Los autores han recibido apoyo de la Avner Pancreatic Cancer Foundation.

Todos los procedimientos fueron aprobados por el Comité de Cuidado y Ética animal de la Universidad de Nueva Gales del Sur (17/109A). Los ratones desnudos athymic femeninos de Balb/c, envejecidos 8-10 semanas que pesaban 16-19 g, fueron utilizados para este protocolo. Los ratones fueron alojados en jaulas de micro-aisladores y alimentados con alimentos peletados disponibles en el comercio y agua ad libitum.
1. Implantación ortotópica de cáncer de páncreas
<ol><li>Prepare las células para la implantación. Primero, calcule el número de células requeridas para el procedimiento (1 x 106 células Luciferase-marcadas AsPC-1 y 1 x 106 células radiarias pancreáticas humanas cáncer-asociadas [CAhPSCs] se requieren para cada animal).<ol>
<li>Mantenga estas células en una incubadora humidificada de CO2 con temperatura controlada y realice pruebas rutinarias de micoplasma. El medio de cultivo utilizado para AsPC-1 y CAhPSCs son RPMI 1640 (con L-glutamina de 300 mg/L, suero bovino fetal al 20%, penicilina/estreptomicina al 1% v/v) e IMDM (con L-glutamina de 4 mM, suero bovino fetal del 10%, penicilina/estreptomicina del 1%v/v).</li>
		<li>Utilice técnicas estándar de cultivo celular para tripinizar las células en una suspensión celular. Neutralice la tripsina utilizando el medio de cultivo completo respectivo a un volumen dos veces superior al de la solución de tripsina utilizada.</li>
		<li>Lave estas células dos veces con solución salina tamponada con fosfato (PBS) y resuspend en una mezcla que contenga 1 x 106 células AsPC-1 y 1 x 106 CAhPSCs en una suspensión celular de 50 μL.</li>
		<li>Mantenga esta suspensión sobre hielo hasta su uso.</li>
	</ol></li>
	<li>Prepare un gabinete de bioseguridad de clase II para el procedimiento. Use una estera de calefacción superpuesta por una cortina de plástico estéril. Para la magnificación durante el procedimiento, use un par de lupa quirúrgicas de aumento de 2.5x a 3.5x.</li>
	<li>Prepare hisopos de cadena de bolsa cortando un agujero, de 1 cm de diámetro, en un hisopo de gasa. Asegure este agujero con una sutura de cadena de monedero. Cualquier sutura trenzada fina se puede utilizar para esto (por ejemplo, sutura de ácido poliglicólico 5/0). Se recomienda el material de sutura trenzado, ya que permite que el nudo suelto permanezca en su lugar después del apriete. Esto se ilustra en la Figura 1a.</li>
	<li>Anestesiar el ratón con 80 mg/kg de ketamina y 10 mg/kg de xilazina mediante inyección intraperitoneal.</li>
	<li>Administrar 5 mg/kg de profilaxis antibiótica de enrofloxacina, 2,5 mg/kg de analgesia de flunixina y 1 mL de solución salina al 0,9% por vía subcutánea.</li>
	<li>Una vez anestesiado, coloque el ratón en el campo estéril en una posición supina y aplique povidona-yodo seguido de etanol al 70% para la preparación de la piel.</li>
	<li>Haga una incisión longitudinal en la piel del cuadrante craneal izquierdo del abdomen y luego ingrese al abdomen incisando la capa muscular entre los pórceps.</li>
	<li>Cargue una jeringa de insulina de 29 G con 50 μL de suspensión celular, lo que equivale a 1 x10 6 CAhPSCs y 1 x 106 células AsPC-1 etiquetadas con luciferasa por inyección. Montarlo en el dispositivo de inyección. El diseño y la función de este dispositivo de inyección se explica en detalle en la Figura 1b y su leyenda.</li>
	<li>Coloque el hisopo de la cuerda del bolso sobre la incisión de laparotomía y después exteriorice el bazo y la cola pancreática a través de la abertura de este hisopo. Apriete la cadena del monedero para rodear suavemente el cuerpo del páncreas, exponiendo la cola pancreática para inyección. Es importante estar lo suficientemente apretado como para que la gasa entre en contacto con el páncreas circunferencialmente y al mismo tiempo no lo constricte.</li>
	<li>Usando un par de pórceps, agarre la cola del páncreas y coloque suavemente la tensión lateral sobre ella. Perfore la superficie peritoneal ventral con la aguja en un ángulo poco profundo y luego inyecte la suspensión celular en el páncreas de manera lenta y controlada (más de 10−15 s) con el dispositivo de inyección.</li>
	<li>Durante el proceso de inyección, observe cuidadosamente si hay fugas, tanto alrededor del sitio de inyección (por reflujo) como por el otro lado del lóbulo pancreático (en caso de penetración de paso a través). Si se produce una fuga visible, detenga la inyección y observe el volumen de fuga comprobando el volumen de inyección restante en la jeringa. Si la fuga es de pequeño volumen (<10 μL), y luego absorber cualquier fuga con gasa y reposicionar la aguja en un lóbulo pancreático diferente para completar la inyección.</li>
	<li>Reemplace el bazo y el páncreas y cierre la pared abdominal con 5/0 sutura de ácido poliglicólico de manera continua. Cierre la piel con clips.</li>
	<li>Monitoree al ratón en una jaula calentada hasta que se recupere del anestésico. Una vez despierto y alerta, mueva el ratón de nuevo a su jaula.</li>
