin+suppresses+tumorigenesis+in+a+mouse+model+of+progressive+bladder+cancer.">Intravesical delivery of rapamycin suppresses tumorigenesis in a mouse model of progressive bladder cancer.</a> Cancer Prev. Res. 2 (12), 1008-1014 (2009).</li><li>Chodak, G. W., Shing, Y., Borge, M., Judge, S. M., Klagsbrun, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+heparin+binding+growth+factor+in+mouse+bladder+tumors+and+urine+from+mice+with+bladder+cancer.">Presence of heparin binding growth factor in mouse bladder tumors and urine from mice with bladder cancer.</a> Cancer Res. 46 (11), 5507-5510 (1986).</li><li>De Boer, E. C., Teppema, J. S., Steerenberg, P. A., De Jong, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrovirus+type+C+in+the+mouse+bladder+carcinoma+cell+line+MBT-2.">Retrovirus type C in the mouse bladder carcinoma cell line MBT-2.</a> J. Urol. 163 (6), 1999-2001 (2000).</li><li>Lodillinsky, C., Rodriguez, 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=Novel+invasive+orthotopic+bladder+cancer+model+with+high+cathepsin+B+activity+resembling+human+bladder+cancer.">Novel invasive orthotopic bladder cancer model with high cathepsin B activity resembling human bladder cancer.</a> J. Urol. 182 (2), 749-755 (2009).</li><li>Jurczok, A., Fornara, P., Soling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+to+monitor+bladder+cancer+cell+adhesion+in+vivo:+a+new+approach+to+optimize+a+syngeneic,+orthotopic,+murine+bladder+cancer+model.">Bioluminescence imaging to monitor bladder cancer cell adhesion in vivo: a new approach to optimize a syngeneic, orthotopic, murine bladder cancer model.</a> BJU Int. 101 (1), 120-124 (2008).</li><li>Brocks, C. P., Buttner, H., Bohle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+tumor+implantation+by+intravesical+gemcitabine+in+a+murine+model+of+superficial+bladder+cancer.">Inhibition of tumor implantation by intravesical gemcitabine in a murine model of superficial bladder cancer.</a> J. Urol. 174 (3), 1115-1118 (2005).</li><li>Wu, Q., Esuvaranathan, K., Mahendran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+response+of+orthotopic+bladder+tumors+to+granulocyte+macrophage+colony-stimulating+factor+therapy+using+the+prostate-specific+antigen+gene+as+a+reporter.+Clin.">Monitoring the response of orthotopic bladder tumors to granulocyte macrophage colony-stimulating factor therapy using the prostate-specific antigen gene as a reporter. Clin.</a> Cancer Res. 10 (20), 6977-6984 (2004).</li><li>Wu, Q., Mahendran, R., Esuvaranathan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+cytokine+gene+therapy+on+an+orthotopic+bladder+cancer+model.">Nonviral cytokine gene therapy on an orthotopic bladder cancer model.</a> Clin. Cancer Res. 9 (12), 4522-4528 (2003).</li><li>Bonfil, R. D., Russo, D. M., Binda, M. M., Delgado, F. M., Vincenti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+antitumor+activity+of+vinflunine+than+vinorelbine+against+an+orthotopic+murine+model+of+transitional+cell+carcinoma+of+the+bladder.">Higher antitumor activity of vinflunine than vinorelbine against an orthotopic murine model of transitional cell carcinoma of the bladder.</a> Urol. Oncol. 7 (4), 159-166 (2002).</li><li>Bohle, A., Jurczok, A., 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=Inhibition+of+bladder+carcinoma+cell+adhesion+by+oligopeptide+combinations+in+vitro+and+in.">Inhibition of bladder carcinoma cell adhesion by oligopeptide combinations in vitro and in.</a> 167 (1), 357-363 (2002).</li><li>Gunther, J. H., Jurczok, A., 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=Optimizing+syngeneic+orthotopic+murine+bladder+cancer+(MB49).">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59 (12), 2834-2837 (1999).</li><li>Gunther, J. H., Frambach, 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=Effects+of+acetylic+salicylic+acid+and+pentoxifylline+on+the+efficacy+of+intravesical+BCG+therapy+in+orthotopic+murine+bladder+cancer+(MB49).">Effects of acetylic salicylic acid and pentoxifylline on the efficacy of intravesical BCG therapy in orthotopic murine bladder cancer (MB49).</a> J. Urol. 161 (5), 1702-1706 (1999).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthotopic+model+of+murine+bladder+cancer.">An orthotopic model of murine bladder cancer.</a> J Vis Exp. (48), (2011).</li><li>Tham, S. M., Ng, K. H., Pook, S. H., Esuvaranathan, K., Mahendran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+and+microenvironment+modification+during+progression+of+murine+orthotopic+bladder+cancer.">Tumor and microenvironment modification during progression of murine orthotopic bladder cancer.</a> Clin. Dev. Immunol. 2011, 865684 (2011).