We thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript. This work was supported by the National Cancer Institute at the National Institutes of Health through grant numbers RO1CA160079, RO1CA138642, UO1CA184966 and VA funded program project number 1P1 BX001604.

Los protocolos para todos los procedimientos con animales deben ser revisados ​​y aprobados por un Comité para el Cuidado y Uso de Animales Institucional (IACUC). Siga los procedimientos aprobados oficialmente para el cuidado y uso de animales de laboratorio. La inyección intra-prostática requiere cirugía abdominal abierta y animales debe mantenerse en un entorno libre de patógenos de una sala de cirugía designada donde se utilizan técnicas asépticas quirúrgicos adecuados durante todo el procedimiento. 1. Preparación de las células para la implantación NOTA: Basado en la necesidad de investigación, cualquier línea celular de cáncer de próstata se puede utilizar. Las líneas celulares se cultivaron de acuerdo con las instrucciones del proveedor. <ol><li> Para la línea celular PC3M-Luc-C6 que expresa de forma estable el gen de luciferasa de luciérnaga, células de cultivo en medio esencial mínimo (MEM) suplementado con 10% suero bovino fetal (FBS), aminoácidos 1x no esenciales, 1x fleomicina D1 y 1 mM de piruvato de sodio . Mantener las células en una incubadora wITH una atmósfera humidificada de 95% de aire y 5% de CO2 a 37 ° C. células PC3M-Luc-C6 se adquirieron a partir facilidad de la base de la UCSF. </li><li> Cosecha células por tripsinización. Lavar las placas de cultivo una vez con solución salina tamponada con fosfato (PBS). Añadir 2 ml de tripsina 0,05% a una placa de 10 cm y se incuba durante 3 a 5 min en una incubadora con una atmósfera humidificada de 95% de aire y 5% de CO2 a 37 ° C hasta que se separan las células. <ol><li> Para evitar la formación de grumos, no agitar las células golpeando o sacudiendo el plato a la espera de las células se despeguen. Recoger las células en 5 ml de medio completo y centrifugar durante 5 minutos a 200 x g. Lavar el sedimento celular con PBS para eliminar la tripsina. </li></ol></li><li> Enumerar las células vivas mediante el ensayo de exclusión de azul de tripano. Mezclar 10 l de la suspensión de células en PBS con 10 l de 0,4% (v / p) de solución de azul de tripano. Cargar la mezcla en un hemocitómetro o cámara de recuento de diapositivas y contar las células inmediatamente bajo un microscopio o leer la diapositiva cámara en un cou célulanter. <ol><li> Preparar una suspensión celular que contenía 2,5 x 10 5 células en 10 l de medios. Mezclar la suspensión de células con el extracto de matriz extracelular 10 l membrana basal-like y colocar las células en hielo. Añadir luciferina a la suspensión celular (1: 200 l; de stock 30 mg / ml de concentración) antes de la inyección en ratones. NOTA: Esto permite inmediata bio-imágenes de los animales para comprobar la coherencia de las inyecciones de células entre los diferentes grupos experimentales. Inyectar las células tan rápidamente como sea posible, preferiblemente dentro de 30 min después de tripsinización desde la viabilidad celular disminuye rápidamente después de la separación. </li></ol></li></ol> 2. Preparación del área quirúrgica <ol><li> Realizar la cirugía en un área despejada, desinfectada que promueve la asepsia. </li><li> Desinfectar el banco contador / laboratorio con una solución de cloro antes de la cirugía. NOTA: El uso de alcohol no se recomienda debido al largo tiempo de contacto requerido para entrar en vigor (15 min). </li><li> Use paños estériles,limpiar las almohadillas absorbibles o toallas, y reemplazar estos materiales después de cada sesión quirúrgica. Esterilizar todos los instrumentos antes de su uso. Los métodos preferidos son un autoclave de vapor, vidrio esterilizador de cuentas, gas óxido de etileno, o la esterilización con vapor de peróxido de hidrógeno. </li><li> Utilizar un microscopio de disección aséptica limpiado para realizar el procedimiento quirúrgico o investigadores experimentados pueden realizar sin un microscopio. </li></ol> 3. Implantación de células tumorales <ol><li> Utilice macho de 6-8 semanas de edad inmunocomprometidos Balb / c / o ratones SCID NOD. NOTA: Es más difícil de operar en los animales más pequeños y animales más grandes tienden a tener más lenta cinética de crecimiento del tumor y la metástasis. </li><li> Inyectar antes de la cirugía medicamentos para el dolor según las instrucciones de la instalación para animales. Por ejemplo, buprenorfina