We thank the Western University of Health Sciences for the Intramural Grant.

Il protocollo descritto di seguito viene eseguito sotto la guida e l&#39;approvazione del Comitato di cura e l&#39;uso di animali di Western University. Tutti gli esperimenti sono eseguiti in conformità con tutte le linee guida, dei regolamenti e agenzie di regolamentazione. Cultura 1. cellulare <ol><li> Preparazione del terreno completo <ol><li> Utilizzando una cappa di sicurezza biologica Classe II, preparare terreno completo aggiungendo asetticamente siero fetale bovino (FBS) e la penicillina streptomicina (P / S) ad una bottiglia da 500 ml di terreno RPMI. Mescolare delicatamente agitando. La concentrazione finale di ogni supplemento è del 10% FBS e 1% P / S (v / v). Ad esempio, integrare 500 ml di media con 56 ml di FBS e 6 ml P / S. </li><li> Posizionare terreno completo in un bagno d&#39;acqua a 37 ° C. </li></ol></li><li> Scongelamento e cellule di moltiplicazione <ol><li> Recupera le cellule PANC-1 GFP da azoto liquido. Scongelare il flacone delicatamente l&#39;agitazione in un bagno d&#39;acqua a 37 ° C. Nota:Scongelamento dovrebbe essere rapida (circa 2 - 3 min). </li><li> Label T-75 palloni da utilizzare con (a) nome di cella-line, la data (b) il numero di passaggio, (c), e le iniziali (d), del ricercatore. </li><li> Pulire la fiala con il 70% di etanolo e il trasferimento fiala di un armadio biosicurezza. </li><li> Per propagare le cellule, aggiungere 9,0 ml di mezzo completo in un tubo da 15 ml. Usando una pipetta 1,0 ml, trasferire accuratamente la sospensione cellulare e sospenderlo in 9 ml di mezzo completo. </li><li> Centrifugare a 125 xg per 8 minuti a temperatura ambiente. Trasferire la fiala conica di un armadio biosicurezza e aspirare il surnatante senza disturbare il pellet. </li><li> Sospendere il pellet in 10 ml di mezzo fresco completa pipettando delicatamente la sospensione su e giù. </li><li> Contare le cellule con un emocitometro e piastra in T-75 flaconi ad una densità di 2,1 x 10 6 cellule / fiasco. Inserire pallone in un incubatore a 37 ° C e 5% CO 2. </li><li> Monitorare le cellule giornaliera a occhio e al microscopio. Se l&#39;influenzaid è necessario il rinnovo, in modo asettico aspirare il mezzo di crescita completo dal pallone e scartare. Aggiungere uguale volume di mezzo fresco completa. </li></ol></li><li> propagazione Cells <ol><li> Asetticamente rimuovere media dal pallone. Aggiungere 5 ml di tampone fosfato salina Dulbecco (DPBS) per lavare le cellule e scartare. </li><li> Staccare le cellule dal pallone con l&#39;aggiunta di 3 ml di 0,25% tripsina. Incubare pallone contenente tripsina a 37 ° C e 5% CO 2 per 5 a 10 min. </li><li> Osservare le cellule al microscopio (ingrandimento 10x) e assicurarsi che sono rotondi e sloggiato. </li><li> Neutralizzare tripsina aggiungendo un volume uguale di mezzo completo e rompere i grumi delicatamente pipettando su e giù. </li><li> Contare le cellule utilizzando un emocitometro e trasferire il volume di sospensione cellulare opportuno nuovi palloni alla densità cellulare di cui al 1.2.7; aggiungere abbastanza mezzi freschi in modo che il volume finale è di 10 ml per pallone. </li></ol></li><li> Suspens cellulariione Preparazione per l&#39;iniezione Nota: Dal momento che la matrice di membrana basale, ad alta concentrazione, solidifica a temperatura ambiente, conservare i materiali sul ghiaccio. Mettere tutti puntali sterili e fiale nel congelatore durante la notte, e tenere in ghiaccio mentre si lavora sulla sospensione. <ol><li> Tenere una bottiglia di RPMI mezzi senza additivi su ghiaccio. Scongelare la matrice di membrana basale, ad alta concentrazione, seguendo le istruzioni del produttore. Preferibilmente, aliquota in fiale piccole per evitare più cicli di gelo-disgelo 10. </li><li> supporti Aspirare da tutti i palloni, e risciacquare con 5 ml di DPBS. Ripetere tutti i passaggi nella sezione precedente, fino al punto 1.3.4. </li><li> Trasferire contenuto in una provetta conica da 15 ml e centrifugare a 125 xg per 8 minuti a temperatura ambiente. Trasferimento fiala conica all&#39;armadietto biosicurezza e risospendere pellet con 10 ml di DPBS. </li><li> pipettare delicatamente la sospensione su e giù per rompere il pellet. Contare le cellule utilizzando un emocitometro. </li><li> ancora Centrifugare come indicato in sTep 1.4.3. Aspirare il surnatante e in funzione del numero di cellule, aggiungere diluente appropriato produrre 3 x 10 6 cellule in un volume di 50 microlitri. Il diluente sarà una miscela di siero media liberi e alta concentrazione