</ol>2. Cirugía de resección del cáncer: pancreatectomía distal y esplenectomía
<ol><li>El momento de la resección en relación con la implantación puede variar dependiendo del protocolo experimental. En general, permita que los tumores crezcan al menos durante 3 semanas antes de la resección, pero optimice esto empíricamente para la línea celular de cáncer implantada en particular.</li>
	<li>El día anterior a la cirugía de resección, realice imágenes de bioluminiscencia en los animales para confirmar la presencia de un tumor primario localizado. Tenga en cuenta que este estudio por imágenes se utiliza simplemente para excluir ratones con enfermedad extracreática obvia de la resección. Ni el tamaño ni el flujo radiante deben utilizarse como umbrales para determinar la elegibilidad para la resección.<ol>
<li>Pesar ratones e inyectar con D-luciferina por vía intraperitoneal (150 mg/kg).</li>
		<li>Determinar el momento de la etapa de obtención de imágenes en relación con la inyección de luciferina para cada experimento mediante la realización de una curva cinética de luciferina. El período de tiempo en el que el flujo radiante está por encima del 90% de su máximo representa el tiempo óptimo para la obtención de imágenes de bioluminiscencia (en este experimento, de 18 a 26 minutos después de la inyección)</li>
		<li>Induzca anestesia y mantenga el uso de isoflurano (4% y 3% con oxígeno, respectivamente) y realice imágenes utilizando un dispositivo de imágenes bioluminiscente (por ejemplo, IVIS Lumina II). Utilice la configuración automática de exposición y binning (sin embargo, esto se puede optimizar para el flujo radiante esperado).</li>
	</ol>
</li>
	<li>Prepare el gabinete de bioseguridad de clase II para el procedimiento. Use una estera de calefacción superpuesta por una cortina de plástico estéril. Para la magnificación durante la disección, use un par de lupa quirúrgicas de aumento de 2.5x a 3.5x.</li>
	<li>Anestesiar el ratón con 80 mg/kg de ketamina y 10 mg/kg de xilazina mediante inyección intraperitoneal.</li>
	<li>Administrar 5 mg/kg de profilaxis antibiótica de enrofloxacina, 2,5 mg/kg de analgesia de flunixina y 1 mL de solución salina al 0,9% por vía subcutánea.</li>
	<li>Coloque el ratón en el campo estéril en una posición supina y aplique povidona-yodo seguido de etanol al 70% para la preparación de la piel.</li>
	<li>Haga una incisión longitudinal en la piel del cuadrante craneal izquierdo del abdomen, preferiblemente a través del sitio de la incisión anterior.</li>
	<li>Diseccione embotadamente la piel de la pared abdominal muscular subyacente, y luego coloque un retractor de autocontención de Alm para mantener la herida de la piel abierta.</li>
	<li>Incise la capa muscular entre las autorórceps justo a un lado de la línea de sutura de la operación anterior, y luego extienda la incisión para eliminar toda la línea de sutura anterior.</li>
	<li>Exteriorizar el bazo y el páncreas distal y retraerlo cranealmente. En el aspecto caudal del páncreas, los dos puntos se pueden encontrar unidos por adherencias fílmicas. Si esto se encuentra, diseccione sin rodeos los dos puntos.</li>
	<li>Pase con cuidado un par de tórceps dorsales al cuerpo del páncreas y los vasos esplénicos y abra este espacio. Esto libera un segmento del páncreas para la ligadura posterior.</li>
	<li>Ligate el cuerpo del páncreas próximo al tumor con un clip titanium de la ligadura, y después transecte el páncreas distal a esto con cautery. Una forma alternativa de controlar el tocón pancreático es ligarlo en continuidad con la sutura de ácido poliglicólico 5/0 antes de la transección.</li>
	<li>Retraiga el páncreas caudalmente y cauterice los vasos gastroesplénicos entre el polo craneal del bazo y el estómago.</li>
	<li>Retire el espécimen y confirme la hemostasia.</li>
	<li>Cierre la pared abdominal con 5/0 de sutura de ácido poliglicólico de manera continua. Cierre la piel con clips.</li>
</ol>3. Manejo postoperatorio
<ol><li>En el período anestésico inmediatamente posterior (para los dos procedimientos anteriores), monitoree el ratón en una jaula calentada hasta que se recupere del anestésico. Una vez despierto y alerta, mueva el ratón de nuevo a su jaula. En el período postoperatorio, vigile a los animales para el dolor y las muestras de la señal de socorro. Administrar 0,05 mg/kg de buprenorfina por inyección subcutánea y observar de cerca a los animales durante 12 horas.</li>
	<li>Posteriormente, monitoree los ratones diariamente para el peso, la ingesta de alimentos y la actividad. Examine los sitios de la incisión y palpate para el tamaño del tumor. Retire los clips de la piel en el séptimo día postoperatorio.</li>
	<li>Eutanasiar el ratón si se alcanzan extremos humanos. Estos criterios de valoración humanos incluyen: pérdida de peso corporal >20%, características de sufrimiento intratable (incluida la postura encorvada, la falta de movimiento o el acicalamiento) y el tamaño del tumor superior a 1 cm3 según lo estimado por la palpación externa.</li>
</ol>