</li><li>Seow, S. W., Cai, 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=Lactobacillus+rhamnosus+GG+induces+tumor+regression+in+mice+bearing+orthotopic+bladder+tumors.">Lactobacillus rhamnosus GG induces tumor regression in mice bearing orthotopic bladder tumors.</a> Cancer Sci. 101 (3), 751-758 (2009).</li><li>Mangsbo, S. M., Ninalga, C., Essand, M., Loskog, A., Totterman, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+therapy+is+superior+to+BCG+in+an+orthotopic+bladder+cancer+model+and+generates+CD4++T-cell+immunity.">CpG therapy is superior to BCG in an orthotopic bladder cancer model and generates CD4+ T-cell immunity.</a> J. Immunother. 31 (1), 34-42 (2008).</li><li>Loskog, A. S., Fransson, M. E., Totterman, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AdCD40L+gene+therapy+counteracts+T+regulatory+cells+and+cures+aggressive+tumors+in+an+orthotopic+bladder+cancer+model.">AdCD40L gene therapy counteracts T regulatory cells and cures aggressive tumors in an orthotopic bladder cancer model.</a> Clin. Cancer Res. 11 (24 Pt 1), 8816-8821 (2005).</li><li>Loskog, A., Ninalga, C., 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=Optimization+of+the+MB49+mouse+bladder+cancer+model+for+adenoviral+gene+therapy.">Optimization of the MB49 mouse bladder cancer model for adenoviral gene therapy.</a> Lab Anim. 39 (4), 384-393 (2005).</li><li>Bockholt, N. A., Knudson, M. 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=Anti-Interleukin-10R1+Monoclonal+Antibody+Enhances+Bacillus+Calmette-Guerin+Induced+T-Helper+Type+1+Immune+Responses+and+Antitumor+Immunity+in+a+Mouse+Orthotopic+Model+of+Bladder+Cancer.">Anti-Interleukin-10R1 Monoclonal Antibody Enhances Bacillus Calmette-Guerin Induced T-Helper Type 1 Immune Responses and Antitumor Immunity in a Mouse Orthotopic Model of Bladder Cancer.</a> J. Urol. 187 (6), 2228-2235 (2012).</li><li>Watanabe, F. T., Chade, D. C., 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=Curcumin,+but+not+Prima-1,+decreased+tumor+cell+proliferation+in+the+syngeneic+murine+orthotopic+bladder+tumor+model.">Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model.</a> Clinics. 66 (12), 2121-2124 (2011).</li><li>Chade, D. C., Andrade, P. 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=Histopathological+characterization+of+a+syngeneic+orthotopic+murine+bladder+cancer+model.">Histopathological characterization of a syngeneic orthotopic murine bladder cancer model.</a> Int. Braz. J. Urol. 34 (2), 220-226 (2008).</li><li>Luo, Y., Chen, X., O'Donnell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+prostate+specific+antigen+to+measure+bladder+tumor+growth+in+a+mouse+orthotopic+model.">Use of prostate specific antigen to measure bladder tumor growth in a mouse orthotopic model.</a> J. Urol. 172, 2414-2420 (2004).</li><li>Zhang, Z., Xu, X., 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+therapeutic+potential+of+SA-sCD40L+in+the+orthotopic+model+of+superficial+bladder+cancer.">The therapeutic potential of SA-sCD40L in the orthotopic model of superficial bladder cancer.</a> Acta Oncol. 50 (7), 1111-1118 (2011).</li><li>Kohno, S., Luo, C., 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=Herpes+simplex+virus+type+1+mutant+HF10+oncolytic+viral+therapy+for+bladder+cancer.">Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer.</a> Urology. 66 (5), 1116-1121 (2005).</li><li>Kikuchi, E., Menendez, 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=Highly+efficient+gene+delivery+for+bladder+cancers+by+intravesically+administered+replication-competent+retroviral+vectors.">Highly efficient gene delivery for bladder cancers by intravesically administered replication-competent retroviral vectors.</a> Clin. Cancer Res. 13 (15 Pt 1), 4511-4518 (2007).</li><li>Siemens, D. R., Austin, J. C., See, W. A., Tartaglia, J., Ratliff, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene+transfer+efficiency+by+viral+vectors+to+murine+bladder+epithelium.">Evaluation of gene transfer efficiency by viral vectors to murine bladder epithelium.</a> J. Urol. 165 (2), 667-671 (2001).</li><li>Siemens, D. R., Crist, S., Austin, J. C., Tartaglia, J., Ratliff, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+viral+vectors:+gene+transfer+efficiency+and+tissue+specificity+in+a+bladder+cancer+model.">Comparison of viral vectors: gene transfer efficiency and tissue specificity in a bladder cancer model.</a> J. Urol. 170 (3), 979-984 (2003).</li><li>Black, P. C., Shetty, A., 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=Validating+bladder+cancer+xenograft+bioluminescence+with+magnetic+resonance+imaging:+the+significance+of+hypoxia+and+necrosis.">Validating bladder cancer xenograft bioluminescence with magnetic resonance imaging: the significance of hypoxia and necrosis.</a> BJU Int. 106 (11), 1799-1804 (2010).</li></ol>