membrana matrice interrato in 1: (v / v) rapporto 1. </li><li> Agitare la sospensione delicatamente e tenere in ghiaccio in ogni momento. La sospensione è ora pronto per l&#39;iniezione. </li></ol></li><li> impianto chirurgico Nota: Assicurarsi che tutti i materiali e gli strumenti chirurgici sono sterili. Pratica tecniche asettiche in ogni momento. <ol><li> Posizionare una piastra elettrica sul tavolo e coprire con un telo sterile. Impostare la macchina per anestesia e garantire tutte le forniture sono a portata di mano. </li><li> Posizionare muso dell&#39;animale nella maschera anestesia e impostare l&#39;anestesia a 1 L / min di ossigeno e 2,5% isoflurano. </li><li> Pulire delicatamente il fianco dell&#39;animale con iodio, e risciacquare con il 70% di etanolo. Ripetete tre volte. </li><li> confermare laanimale è completamente anestetizzato pizzicando la zampa posteriore. L&#39;animale è completamente anestetizzato e pronta per la chirurgia se non si osserva alcuna risposta. Tuttavia, se l&#39;animale indietreggia, assicurarsi che ci sia sufficiente l&#39;anestesia vaporizzatore e permettere all&#39;animale più tempo per andare completamente sotto anestesia. </li><li> Caricare circa 200 ml di sospensione cellulare in una siringa 1,0 ml TB con un ago da 18 G (precedentemente raffreddato). Sostituire l&#39;ago con un ago G 27 e tornare al ghiaccio fino al momento dell&#39;uso. </li><li> Individuare l&#39;area generale della milza (quadrante superiore sinistro dell&#39;addome) e l&#39;utilizzo di pinze pizzicare la pelle sulla parte superiore di quella regione. Utilizzando le forbici chirurgiche fare un&#39;incisione di circa 1,0 centimetri per creare una tasca. Analogamente, pizzicare la muscolatura liscia sulla parte superiore della milza e tagliare per accedere alla cavità peritoneale. </li><li> afferrare delicatamente l&#39;estremità caudale della milza e tirarlo fuori dal corpo. Il pancreas verrà allegato alla milza. Stendere il pancreas con una st bagnatoerile Q-tip e individuare la coda del pancreas. </li><li> Fornire l&#39;iniezione di 50 ml nella coda del pancreas, lasciare l&#39;ago dentro per 10 secondi e ruotare lentamente l&#39;ago del pancreas. Un impianto di successo sarà simile a una bolla superficiale, senza perdite. </li><li> Ritorno il pancreas e la milza alla cavità peritoneale. In primo luogo racchiudere il muscolo e quindi racchiudere la pelle separatamente. Utilizzare una sutura 6-0 o fiocco per chiudere l&#39;incisione. Per evitare il dolore negli animali, amministrare ketoprofene per via sottocutanea (SC) (5 mg / kg) oltre 24 ore. In alternativa, amministrare buprenorfina sc (0,05 - 0,1 mg / kg) ogni 12 ore in un periodo di 36 ore. </li><li> Recuperare l&#39;animale dall&#39;anestesia e restituirlo alla sua gabbia. monitorare anche per il dolore. Gli animali devono essere dotati di sollievo dal dolore al momento della (o anche prima - preventiva) un intervento chirurgico. sollievo dal dolore supplementare deve essere fornita agli animali che soffrono di dolore in base alla IACCUC-protocollo o come prescritto dal altendente veterinario o designato. </li></ol></li><li> Imaging in vivo Nota: la cattura immagine è stata effettuata utilizzando un sistema di imaging commerciale dotato di una camera oscura ei filtri adeguati per l&#39;imaging GFP. acquisizione delle immagini è stato realizzato con una telecamera CCD e un sistema ottico composto da lenti intercambiabili di eccitazione / emissione (eccitazione: 455-495 nm di emissione: 513-557 nm). immagini in campo chiaro sono stati catturati senza un filtro 1 x 1 binning per ogni punto di tempo. Eccitazione di GFP utilizzata una sorgente di luce multispettrale allo xeno. Gli animali sono stati mantenuti in anestesia durante il processo di imaging collegando la macchina anestesia per un collettore anestesia gas integrato nella camera oscura del sistema di imaging. <ol><li> Anestetizzare l&#39;animale come indicato nel 1.5.2. Una maschera anestesia è all&#39;interno della camera oscura del sistema di imaging commerciale al fine di mantenere l&#39;animale in anestesia durante l&#39;imagingProcesso. </li><li> Impostare i corretti filtri eccitazione e di emissione. </li><li> Iniziare ottenendo un&#39;immagine iniziale utilizzando solo la luce bianca. Mantenere la stessa posizione dell&#39;animale per tutta la sessione di imaging e passare ai filtri GFP prendere una seconda immagine. </li><li> Per ottenere i migliori risultati si sovrappongono due immagini. Analizzare l&#39;immagine per l&#39;area fluorescenti e intensità. </li></ol></li></ol>