<ol>
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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=Cell+migration:+a+novel+aspect+of+pancreatic+stellate+cell+biology.">Cell migration: a novel aspect of pancreatic stellate cell biology.</a> Gut. 52 (5), 677-682 (2003).</li><li>Boj, S. F., 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Egberts, J. H., 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=Superiority+of+extended+neoadjuvant+chemotherapy+with+gemcitabine+in+pancreatic+cancer:+a+comparative+analysis+in+a+clinically+adapted+orthotopic+xenotransplantation+model+in+SCID+beige+mice.">Superiority of extended neoadjuvant chemotherapy with gemcitabine in pancreatic cancer: a comparative analysis in a clinically adapted orthotopic xenotransplantation model in SCID beige mice.</a> Cancer Biology &amp; Therapy. 6 (8), 1227-1232 (2007).</li></ol>

Los autores no tienen nada que revelar con respecto a este proyecto.

Un modelo orthotopic resectional del ratón del cáncer pancreático es importante porque permite la prueba de tratamientos ayudantes y neoadjuvant. Esto es particularmente importante en el cáncer de páncreas, donde la cirugía sigue siendo el tratamiento más eficaz, pero se asocia con un alto riesgo de recurrencia. Este papel describe un método que produzca confiablemente un cáncer pancreático que sea potencialmente curable con la resección, replicando el escenario clínico donde se requiere la terapia neoadjuvan...

El adenocarcinoma ductal pancreático (cáncer pancreático [PC]) se asocia a un pronóstico pobre1. La resección quirúrgica sigue siendo el único tratamiento potencialmente curativo para la PC y se debe considerar para los pacientes que presentan con enfermedad del primero tiempo. Desafortunadamente, incluso con la resección R0 (es decir, márgenes de la resección libres de tumor), la tarifa de la repetición (local o de la enfermedad metastática no detectada) es alta2,3. Por lo tanto, la terapia adyuvante sistémica está indicada en casi todos los pacientes que se someten a la resección4. Además, mientras que la terapia neoadyuvante ahora se recomienda solo para cánceres resecables limítrofes, sus indicaciones se están expandiendo de tal manera que su uso rutinario es el foco de mucha investigación clínica5,6,7,8. Para desarrollar los acercamientos terapéuticos nuevos para la PC que implica la resección, estos acercamientos necesitan primero ser evaluados en los modelos preclínicos que recapitulan exactamente ajustes clínicos.
Los modelos ortotópicos de ratón de PC se han utilizado con frecuencia en el pasado para probar tratamientos farmacológicos9,10. Muchos de éstos fueron producidos por la inyección de células cancerosas solamente en el páncreas del ratón, dando por resultado los tumores que carecían del tejido conectador prominente que es característico de la PC. Más recientemente, los modelos ortotópicos de coinyección, como el que desarrollamos por primera vez inyectando una mezcla de PC humana y células estrelladas pancreáticas humanas (PSC, los principales productores del estroma colagenoso en la PC), han entrado en uso regular11,12. Los tumores producidos por tal co-inyección del cáncer y de las células stromal exhiben (i) los elementos del cáncer y el componente (desmoplastic) stromal característico de la PC, y (ii) proliferación y metástasis de célula cancerosasaumentadas 11. Por lo tanto, este modelo se asemeja mucho a la PC humana. Mientras que un número de modelos reseccionales de la PC orthotopic se han descrito13,14,15,16,ningunos han reflejado las realidades clínicas de la resección pancreática en seres humanos tan exactos como este modelo, y por lo tanto han sido subóptimos para probar tratamientos ayudantes o neoadyuvantes.
Los objetivos del modelo del ratón presentado eran demostrar cómo: (i) implantar con éxito el cáncer pancreático orthotopic mientras que minimizaba la difusión peritoneal inadvertida y (ii) resecar posteriormente totalmente el cáncer. El documento destaca los consejos y posibles escollos de esta técnica.