Den primära metod som beskrivs i detta protokoll är kateterisering av mus blåsor, som har breda applikationer för instillation av celler eller någon agent är avsedd för lokal leverans till epitelcellerna i urinblåsan. Den specifika protokoll som beskrivs ovan har optimerats för korttidsstudier (~ 10 dagar). Implantation det exakta antalet celler är kritisk, eftersom en högre cell nummer kommer att leda till en snabbare tumörtillväxt och eventuellt förlust av djur på grund av stor tumörbörda. Med hjälp ...

Urinblåsecancer är den näst vanligaste cancerformen i den urogenitala kanalen med nästan 75.000 nya fall och 15.000 dödsfall som förväntas under 2012 1. Hög frekvens av återfall kräver livslång uppföljning, vilket gör blåscancer en av de mest kostsamma cancer att behandla. Cancer i urinblåsan som har invaderat muskeln skiktet kan metastasera till lever, lunga eller ben via det lymfatiska systemet. Multimodal behandling av avancerade tumörer resulterar i endast 20-40% överlevnad efter 5 år. Därför finns ett stort behov effektiva behandlingsstrategier för att minska återfall och progression av ytlig blåscancer och förbättra terapeutiska resultat hos patienter med avancerad sjukdom. Utveckling av nya läkemedel krävs prekliniska modeller för att utvärdera effekten efter inledande in vitro-utvärdering. Tumormicroenvironmenten kan avsevärt påverka cancerutveckling och lyhördhet, vilket understryker behovet av preclinical modeller där tumörer uppstår eller kan fastställas i det organ i ursprung. Ett tillvägagångssätt är att utveckla transgena modeller i vilka tumörer uppstå spontant eller kan induceras i en organspecifikt sätt. En utmärkt protokoll av en transgen blåscancer modell har nyligen publicerats 2. Nackdelen med transgena modeller är att tumörer tenderar att utvecklas långsamt och med mindre enhetlighet än önskat. Dessutom har kostnaden för att upprätthålla en avelskoloni vägas. Ett alternativ till transgena modeller är orthotopic implantation av tumörceller, som har fördelen av korta tidsramar för tumör etablering i kommersiellt tillgängliga möss. Medan vissa humana cancer i urinblåsan cellinjer kan odlas orthotopically (vi har med framgång använt UM-UC-3), kan det vara önskvärt att upprätta tumörer i immunkompetenta möss. Två murina blåscancer cellinjer, som växer orthotopically är MBT-2 och MB49 3. Eftersom MBT-2-celler är förorenade med replikeratyp C retrovirus 4, har vi valt MB49 celler för våra studier. Det är viktigt att notera att MB49-celler isolerades från en manlig mus och orthotopic implantationer är endast för anatomiska skäl utförs i honmöss. Detta har fördelen av enkel identifiering av de implanterade cellerna genom markörer på Y-kromosomen, men obalansen kön kan vara en nackdel för immunologiska studier. Blåsan epitel kantas av en glykosaminoglykan (GAG)-skikt, som fungerar som en barriär för infektion av mikroorganismer. Denna barriär kan också störa implantation av tumörceller och ett flertal metoder har utvecklats för att övervinna denna svårighet (tabell 1). Diatermi har använts i stor utsträckning som ett fysiskt sätt att störa GAG lagret 5-13 och ett protokoll som visar diatermi har nyligen publicerats i JUPITER 14. Dock bör en diatermi enhet inte är tillgängliga, kemiska medel för att förstöra GAG skikt, såsom silvernitrat eller poly-L-lysin kan också användas 15 till 24. Tumörer som tillsatts genom en kort exponering av blåsan till en liten volym av silvernitrat (5 till 10 | il, 0,15 till 1,0 M, ~ 10 sek) eller längre kontakt med poly-L-lysin (100 pl av 0,1 mg / ml under 20 min) (tabell 1). Här beskriver vi en metod som använder trypsin för att underlätta implantation av MB49 celler. I ett försök att förbättra terapeutiska metoder för cancer i urinblåsan har genterapi rönt betydande uppmärksamhet. Ur klinisk synvinkel är blåscancer ett perfekt mål för genterapi på grund av lätt tillgänglighet av orgeln och förmågan att lokalt leverera nyttolasten. Virala vektorer som har utforskats för blåscancer genterapi inkluderar ett onkolytiskt herpes simplex virus 25, retrovirus 26, canarypoxvirus 27, vaccinia-virus, AAV, och adenovirus 28. I den andra delen av vår protokoll, beskriver vi en metod för viral leverans som är praktiskt taget identisk med instillation av tumörcellerna. Av intresse i vårt labb är att utveckla nya metoder för gen leverans, vilket vi bedömer via mareld med hjälp av en adenovirusvektor som uttrycker en luciferas transgen. Emellertid kan metoden i blåsan kateterisering användas för tillförsel av olika medel och har bred tillämpbarhet därför.