<ol>
	<li>Smyth, E., Cunningham, D., Kasper, D., 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=.">.</a> Harrison's Principles of Internal Medicine. , (2015).</li><li>Mahipal, A., Frakes, J., Hoffe, S., Kim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+borderline+resectable+pancreatic+cancer.">Management of borderline resectable pancreatic cancer.</a> World J Gastrointest Oncol. 7, 241-249 (2015).</li><li>De La Cruz, M. S., Young, A. P., Ruffin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+pancreatic+cancer.">Diagnosis and management of pancreatic cancer.</a> Am Fam Physician. 89, 626-632 (2014).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nat Rev Cancer. 7, 645-658 (2007).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+orthotopic+xenografts:+better+mimic+of+metastasis+than+subcutaneous+xenografts.">Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts.</a> Nat Rev Cancer. 15, 451-452 (2015).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+uses+of+fluorescent+proteins+to+visualize+cancer+in+vivo.">The multiple uses of fluorescent proteins to visualize cancer in vivo.</a> Nat Rev Cancer. 5, 796-806 (2005).</li><li>Jiang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+orthotopic+pancreatic+cancer+mouse+model:+cells+suspended+and+injected+in+Matrigel.">Establishment of an orthotopic pancreatic cancer mouse model: cells suspended and injected in Matrigel.</a> World J Gastroenterol. 20, 9476-9485 (2014).</li><li>Arranz, A., Ripoll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+optical+imaging+for+pharmacological+studies.">Advances in optical imaging for pharmacological studies.</a> Front Pharmacol. 6, 189 (2015).</li><li>Metildi, C. A., Kaushal, S., Hoffman, R. M., Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+serial+selection+of+human+pancreatic+cancer+cells+in+orthotopic+mouse+models+produces+high+metastatic+variants+irrespective+of+Kras+status.">In vivo serial selection of human pancreatic cancer cells in orthotopic mouse models produces high metastatic variants irrespective of Kras status.</a> J Surg Res. 184, 290-298 (2013).</li><li>Kim, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nat Protoc. 4, 1670-1680 (2009).</li><li>Katz, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+efficacy+of+adjuvant+cytosine-analogue+CS-682+in+a+fluorescent+orthotopic+model+of+human+pancreatic+cancer.">Survival efficacy of adjuvant cytosine-analogue CS-682 in a fluorescent orthotopic model of human pancreatic cancer.</a> Cancer Res. 64, 1828-1833 (2004).</li><li>Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optical+imaging+of+primary+tumor+growth+and+multiple+metastatic+events+in+a+pancreatic+cancer+orthotopic+model.">Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model.</a> Cancer Res. 62, 1534-1540 (2002).</li></ol>

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Descriviamo un modello murino ortotopico di cancro pancreatico che esprime GFP, consentendo così il monitoraggio non invasivo della crescita tumorale mediante intero corpo imaging fluorescente vivo (Figura 1). Questa tecnica permette di monitorare lo sviluppo del tumore in tempo reale (figura 3); può essere uno strumento importante per i ricercatori di studiare l&#39;efficacia terapeutica dei nuovi farmaci contro il cancro al pancreas. Un altro aspetto importante di ...