<table>
	<tbody>
		<tr>
			<td>Animals, Materials and Equipment for Implantation Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid)</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td></td>
			<td>supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan</td>
		</tr>
		<tr>
			<td>Autoclip wound clips, 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW, Australia</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cancer associated human pancreatic stellate cells</td>
			<td>Pancreatic Research Group cell bank</td>
			<td></td>
			<td>In house cell bank</td>
		</tr>
		<tr>
			<td>Cryogenic tubes, 1.0 mL</td>
			<td>Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia</td>
			<td>366656</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS)</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female</td>
			<td>Australian Bioresources, Moss Vale, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) with 4mM L-glutamine and no phenol red</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>21056023</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Penicillin 10,000 U/mL, streptomycin 10,000 &#956;g/mL</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>11875085</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25300054</td>
			<td>For pancreatic stellate cells</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25200056</td>
			<td>For ASPC-1 cells</td>
		</tr>
		<tr>
			<td>U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) &#215; 13 mm needle</td>
			<td>Terumo Medical Corporation, Elkton, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment for Resection Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alm self-retaining retractor</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Autoclip wound clips 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td>08-559NP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Hand-held high temperature fine tip cautery</td>
			<td>Bovie Medical Corporation, Melville, NY, USA</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II Bioluminescent Imaging Device</td>
			<td>Caliper Life Sciences, Hopkinton, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex wound clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Titanium &#8220;Weck style&#8221; Ligaclip, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Ligaclip applier for open surgery, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volatile anaesthetic machine, including vapouriser and induction chamber</td>
			<td>Generic</td>
			<td></td>
			<td>Generic vapouriser and induction chamber</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for Procedures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% w/w ethanol solution</td>
			<td>Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia</td>
			<td></td>
			<td>Applied topically as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 0.05 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>D-Luciferin (1 U/g)</td>
			<td>PerkinElmer, Inc., Waltham, MA, USA</td>
			<td>122799</td>
			<td>diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p</td>
		</tr>
		<tr>
			<td>Enrofloxacin 50 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Flunixin 50 mg/mL</td>
			<td>Norbrook Laboratories Australia, Tullamarine, VIC, Australia</td>
			<td></td>
			<td>Dose: 2.5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis Australia Pty Ltd., Rhodes, NSW, Australia</td>
			<td></td>
			<td>Dose (vapourised with oxygen): 4% induction, 3% maintenance</td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Maylab, Slacks Creek, QLD, Australia</td>
			<td></td>
			<td>Dose: 80 mg/kg i.p.</td>
		</tr>
		<tr>
			<td>Povidone-Iodine 10% w/v solution</td>
			<td>Perrigo Australia, Balcatta, WA, Australia</td>
			<td>RIO00802F</td>
			<td>Applied topically to the anterior abdomen as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w)</td>
			<td>Allergan Australia Pty Ltd, Gordon, NSW, Australia</td>
			<td></td>
			<td>Applied to both eyes</td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% w/v</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>9481P</td>
			<td>Dose: 900 &#956;L s.c.</td>
		</tr>
		<tr>
			<td>Water for injections BP</td>
			<td>Pfizer Australia, Sydney, NSW, Australia</td>
			<td></td>
			<td>For dilution of drugs</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 10 mg/kg i.p.</td>
		</tr>
	</tbody>
</table>