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="height:25px;">
			Name of reagent
			</td>
			<td style="height:25px;">
			Company
			</td>
			<td style="width:188px;height:25px;">
			Catalog number
			</td>
			<td style="width:193px;height:25px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="height:40px;">
			6-8 week old female mice
			</td>
			<td style="height:40px;">
			Jackson Laboratories
			</td>
			<td style="width:188px;height:40px;">
			Strain Name: C57BL/6J
			Stock Number: 000664
			</td>
			<td style="width:193px;height:40px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			Trypsin*
			</td>
			<td style="height:25px;">
			MediaTech
			</td>
			<td style="width:188px;height:25px;">
			MT25-053-CI
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			DMEM*
			</td>
			<td style="height:25px;">
			MediaTech
			</td>
			<td style="width:188px;height:25px;">
			MT10-017-CV
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			FBS
			</td>
			<td style="height:25px;">
			Hyclone
			</td>
			<td style="width:188px;height:25px;">
			SH30071.03
			</td>
			<td style="width:193px;height:25px;">
			Heat-inactivated
			</td>
		</tr>
		<tr>
			<td style="height:25px;">
			T25 flasks*
			</td>
			<td style="height:25px;">
			Corning Costar
			</td>
			<td style="width:188px;height:25px;">
			Corning No.:3056
			Fisher: 07-200-63
			</td>
			<td style="width:193px;height:25px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:42px;">
			MB49 cells
			</td>
			<td style="height:42px;">
			N/A
			</td>
			<td style="width:188px;height:42px;">
			N/A
			</td>
			<td style="width:193px;height:42px;">
			Obtained from Dr. Boehle (see reference11)
			</td>
		</tr>
		<tr>
			<td style="height:46px;">
			Puralube Vet Ointment*
			</td>
			<td style="height:46px;">
			Pharmaderm
			</td>
			<td style="width:188px;height:46px;">
			Henry Schein Company
			No.:036090-6050059
			Fisher: NC9676869
			</td>
			<td style="width:193px;height:46px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:53px;">
			Depilatory cream: Veet
			</td>
			<td style="height:53px;">
			local pharmacy
			</td>
			<td style="width:188px;height:53px;">
			