Il tumore al pancreas viene diagnosticato con maggior frequenza rispetto ad altri tipi di cancro ed è la 4 ° causa principale di decessi correlati al cancro negli Stati Uniti. Dal momento della diagnosi, oltre il 90% dei pazienti muoiono entro cinque anni 1,2. Attualmente, la rimozione del tumore chirurgica è l&#39;unica cura per il cancro al pancreas, ma meno del 20% dei pazienti hanno diritto a sottoporsi ad intervento chirurgico soprattutto perché al momento della diagnosi della malattia è in fase avanzata e ha metastatizzato 3,4. La mancanza di sintomi specifici rende cancro al pancreas una malattia silenziosa; alcuni dei sintomi includono dolore addominale, mal di schiena, perdita di appetito, ittero e nausea; che può essere facilmente interpretato come malattie digestive comuni 4. Per questo motivo, è importante sviluppare nuovi strumenti farmacologici per aiutare nella diagnosi e nel trattamento del cancro pancreatico. L&#39;uso di modelli animali ci permette di capire la biologia del Pancrécancro e ATIC fornisce una panoramica di applicare questa conoscenza per gli esseri umani. Xenotrapianto modelli ortotopici del cancro del pancreas sono realistici, perché i tumori crescono nell&#39;organo di origine 5. A differenza dei modelli eterotopici, in cui le linee di cellule o frammenti di tumore vengono impiantati per via sottocutanea, la modellazione ortotopico permette per la ricreazione del microambiente tumorale e imita l&#39;interazione delle cellule tumorali con l&#39;ambiente circostante 6. Il modello di xenotrapianto descritto qui deriva tumori dal pancreas linea di cellule umane di cancro PANC-1 GFP, che è geneticamente per esprimere la proteina fluorescente verde (GFP). Rilevamento GFP permette di imaging e monitoraggio della crescita tumorale e metastasi 7 non invasivo. Lo sviluppo del tumore avviene rapidamente, spontaneamente, e assomiglia molto a quello dei tumori primari di umani malati di cancro del pancreas 8. modelli ortotopico fornire una previsione più accurata della efficacia del farmaco in risposta ad agenti terapeutici, mentreimitando il microambiente tumorale. Come accennato in precedenza, questo modello animale permette la rilevazione fluorescente della crescita tumorale e metastasi in tempo reale. rilevazione fluorescente consente una formazione immagine più diretta / dal vivo rispetto a luminescenza. Con la fluorescenza della luce emessa è il risultato di una eccitazione da un&#39;altra luce di una lunghezza d&#39;onda; mentre in luminescenza, la luce emessa è il risultato di una reazione chimica e non può avere forti emissioni 9. Inoltre, tutto il corpo imaging in vivo fluorescente non è dannoso per l&#39;animale e permette ai ricercatori di monitorare la crescita del tumore nel tempo in risposta a trattamenti terapeutici.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI media 1640&#160;</td>
			<td>Caisson Labs&#160;</td>
			<td>RPL03-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco</td>
			<td>10437-077</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;&#160;</td>
			<td>Thermo Ficher Sci</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel HC&#160;</td>
			<td>Corning&#160;</td>
			<td>354248</td>
			<td></td>
		</tr>
		<tr>
			<td>SutureVet PGA 6-0 PGA</td>
			<td>Henry Schein</td>
			<td>39010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcare or Foamed Antiseptic Handrub</td>
			<td>Steris</td>
			<td>639680</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dubelcco&#39;s Phosphate-Buffered saline)&#160;</td>
			<td>Thermo Ficher Sci</td>
			<td>21300025</td>
			<td></td>
		</tr>
		<tr>
			<td>TB Syringe 27G1/2</td>
			<td>Becton Dickinson</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Blutler Schein</td>
			<td>50562</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketoprofen&#160;</td>
			<td>Fort Dodge Animal Health&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors, 5.5&#34;straight mayo&#160;</td>
			<td>Henry Schein</td>
			<td>22-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1 GFP cell line&#160;</td>
			<td>Anticancer, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Imaging System:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iBOx Scientia, UVP :</td>
			<td>UVP, LLC&#160; Upland, CA.&#160;</td>
			<td colspan="2">Small Animal Imaging System to observe the fluorescent tumor in live animals</td>
		</tr>
	</tbody>
</table>