an orthotopic resectional mouse model of pancreatic cancer

Hay una carencia de modelos animales satisfactorios para estudiar terapia ayudante y/o neoadjuvant en los pacientes que son considerados para la cirugía del cáncer pancreático (PC). Para tratar esta deficiencia, describimos un modelo del ratón que implica la implantación orthotopic de la PC seguida por pancreatectomy y el splenectomy distales. Se ha demostrado que el modelo es seguro y convenientemente flexible para el estudio de diversos enfoques terapéuticos en entornos adyuvante y neoadyuvante.
En este modelo, un tumor pancreático primero es generado implantando una mezcla de células cancerosas pancreáticas humanas (luciferasa-marcada AsPC-1) y de células radiadas pancreáticas asociadas cáncer humano en el páncreas distal de los ratones desnudos athymic de Balb/c. Después de tres semanas, el cáncer es resecado por re-laparotomía, pancreatectomía distal y esplenectomía. En este modelo, las imágenes de bioluminiscencia se pueden utilizar para seguir el progreso del desarrollo del cáncer y los efectos de la resección/tratamientos. Después de la resección, se puede administrar terapia adyuvante. Alternativamente, el tratamiento neoadyuvante se puede dar antes de la resección.
Los datos representativos a partir de 45 ratones se presentan. Todos los ratones experimentaron pancreatectomy/splenectomy distal acertado sin las ediciones del hemostasis. Un margen pancreático próximo macroscópico mayor de 5 milímetros fue alcanzado en 43 (el 96%) ratón. El índice de éxito técnico de la resección pancreática era el 100%, con la mortalidad y la morbosidad tempranas del 0%. Ningunos de los animales murieron durante la semana después de la resección.
En resumen, describimos una técnica robusta y reproducible para un modelo quirúrgico de la resección del cáncer pancreático en los ratones que mímico el escenario clínico. El modelo puede ser útil para la prueba de tratamientos adyuvantes y neoadyuvantes.

Cincuenta y nueve ratones consecutivos se sometieron a cirugía de implantación. La fuga bruta ocurrió en ocho (14%) ratón. El grado de fuga en el momento de la inyección se estima como se describió anteriormente en la sección de protocolo. Después de tres semanas para permitir que estos tumores implantados crezcan, la proyección de imagen de la bioluminiscencia de la pre-resección fue realizada para excluir ratones con enfermedad metastática gruesa antes de la resección. Cuarenta y cinco (76%) los ratones exp...

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

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

En el contexto clínico, los pacientes con cáncer de páncreas localizado se someterán a pancreatectomía seguida de tratamiento adyuvante. Este protocolo divulgado aquí apunta establecer un método seguro y de manera efectiva de modelar este escenario clínico en ratones desnudos, con la implantación orthotopic del cáncer pancreático seguido por pancreatectomy y splenectomy distales.

Un modelo ortotópico de ratón reseccional de cáncer de páncreas

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

an-orthotopic-resectional-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

10.0K vistas.  University of New South Wales. En el contexto clínico, los pacientes con cáncer de páncreas localizado se someterán a pancreatectomía seguida de tratamiento adyuvante. Este protocolo divulgado aquí apunta establecer un método seguro y de manera efectiva de modelar este escenario clínico en ratones desnudos, con la implantación orthotopic del cáncer pancreático seguido por pancreatectomy y splenectomy distales.

Video: An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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 survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

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.

Fluorescente modelo de ratón ortotópico de cáncer de páncreas

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 ...

Investigación

Investigación sobre el cáncer

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.

Aislamiento de células tumorales circulantes en un modelo de ratón ortotópico de cáncer colorrectal

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

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 modelo de ratón de cáncer de páncreas Syngeneic para estudiar los efectos de la electroporación Irreversible

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

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 de Orthotopic la homoplastia (Syngeneic) de Adenocarcinoma Ductal pancreática KPC primaria murinas por citometría de flujo

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

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Un modelo de cáncer de endometrio ortotópico con linfadenopatía retroperitoneal hecha de células VX2 cultivadas y cultivadas en Vivo

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

Un modelo de ratón ortotópico inducido por hepatocitos oncogénicos que surge en el ajuste de la inflamación hepática y la fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

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.

Estudio del triple cáncer de mama negativo con el modelo de cáncer de mama ortotópico

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

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.

Ultrasonido-Guiado Implantación Ortotópica de Adenocarcinoma Ductal Pancreático Murino

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.

Generación de tumores pancreáticos ortotópicos y caracterización ex vivo de la citotoxicidad de células T infiltrantes en tumores

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

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Generación de un xenoinjerto ortotópico de células de cáncer de páncreas mediante inyección guiada por ultrasonido

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...