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

Este trabalho foi financiado pelo NIH R21 CA143505 para Christina Voelkel-Johnson.

Todos os procedimentos envolvendo animais foram revisadas e foram aprovados pelo Comitê Institucional Animal Care e Use na Medical University of South Carolina. O protocolo foi aprovado sob USDA categoria D para a dor. 1. Implantação celular <ol><li> Dois dias antes de realizar o procedimento, placa 1 x 10 6 MB49 células em frascos T-25. Use alta DMEM glucose suplementado com 10% FBS (e antibióticos, se desejar). Um frasco é suficiente para cada grupo de até cinco ratinho...

<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>

A metodologia principal descrito neste protocolo é o cateterismo de bexigas de rato, que tem amplas aplicações para a instilação de células ou qualquer agente destinado para entrega local para o epitélio da bexiga. O protocolo específico descrito acima foi otimizado para estudos de curto prazo (~ 10 dias). Implantar o número exato de células é fundamental, uma vez que um número de células maior resultará em crescimento do tumor e, possivelmente, mais rápida perda de animais devido a grande carga tumoral. ...

O câncer de bexiga é o segundo tipo de câncer mais comum do trato urogenital, com cerca de 75.000 novos casos e 15.000 mortes esperadas em 2012 1. As altas taxas de recorrência ao longo da vida requerem acompanhamento, o que faz com que o câncer de bexiga um dos cancros mais caras para tratar. Cancro da bexiga que invadiu a camada muscular podem metastizar para o fígado, o pulmão ou o osso através do sistema linfático. Terapia Multimodal de tumores avançados resultados em apenas 20-40% de sobrevida após 5 anos. Portanto, as estratégias de tratamento eficazes destinadas a reduzir a recorrência ea progressão do câncer superficial da bexiga, bem como melhorar os resultados terapêuticos em pacientes com doença avançada são urgentemente necessários. Desenvolvimento de novas terapias requer modelos pré-clínicos para avaliar a eficácia após a avaliação inicial in vitro. O microambiente do tumor pode influenciar significativamente o desenvolvimento do câncer e capacidade de resposta, o que evidencia a necessidade de pré-clínicosal modelos em que os tumores surgem ou pode ser estabelecido no órgão de origem. Uma das abordagens é o desenvolvimento de modelos transgénicos em que os tumores surgem espontaneamente ou pode ser induzida de uma forma específica do órgão. Um excelente protocolo de um modelo de câncer de bexiga transgênico foi recentemente publicado 2. A desvantagem de modelos transgénicos é que os tumores tendem a desenvolver-se lentamente e com menos uniformidade do que o desejado. Além disso, o custo de manutenção de uma colónia de reprodução tem de ser considerado. Uma alternativa aos modelos transgénicos é o implante ortotópico de células tumorais, o que tem a vantagem de prazos curtos para o estabelecimento do tumor em ratinhos disponíveis comercialmente. Enquanto algumas linhas celulares de cancro da bexiga humano pode ser cultivado ortotopicamente (temos utilizado com sucesso UM-UC-3), pode ser desejável estabelecer tumores em ratinhos imunocompetentes. Duas linhas celulares de cancro da bexiga murino, que crescem ortotopicamente são MBT-2 e MB49 3. Desde MBT-2 células estão contaminados com replicandotipo C retrovírus 4, escolhemos MB49 células para os nossos estudos. É importante notar que a MB49 células foram isoladas a partir de um rato macho e implantes ortotópicos são por razões anatómicas realizados em ratos fêmeas. Este tem a vantagem de facilitar a identificação das células implantadas por meio de marcadores do cromossomo Y, mas a incompatibilidade de gênero pode ser um inconveniente para os estudos imunológicos. O epitélio da bexiga é revestida por um (GAG) da camada de glicosaminoglicano, o qual funciona como uma barreira para a infecção por micro-organismos. Esta barreira também pode interferir com a implantação de células tumorais e de vários métodos têm sido desenvolvidos para superar esta dificuldade (Tabela 1). Eletrocautério tem sido amplamente utilizado como um meio físico para romper a camada de GAG 5-13 e um eletrocautério demonstrando protocolo foi recentemente publicado na Jove 14. No entanto, se uma unidade de electrocautério não estar disponível, meios químicos para destruir o GAG camada tais como nitrato de prata, ou poli-L-lisina também pode ser utilizada 15-24. Os tumores são estabelecidos de forma eficaz através de uma breve exposição da bexiga a um pequeno volume de nitrato de prata (5-10 ul, 0,15-1,0 M, ~ 10 seg) ou mais, o contacto com poli-L-lisina (100 ul de 0,1 mg / ml durante 20 min) (Tabela 1). Aqui, descrevemos um método que utiliza a tripsina para facilitar a implantação de células MB49. Em uma tentativa de melhorar as abordagens terapêuticas para o câncer de bexiga, a terapia gênica tem atraído atenção significativa. De um ponto de vista clínico, o cancro da bexiga é um alvo ideal para terapia génica devido ao fácil acesso do órgão e a capacidade para distribuir localmente a carga útil. Os vetores virais que foram exploradas para a terapia genética câncer de bexiga incluem um vírus herpes simplex oncolítico 25, 26 retrovírus, vírus de canários 27, o vírus vaccinia, AAV, e adenovirus 28. Na segunda parte do nosso protocolo, que descrevem um método para a entrega viral que é virtualmente idêntico ao da instilação das células tumorais. De interesse no nosso laboratório é o desenvolvimento de novas abordagens para a entrega do gene, que avaliam através bioluminescência, utilizando um vector adenoviral que expressa um transgene da luciferase. No entanto, a metodologia de cateterização da bexiga pode ser utilizada para a entrega dos vários agentes e, portanto, tem ampla aplicabilidade.

