Questo lavoro è stato sostenuto da NIH R21 CA143505 a Christina Voelkel-Johnson.

Tutte le procedure che coinvolgono gli animali sono stati esaminati e sono stati approvati dal Comitato Istituzionale cura degli animali ed uso presso la Medical University of South Carolina. Il protocollo è stato approvato in base USDA categoria D per il dolore. 1. Impianto cellulare <ol><li> Due giorni prima di eseguire la procedura, piatto 1 x 10 6 MB49 cellule in T-25 palloni. Utilizzare alta DMEM glucosio supplementato con 10% FBS (e antibiotici, se lo si desidera). Una beuta è sufficiente per ciascun gruppo di fino a cinque topi che verrà anestetizzato e impiantato in parallelo. </li><li> Il giorno della procedura, preparare tripsina per instillazione diluendo sterile 0,25% tessuto coltura grado tripsina allo 0,125% con terreno base DMEM sterile (diluizione 1:2). 1,2 ml è sufficiente per 10 topi. Mettere in bagnomaria a 37 ° C per equilibrare. </li><li> Preriscaldare una piastra elettrica e posizionarlo sotto le nosecones del sistema di anestesia che verranno utilizzati per fornire isoflurane. Posizionare un tampone assorbente pulito sopra il pad di riscaldamento. </li><li> Topi posto nella camera di induzione del sistema di anestesia e indurre anestesia con isoflurano 3%. Quando i topi hanno perso la coscienza, che è verificata la perdita del riflesso toe-pinch, trasferirli ai nosecones. Assicurarsi che ogni animale è in posizione supina e correttamente posizionato sulla piastra elettrica per mantenere la temperatura corporea. </li><li> Ridurre la concentrazione di isoflurano al 2% per mantenere l&#39;anestesia. </li><li> Applicare una pomata oftalmica per entrambi gli occhi per evitare l&#39;essiccazione. </li><li> Passaggio facoltativo: Eliminare i peli addominali. Parrucchieri sull&#39;addome inferiore deve essere rimossa se ​​verrà effettuata in vivo bioluminescente imaging. <ol><li> Applicare uno strato abbondante di crema depilatoria, come Veet, al più basso 1 in piazza della pelliccia sull&#39;addome, facendo attenzione a non contattare i genitali esterni. Attendere 3 min. </li><li> Rimuovere i capelli strofinando la zona con una spugna asciutta, e quindi utilizzare una spugna umida per pulire la pelle. Ripetere una volta, se necessario. </li></ol></li><li> Cateterizzare la vescica <ol><li> Lubrificare un nuovo, sterile 24 G catetere venoso pediatrica con un gel lubrificante non irritante come KY. Rimuovere e gettare l&#39;ago. Se fuoriuscita di fluido intorno al catetere è un problema comune, vale a dire. si verifica più frequentemente che in una delle dieci topi, cateteri diametro maggiore (20 g) possono essere utilizzati. Questo può essere una considerazione importante in topi di età superiore a 8 settimane. </li><li> Tenendo il fulcro del catetere, utilizzare il pollice e l&#39;indice della mano opposta a diffondere le zampe posteriori del mouse ed esporre il meato uretrale. Inserire delicatamente il catetere nell&#39;uretra con un angolo di 45 °, modificando l&#39;angolo di una parallela con il banco per inserirlo completamente. </li><li> Dopo inserimento completo, sollevare il catetere delicatamente, mantenendolo parallelo al banco, e confermare visivamente che viene inserito nell&#39;uretra e non la vagina, che è al di sotto di esso. Se posizionato correttamente, il catetere potrà essereserisce pienamente poiché la vagina è più corta della uretra. </li><li> Ridurre la concentrazione di isoflurano al 1%. </li></ol></li><li> Attaccare una punta di una pipetta P200 e rimuovere urina dalla vescica applicando aspirazione all&#39;estremità esterna del catetere. Rimuovere eventuali residui di urina dal mozzo del catetere. Eliminare l&#39;urina e lasciare catetere. </li><li> Pipetta con cautela 80 ml soluzione di tripsina 0,125% nel mozzo del catetere, evitando bolle d&#39;aria. Attaccare una siringa piena d&#39;aria 1 ml di raccordo del catetere e lentamente spingere il pistone 0,1-0,2 ml per consegnare la tripsina nella vescica. </li><li> Lasciare la siringa e gruppo catetere in posizione per 15 min. Ripetere i passaggi 1,8-1,11 per qualsiasi rimanenti topi anestetizzati (fino a 4). Procedere con il passaggio successivo durante il periodo di attesa. SUGGERIMENTO: Inizialmente può essere utile avere qualcuno a preparare le cellule (passo 1.12). </li><li> Preparare le cellule MB49 per l&#39;impianto. Le