intra-peritoneal a una dosis de 0,1 mg / kg de peso se puede utilizar. </li><li> Anestesiar animales colocándolos en una cámara de isoflurano con 1-3% de isoflurano en oxígeno y wahasta que los animales están completamente anestesiados. Asegúrese de que no hay reflejo del dedo del pie de tono muscular en este punto. El uso adecuado de lubricante veterinaria pomada oftálmica para prevenir la ceguera por xeroftalmía durante la anestesia general. NOTA: Anestesie los animales por del investigador método preferido, por ejemplo, por pentobarbital sódico, se administra 0,05 mg por gramo de peso corporal intra-peritoneal o la solución de ketamina / xilazina (concentración: 17,16 mg / ml) a una dosis de 65 mg / kg de peso corporal se usa por vía subcutánea. inhalación de isoflurano es un método preferido de la anestesia. La pomada ocular se debe aplicar suavemente sin frotar contra la córnea. </li><li> Retire el animal de la cámara de isoflurano y colocar en un aparato de cono de la nariz con flujo continuo de 1 a 2% de isoflurano en oxígeno para asegurar que el animal está bajo anestesia completa antes de continuar. <ol><li> Quitar el pelo de los ratones por el afeitado o usar una crema de eliminación de vello antes de comenzar el procedimiento. NOTA: Sería preferible colocar el ratón sobre una almohadilla caliente estéril durante la cirugía. </li></ol></li><li> Coloque el ratón en una posición supina. Limpiar la parte inferior del abdomen con 10% w / w solución de povidona-yodo seguido por 70% de etanol hisopos. </li><li> Con un par de pinzas finas, levante un área de piel de 2 mm por encima de la glándula prepucial, alrededor de 1-2 cm por encima de la vaina del pene, y alrededor de 2 a 3 cm por debajo de la parte inferior de la caja torácica. </li><li> Hacer un 1 cm de la línea media de la incisión de longitud, primero a través de la piel con un escalpelo y luego a través de la capa muscular con una tijera (Figura 1). </li><li> Ubicar la vejiga en la cavidad corporal. Es un órgano esférico de color marrón-amarillo claro, que se encuentra directamente debajo de la incisión (Figura 1). </li><li> Con un par de agarre pinzas finas de la vejiga y levantar hacia arriba luego hacia abajo fuera de la cavidad del cuerpo hacia la vaina del pene. Esto expondrá las dos vesículas seminales, que son un par de órganos en forma de saco blanco y claramente distinto. </li><li> Con unhisopo de algodón en cada mano, exteriorizar las vesículas seminales, uno por uno, y sacarlos de la cavidad del cuerpo y yacía boca abajo sobre la superficie exterior del abdomen con la vejiga en el medio (Figura 2). </li><li> El uso de los bastoncillos de algodón, suavemente inclinación hacia atrás de las vesículas seminales en el punto de inserción cerca del cuello de la vejiga, hacia la vaina del pene, de manera que los dos lóbulos de la próstata dorsal son claramente visibles. Use hisopos de algodón húmedo para evitar daños en los tejidos (Figura 3). </li><li> Agitar la suspensión de células con una micropipeta antes de cargar en la jeringa. </li><li> Mientras que la colocación de las vesículas seminales en su posición con un hisopo de algodón, inserte la aguja de la jeringa en el lóbulo dorsal de la próstata bajo el microscopio (Figura 4). inyectar lentamente 20 l de suspensión celular hasta que se identifica una formación de bullas. Una ampolla abultada indica que la inyección es correcta. </li><li> Mientras se retrae la aguja, presione ligeramente en la zona de inyección conun hisopo de algodón y mantener durante unos segundos para evitar fugas. </li><li> Levante con cuidado las vesículas seminales con hisopos de algodón e insertarlos de nuevo a la cavidad del cuerpo, uno por uno, seguido de la vejiga. Evitar &#39;&#39; girando los órganos internos mientras realiza este procedimiento. </li><li> Después de colocar de nuevo los órganos en la cavidad corporal, la sutura de la capa muscular primero en un patrón interrumpido con 4-0 suturas de catgut crómico absorbibles seguido por el cierre de la piel con no absorbible 4-0 nylon sutura quirúrgica. La piel también se puede tirar juntos y se cerró con pinzas quirúrgicas para cerrar la incisión por completo. NOTA: Los ratones tienen un hábito de arañar y morder a su herida, lo que puede dar lugar a la reapertura de la herida, por lo tanto, el uso del adhesivo tisular también se recomienda junto con suturas. animales imagen de inmediato para asegurar que no es igual intensidad bioluminiscente entre todos los grupos experimentales. </li><li> Devolver