			</td>
			<td style="width:193px;height:53px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:53px;">
			Lubricant:
			K-Y Jelly
			</td>
			<td style="height:53px;">
			local pharmacy
			</td>
			<td style="width:188px;height:53px;">
			
			</td>
			<td style="width:193px;height:53px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:72px;">
			Catheters*
			</td>
			<td style="height:72px;">
			Exel International
			</td>
			<td style="width:188px;height:72px;">
			Exel International
			No.:26751;
			Fisher: 14-841-21
			</td>
			<td style="width:193px;height:72px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="height:45px;">
			Isoflurane
			</td>
			<td style="height:45px;">
			Terrell
			</td>
			<td style="width:188px;height:45px;">
			NDC 66794-011-25
			</td>
			<td style="width:193px;height:45px;">
			Obtained though hospital pharmacy
			</td>
		</tr>
		<tr>
			<td style="height:52px;">
			1 ml slip tip TB syringes
			</td>
			<td style="height:52px;">
			Becton Dickinson
			</td>
			<td style="width:188px;height:52px;">
			BD309659
			Fisher:14-823-434
			</td>
			<td style="width:193px;height:52px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:27px;">
			D-Luciferin
			</td>
			<td style="height:27px;">
			Gold Biotechnologies
			</td>
			<td style="width:188px;height:27px;">
			L-123-1
			</td>
			<td style="width:193px;height:27px;">
			
			</td>
		</tr>
		<tr>
			<td style="height:76px;">
			Ad-CMV-Luc
			</td>
			<td style="height:76px;">
			VectorBiolabs
			</td>
			<td style="width:188px;height:76px;">
			1000; Request large scale amplification and CsCl purification for in vivo use
			</td>
			<td style="width:193px;height:76px;">
			Infectious agent that requires BSL2 containment
			</td>
		</tr>
		<tr>
			<td style="height:76px;">
			Steady-Glo&#160;Luciferase Assay System
			</td>
			<td style="height:76px;">
			Promega
			</td>
			<td style="width:188px;height:76px;">
			E2510 (10 ml), E2520 (100 ml), or E2550 (10 x 100 ml)
			</td>
			<td style="width:193px;height:76px;">
			