cellule sono state piastrate a 1 x 10 6 per T-25 pallone 48 ore prima dell&#39;impianto (punto 1.1). <ol><li> Rimuovere i supporti e aggiungere 500 ml di 0,25% tripsina al pallone. Quando le cellule si staccano aggiungere 5 ml di DMEM completo di riattivare le cellule. Rimuovere una aliquota di 50 microlitri di contare, e centrifugare il resto a 1.000 rpm per 5 min. Durante la fase di centrifugazione continuare a passi 1.12.2 e 1.12.3. </li><li> Contare le cellule utilizzando un emocitometro. Calcolare le cellule / ml. Quindi calcolare il volume necessario per portare il pellet cellulare a 4 x 10 6 cellule / ml (2 x 10 5 cellule per 50 microlitri). </li><li> Versare il surnatante dalle cellule centrifugate e risospendere il pellet in terreno di base DMEM con il volume richiesto di sospendere le cellule a 4 x 10 6 cellule / ml (come calcolato al punto 1.12.2). Mantenere le cellule a temperatura ambiente. </li></ol></li><li> Quando viene raggiunto il punto di tempo di 15 min per trattamento con tripsina di vesciche, staccare la siringa piena d&#39;aria dal catetere lasciando il catetere in posizione. Tripsina speso sarà normalely rifluire attraverso il catetere. Rimuovere eventuali tripsina rimanente dalla vescica mediante aspirazione con la pipetta P200 come al punto 1.9 e scartare. </li><li> Pipettare immediatamente 50 ml di sospensione cellulare MB49 nel mozzo del catetere. Attaccare la siringa piena d&#39;aria 1 ml al mozzo del catetere e fornire le cellule alla vescica lentamente lo stantuffo 0,1-0,2 ml. Ripetere questa operazione fino a quattro volte supplementari per impiantare tutti e cinque i topi. Eliminare le celle inutilizzate. </li><li> Lasciando il gruppo catetere-siringa in loco, permettono alle cellule di abitare nella vescica per 50 min. </li><li> Prelevare delicatamente il gruppo catetere-siringa dall&#39;uretra. Girare la concentrazione di isoflurano a zero e spostare ogni mouse di un centimetro o due di distanza dal musetto di recuperare sul pad di riscaldamento. </li><li> Quando un topo ha riacquistato il suo riflesso di raddrizzamento, tornare alla sua gabbia. </li></ol> 2. Consegna intravescicale di Adenovirus (8 giorni dopo l&#39;impianto di cellule) </p&gt; ATTENZIONE: Questa parte del protocollo utilizza adenovirus, che è un agente infettivo e deve essere maneggiato secondo le linee guida BSL2 severe (http://oma.od.nih.gov/manualchapters/intramural/3035/). Tutte le procedure eseguite con agenti infettivi sono stati approvati dal Comitato di Biosicurezza istituzionale della Medical University of South Carolina. <ol><li> Otto giorni dopo l&#39;impianto di topi con cellule di MB49, verificare la presenza di ematuria. Pick up singoli animali su un pezzo di carta assorbente e premere delicatamente sul ventre per cancellare gocce di urina sulla carta. Urina che è rosa al rosso indica ematuria ed è un segno che i tumori hanno stabilito. Tasso di tumore è almeno 80% e con eccellente tecnica può essere fino al 100%. </li><li> Scongelare adenoviral azione sul ghiaccio e diluire a 10 9 PFU/50 microlitri (2 x 10 10 PFU / ml) in sterile, a temperatura ambiente PBS. </li><li> Anestetizzare topi come descritto ai punti 1.3-1.6 sopra. </li><li> Cateterizzarla topi come descritto ai punti 1.8 e rimuovere urina come al punto 1.9 </li><li> Dopo urina viene rimosso, pipettare immediatamente 50 ml di sospensione di virus nel mozzo del catetere. Attaccare la siringa piena d&#39;aria 1 ml al mozzo del catetere e consegnare il virus alla vescica lentamente lo stantuffo 0,1-0,2 ml. </li><li> Lasciando il gruppo catetere-siringa in loco, permettono al virus di abitare nella vescica per 40 min. </li><li> Prelevare delicatamente il gruppo catetere-siringa dall&#39;uretra, e gettare nella soluzione di candeggina al 10% per inattivare i virus rimanente. </li><li> Girare la concentrazione di isoflurano a zero e spostare ogni mouse di un centimetro o due di distanza dal musetto di recuperare sul pad di riscaldamento. </li><li> Quando un topo ha riacquistato il suo riflesso di raddrizzamento, tornare alla sua gabbia. </li><li> Segna gabbie per indicare che i topi sono stati instillato con adenovirus. Virus sarà versato nel letto dei gabbia per diversi giorni e tutte le gabbie e biancheria da letto deve esseremanipolati e disinfettati adeguatamente. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