<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">
	<tbody>
		<tr>
			<td style="width:97px;">
			Material name
			</td>
			<td style="width:114px;">
			Company
			</td>
			<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>
		</tr>
	</tbody>
</table>

an orthotopic bladder cancer model for gene delivery studies

O câncer de bexiga é o segundo tipo de câncer mais comum do trato urogenital e novas abordagens terapêuticas que podem reduzir a recorrência ea progressão são necessários. O microambiente do tumor pode influenciar significativamente o desenvolvimento do tumor e a resposta terapia. Por conseguinte, é muitas vezes desejável para crescer as células de tumor no órgão a partir do qual se originou. Este protocolo descreve um modelo ortotópico de cancro de bexiga, em que as células de carcinoma de bexiga de murino MB49 são instilada na bexiga através do cateterismo. Implantação de células de tumor bem sucedida, neste modelo requer a interrupção da camada de glicosaminoglicano de protecção, que pode ser realizada por meios físicos ou químicos. No nosso protocolo da bexiga é tratada com tripsina antes da instilação de células. Cateterização da bexiga podem também ser usados ​​para entregar agentes terapêuticos uma vez que os tumores estão estabelecidos. Este protocolo descreve o fornecimento de uma construção adenoviral que expressa um gene repórter da luciferase. Enquanto our protocolo foi otimizado para estudos de curto prazo e se concentra na entrega gene, a metodologia de rato cateterismo vesical tem amplas aplicações.

Hematúria é observado em quase todos os ratos num período de 8 dias após a implantação de 200.000 células MB49. Como mostrado na Figura 1, o peso da bexiga mais do que duplica de 34,7 ± 3,3 mg (gama 31-37 mg, n = 4) não tumorais em ratinhos portadores de 87,5 ± 19,2 mg (gama de 77-120 mg, n = 10) em ratinhos que foram implantados com células MB49. Em termos de entrega de genes, descobrimos que imagiologia ratinhos 24 horas após a instilação viral produz um sinal mais forte do que depois de...

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

Implantação de células cancerosas para o órgão de origem pode servir como um modelo pré-clínico útil para avaliar novas terapias. Células de carcinoma da bexiga MB49 pode ser cultivada no interior da bexiga, após a instilação intravesical. Este protocolo demonstra a cateterização da bexiga do rato com a finalidade de implantação do tumor e entrega adenoviral.

Um modelo de cancro da bexiga para Ortotópico Gene Estudos de entrega

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 Cultura Vivo de tecido do paciente e Exame de Entrega Gene

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 ...

Medicina

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.

Um modelo ortotópico de câncer de bexiga murino

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-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

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.

Radioterapia em uma combinação ortotópico modelo de tumor de cérebro de camundongos

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.

Um modelo de tumor de bexiga ortotópico e Avaliação de Tratamento de sarna intravesical

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.

Um modelo de camundongo ortotópico de Carcinoma anaplásico da tiróide

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.

Modelo ortotópico bioluminescente de progressão do câncer de pâncreas

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.

Um Modelo murino de próstata humana Metástase Ortotópico

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.

Minimamente Invasiva Estabelecimento de murino ortotópico bexiga Xenoenxertos

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.