			</td>
		</tr>
	</tbody>
</table>
*available through multiple vendors



EQUIPMENT
<table border="1" cellpadding="0" cellspacing="0">
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			<td style="width:97px;">
			Material name
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			<td style="width:114px;">
			Company
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			<td style="width:330px;">
			Catalog number
			</td>
			<td style="width:97px;">
			Comments
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			Anesthesia system
			</td>
			<td style="width:114px;">
			E-Z Systems, Euthanex Corporation
			</td>
			<td style="width:330px;">
			Anesthesia system: EZ7000
			5-port mouse rebreathing device: EZ109
			</td>
			<td style="width:97px;">
			Obtained through Fisher
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			Xenogen IVIS 200
			</td>
			<td style="width:114px;">
			Caliper Life Sciences
			</td>
			<td style="width:330px;">
			http://www.caliperls.com/products/preclinical-imaging/ivis-imaging-system-200-series.htm
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:97px;">
			FLUOstar Optima
			</td>
			<td style="width:114px;">
			BMG Labtech
			</td>
			<td style="width:330px;">
			http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2
			</td>
			<td style="width:97px;">
			
			</td>
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an orthotopic bladder cancer model for gene delivery studies

Urinblåsecancer är den näst vanligaste cancerformen i den urogenitala kanalen och nya behandlingsmetoder som kan minska återfall och progression behövs. Tumormicroenvironmenten kan avsevärt påverka tumörutveckling och terapisvar. Därför är det ofta önskvärt att odla tumörceller i det organ från vilka de har sitt ursprung. Detta protokoll beskriver en orthotopic modell av cancer i urinblåsan, där MB49 murina blåskarcinom celler ingjutit in i urinblåsan via kateter. Framgångsrik tumörcells implantation i denna modell kräver avbrott i det skyddande glykosaminoglykan skiktet, vilket kan åstadkommas genom fysikaliska eller kemiska medel. I våra protokoll blåsan behandlas med trypsin före cellinstillation. Kateterisering av blåsan kan också användas för att leverera terapeutiska medel när tumörerna är etablerade. Detta protokoll beskriver leverans av en adenoviral konstrukt som uttrycker en luciferas reportergen. Medan our protokoll har optimerats för korttidsstudier och fokuserar på genen leverans, har metodiken för musen blåsan kateterisering breda applikationer.

Hematuri har observerats i nästan alla möss inom 8 dagar efter implantation av 200.000 MB49 celler. Såsom visas i figur 1, blåsa i vikt mer än fördubblas från 34,7 ± 3,3 mg (intervall 31 till 37 mg, n = 4) i nontumor bärande möss till 87,5 ± 19,2 mg (intervall 77 till 120 mg, n = 10) i möss som har implanterats med MB49 celler. När det gäller genen leverans, fann vi att avbildning möss 24 timmar efter virusinstillation ger en starkare signal än efter 48 timmar (Figur 2)....

Watch this Scientific Journal Video about An Orthotopic Bladder Cancer Model for Gene Delivery Studies at JoVE.com

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Implantering av cancerceller in i ursprungsorgan kan fungera som en användbar preklinisk modell för att utvärdera nya terapier. MB49 blåsa cancerceller kan odlas i urinblåsan efter intravesikal instillation. Detta protokoll visar kateterisering av musen blåsan för att tumörimplantation och adenoviral leverans.

En Orthotopic blåscancer modell för Gene Delivery Studier

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

an-orthotopic-bladder-cancer-model-for-gene-delivery-studies

Research

JoVE Journal

Medicine

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

Ex vivo kultur Patient Tissue &amp; Undersökning av Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

En Orthotopic Modell av murina blåscancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-photon avbildning av tumörcellen invasion i ett Orthotopic musmodell av oral skivepitelcancer

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Kombination Strålbehandling i en ortotop mus hjärntumör modell

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

En ortotopisk blåstumör modell och utvärdering av Intravesikal Sarna behandling

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

En Orthotopic musmodell av Anaplastisk thyroidcarcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Bioluminescent Orthotopic Modell av bukspottkörtelcancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

En Orthotopic Murina Model of Human Prostate cancer metastaser

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimalinvasiva Etablering av murina Orthotopic Bladder implanterad

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.