La metodologia primario descritto in questo protocollo è cateterizzazione di vesciche topo, che ha applicazioni di massima per instillazione di cellule o di qualsiasi agente destinati per il recapito locale all&#39;epitelio vescicale. Il protocollo specifica di cui sopra è stato ottimizzato per studi a breve termine (~ 10 giorni). Impiantare il numero esatto delle cellule è fondamentale, dal momento che un elevato numero di cellule si tradurrà in più rapida crescita del tumore e possibile perdita di animali a causa...

Il cancro della vescica è il secondo tumore più comune del tratto urogenitale con circa 75.000 nuovi casi e 15.000 decessi attesi nel 2012 1. Alti tassi di recidiva richiedono follow-up permanente, che rende il cancro della vescica uno dei tumori più costoso da trattare. Cancro della vescica che ha invaso lo strato muscolare può metastatizzare fegato, polmone o osso attraverso il sistema linfatico. La terapia multimodale dei tumori avanzati risultati solo nel 20-40% di sopravvivenza dopo 5 anni. Pertanto, sono urgentemente necessarie strategie di trattamento efficaci volti a ridurre la recidiva e la progressione del tumore superficiale della vescica, nonché a migliorare il risultato terapeutico nei pazienti con malattia avanzata. Sviluppo di nuove terapie richiede modelli preclinici per valutare l&#39;efficacia seguente valutazione iniziale in vitro. Il microambiente tumorale può influenzare significativamente lo sviluppo del cancro e la risposta, che evidenzia la necessità di preclinicoAL modelli in cui i tumori si presentano o possono essere stabiliti nell&#39;organo di origine. Un approccio è lo sviluppo di modelli transgenici in cui i tumori sorgono spontaneamente o possono essere indotte in maniera organo-specifica. Un protocollo ottimo di un modello di cancro della vescica transgenico è stato recentemente pubblicato 2. Lo svantaggio di modelli transgenici è che i tumori tendono a svilupparsi lentamente e con meno uniformità di quanto desiderato. Inoltre, il costo di mantenimento di una colonia di riproduzione deve essere considerato. Un&#39;alternativa ai modelli transgenici è impianto ortotopico di cellule tumorali, che ha il vantaggio di tempi brevi per lo stabilimento del tumore nei topi disponibili in commercio. Mentre alcune linee cellulari di cancro della vescica umane possono essere coltivate ortotopicamente (abbiamo utilizzato con successo UM-UC-3), può essere auspicabile stabilire tumori in topi immunocompetenti. Due linee di cellule di cancro alla vescica murini, che crescono ortotopicamente sono MBT-2 e MB49 3. Poiché MBT-2 celle sono contaminati da replicaretipo C retrovirus 4, abbiamo scelto MB49 celle per i nostri studi. È importante notare che MB49 cellule sono state isolate da un topo maschio e implantologia ortotopiche sono per ragioni anatomiche eseguite nei topi femmina. Questo ha il vantaggio di una facile identificazione delle cellule impiantate mediante marcatori del cromosoma Y, ma il disadattamento genere può essere uno svantaggio per studi immunologici. L&#39;epitelio vescicale è fiancheggiato da un glicosaminoglicani (GAG) strato, che funziona come una barriera per l&#39;infezione da microrganismi. Questa barriera può anche interferire con impianto di cellule tumorali e diversi metodi sono stati sviluppati per superare questa difficoltà (Tabella 1). Elettrocauterizzazione è stato ampiamente utilizzato come un mezzo fisico di perturbare lo strato di GAG 5-13 e dimostrando elettrobisturi protocollo è stato recentemente pubblicato in JoVE 14. Tuttavia, se una unità elettrocauterizzazione non essere disponibile, mezzi chimici per distruggere il GAG strato come il nitrato di argento o poli-L-lisina può essere utilizzato anche 15-24. I tumori sono stabiliti efficacemente da una breve esposizione della vescica di un piccolo volume di nitrato d&#39;argento (5-10 microlitri, 0,15-1,0 M, ~ 10 sec) o contatto prolungato con poli-L-lisina (100 pl di 0,1 mg / ml per 20 min) (Tabella 1). Qui si descrive un metodo che utilizza tripsina per facilitare l&#39;impianto di MB49 cellule. Nel tentativo di migliorare gli approcci terapeutici per il cancro della vescica, la terapia genica ha raccolto notevole attenzione. Da un punto di vista clinico, cancro della vescica è un bersaglio ideale per la terapia genica a causa di facile accessibilità dell&#39;organo e la capacità di fornire localmente il carico utile. Vettori virali che sono stati esplorati per la terapia genica del cancro della vescica includono una oncolytic virus herpes simplex 25, 26 retrovirus, virus del canarino 27, virus vaccinico, AAV, e adenovirus 28. Nella seconda parte del nostro protocollo, si descrive un metodo per la consegna virale che è praticamente identico a instillazione delle cellule tumorali. Di interesse nel nostro laboratorio è lo sviluppo di nuovi approcci alla consegna del gene, che valutiamo tramite bioluminescenza utilizzando un vettore adenovirale che esprime un transgene luciferasi. Tuttavia, la metodologia di cateterismo vescicale può essere utilizzato per la consegna di vari agenti e quindi ha vasta applicabilità.