fluorescent orthotopic mouse model of pancreatic cancer

Il tumore al pancreas rimane uno dei tumori per i quali la sopravvivenza non è migliorata notevolmente negli ultimi decenni. Solo il 7% dei pazienti diagnosticati sopravviverà più di cinque anni. Al fine di comprendere e imitare il microambiente dei tumori pancreatici, abbiamo utilizzato un modello murino ortotopico di cancro al pancreas che permette l&#39;imaging non invasivo di progressione del tumore in tempo reale. Le cellule tumorali pancreatiche che esprimono la proteina fluorescente verde (PANC-1 GFP) sono state sospese in matrice di membrana basale, ad alta concentrazione, (ad esempio, Matrigel HC) con mezzi privi di siero e poi iniettate nella coda del pancreas attraverso laparotomia. La sospensione cellulare nella matrice alta concentrazione membrana basale diventa una sostanza gelatinosa, una volta raggiunta la temperatura ambiente; Pertanto, si gelifica quando entra in contatto con il pancreas, creando un&#39;occlusione sito di iniezione e da evitare fuoriuscite cellule. la crescita del tumore e metastasi ad altri organi sono monitorati in tempo realeanimali utilizzando la fluorescenza. È fondamentale utilizzare i filtri appropriati per l&#39;eccitazione e l&#39;emissione di GFP. La procedura per l&#39;impianto ortotopico sono descritti in questo articolo per cui i ricercatori possono facilmente replicare la procedura in topi nudi. Le fasi principali di questo protocollo sono preparazione della sospensione cellulare, impianto chirurgico, e tutto il corpo fluorescente imaging in vivo. Questo modello ortotopico è stato progettato per valutare l&#39;efficacia di nuove terapie per i tumori primari e metastatici.

Il metodo descrive un impianto ortotopico chirurgica delle cellule tumorali pancreatiche umane fluorescenti, concentrandosi sulla preparazione della sospensione cellulare iniettabile, anestesia adeguata per roditori, la consegna di sospensione cellulare tramite laparotomia, e l&#39;uso di fluorescenza in vivo piccola dell&#39;imaging animali. Il rilevamento di un segnale verde di fluorescenza (segnale GFP) tra due e tre settimane dopo l&#39;impianto, fornisce ai ricercatori un s...

Watch this Scientific Journal Video about Fluorescent Orthotopic Mouse Model of Pancreatic Cancer at JoVE.com

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Fluorescente ortotopico modello murino di cancro al pancreas

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

fluorescent-orthotopic-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

16.5K Views.  Western University of Health Sciences. A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here. 

Video: Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Isolamento delle cellule tumorali circolanti in un modello di topo ortotopico del tumore del colon-retto

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Ricerca

Ricerca sul cancro

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Un modello murino di cancro del pancreas Syngeneic per studiare gli effetti dell'elettroporazione irreversibile

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Un modello murino Syngeneic Orthotopic bioluminescenti e fluorescente di carcinoma della prostata androgeno-dipendente e castrazione-resistente

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Immunophenotyping di Orthotopic omotrapianto (Syngeneic) dell'Adenocarcinoma Ductal pancreatico murino primario KPC tramite flusso Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Modello Xenotrapianto eterogeneo derivato dal paziente del cancro pancreatico utilizzando larve di pesce zebra come ospiti per la valutazione comparativa del farmaco

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Studiare il cancro al seno triplo negativo utilizzando il modello di cancro al seno ortotopico

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

Cultura, manipolazione e trapianto ortotopico di organidi tumorali della vescica del topo

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Impianto ortotopico a ultrasuoni dell'adenocarcinoma dugalese Murine

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Generazione di tumori pancreatici ortotopici e caratterizzazione ex vivo della citotossicità delle cellule T infiltranti di tumore

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

Un modello ortotopico di topo resezionale del cancro al pancreas

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...