<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

Il cancro della vescica è il secondo tumore più comune del tratto urogenitale e nuovi approcci terapeutici che possono ridurre la recidiva e la progressione sono necessari. Il microambiente tumorale può influenzare significativamente lo sviluppo tumorale e risposta alla terapia. È pertanto spesso desiderabile crescere le cellule tumorali nell&#39;organo da cui sono derivati. Questo protocollo descrive un modello ortotopico di cancro della vescica, in cui murine cellule di carcinoma della vescica MB49 sono instillato nella vescica tramite cateterizzazione. Impianto di cellule tumorali successo in questo modello richiede interruzioni dello strato protettivo glicosaminoglicani, che può essere effettuata con mezzi fisici o chimici. Nel nostro protocollo vescica viene trattata con tripsina prima dell&#39;instillazione cella. Cateterizzazione della vescica può anche essere usati per fornire terapie una volta che i tumori sono stabiliti. Questo protocollo descrive la consegna di un costrutto adenovirale che esprime un gene reporter luciferasi. Mentre oprotocollo ur è stato ottimizzato per studi a breve termine e si concentra sulla consegna del gene, la metodologia di topo cateterismo vescicale ha vaste applicazioni.

Ematuria è osservato in quasi tutti i topi entro 8 giorni dopo l&#39;impianto di 200.000 MB49 cellule. Come mostrato in Figura 1, peso vescica è più del doppio di 34,7 ± 3,3 mg (range 31-37 mg, n = 4) in topi non tumorali cuscinetto a 87,5 ± 19,2 mg (range 77-120 mg, n = 10) in topi che sono stati impiantati con MB49 cellule. In termini di consegna del gene, abbiamo trovato che l&#39;imaging topi 24 ore dopo l&#39;instillazione virale produce un segnale più forte dopo 48 ore (Figura 2).</s...

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

Impianto di cellule tumorali nell&#39;organo di origine può servire come un modello preclinico utile per valutare nuove terapie. Cellule di carcinoma della vescica MB49 possono essere coltivate all&#39;interno della vescica seguente instillazione intravescicale. Questo protocollo dimostra cateterizzazione della vescica topo a scopo di impianto tumorale e consegna adenovirale.

Un ortotopico cancro della vescica Modello di Gene Studi di consegna

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

12.3K Views.  Medical University of South Carolina.  Impianto di cellule tumorali nell'organo di origine può servire come un modello preclinico utile per valutare nuove terapie. Cellule di carcinoma della vescica MB49 possono essere coltivate all'interno della vescica seguente instillazione intravescicale. Questo protocollo dimostra cateterizzazione della vescica topo a scopo di impianto tumorale e consegna adenovirale. 

Video: An Orthotopic Bladder Cancer Model for Gene Delivery Studies

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 del tessuto del paziente e l&#39;esame di consegna 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 ...

Ricerca

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.

Un modello ortotopico di cancro alla vescica Murine

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-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

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.

La radioterapia combinata in un modello di tumore ortotopico cervello di topo

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.

Un modello ortotopico tumore della vescica e la valutazione del trattamento intravescicale sarna

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.

Un modello di mouse ortotopico di anaplastico della tiroide Carcinoma

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.

Bioluminescente modello ortotopico di cancro al pancreas Progressione

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.

Un ortotopico Modello murino di Human Prostate Cancer Metastasi

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.

Mini-Invasiva Istituzione di murini ortotopico vescica Xenotrapianti

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.