los animales para limpiar las jaulas y mantenerlos bajo una lámpara de calor o calentamientoing almohadilla. Monitorear constantemente los animales hasta que se recuperan completamente de la anestesia y mantienen decúbito esternal. </li></ol> 4. Seguimiento de los Animales <ol><li> Monitorear los animales regularmente hasta el final del experimento, de acuerdo con protocolos institucionales. Si el uso de clips metálicos quirúrgicos, retire después de una a dos semanas. Duración del experimento depende de la necesidad de investigación específico. NOTA: Este experimento se llevó a cabo durante 21 días para examinar el establecimiento con éxito de las células cancerosas implantadas en la próstata del ratón. </li><li> Administrar medicamentos para el dolor en base a las instrucciones de las instalaciones de animales. Por ejemplo, buprenorfina intra-peritoneal a una dosis de 0,1 mg / kg de peso se puede utilizar en el momento del procedimiento con una segunda dosis después de dosis 6 hr y adicionales cada 8-12 horas, según sea necesario. </li><li> Vigilar el peso del animal, el consumo de alimentos, el color y la textura de la piel, la actividad y la frecuencia de la micción y la defecación. La eutanasia a los animales immediately si hay una pérdida significativa de peso corporal mayor que 15%. <ol><li> Para la eutanasia, entregar el CO 2 de un tanque a presión dentro de una jaula de afluencia de gente a un caudal de desplazar el 10-30% de la cámara o jaula volumen / minuto, permitiendo que el CO 2 entre en la cámara lentamente para que se produzca la inconsciencia y la narcotización completa antes de la muerte. </li><li> Mantener CO 2 de flujo para al menos un minuto después de la parada respiratoria y dejar a los animales en la cámara durante un tiempo suficiente para que la muerte se produce antes de realizar un método físico. </li><li> Realizar la decapitación, la dislocación cervical o cualquier otro método aprobado por el IACUC física después de la eutanasia de los animales químicamente. NOTA: Se ha reducido significativamente el peso corporal a menudo indica una condición de letargo. animales portadores de tumores deben estar en buen estado de salud a excepción de la presencia de tumores hasta el final del experimento. La eutanasia debe ser coherente con las Directrices sobre la eutanasia AVMA y debe ser listed en el protocolo aprobado IACUC. </li></ol></li></ol> 5. La no-invasivo Bio-imágenes de los Animales <ol><li> Monitorear los animales semanalmente usando una técnica de imágenes no invasivo para el seguimiento de la colonización de las células de cáncer, el crecimiento del tumor y cualquier metástasis distante. NOTA: modalidades de imágenes tales como GFP-imágenes, imágenes de la luciferasa, los rayos X o tomografía 3D micro-computarizada (UCT), etc., pueden ser utilizados en base a la investigación específica necesitan 12-15. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Este manuscrito describe un procedimiento detallado para el establecimiento de un cáncer de próstata modelo de xenoinjerto de ratón ortotópico humano. Este modelo fue establecido por la implantación directa de la línea celular de cáncer de próstata humano PC3M-Luc-C6 en los lóbulos prostáticos dorsal de ratones inmunodeficientes. Los tumores se dejaron desarrollar durante el transcurso del experimento. El crecimiento tumoral se controló semanalmente por un sistema de imágenes de bioluminiscencia no invasiva ...

El cáncer de próstata es la segunda causa más frecuente de muerte por cáncer (9%) entre los hombres en los Estados Unidos, junto con el cáncer de pulmón y de bronquios (28%) 1. Según los últimos datos, se estima que 220, 800 casos de cáncer de próstata recién diagnosticados y 27, 540 muertes ocurrirán en 2015 1. La tasa de supervivencia relativa a cinco años de cáncer de próstata en etapa temprana es&gt; 99%, mientras que la de enfermedad metastásica avanzada es sólo el 28% 1. Un reto importante para el tratamiento de la enfermedad metastásica avanzada es la falta de comprensión de los mecanismos moleculares que subyacen a la propensión de la enfermedad a la metástasis a otros órganos, especialmente en el hueso, que es un sitio frecuente de cáncer de próstata. Por lo tanto, existe una clara necesidad de estudiar la composición molecular de estos tumores de próstata con el fin de desarrollar regímenes terapéuticos eficaces contra la progresión a avanzado 2,3 enfermedad metastásica. Los tumores de próstata hig exhibiciónh heterogeneidad biológica sin un camino bien definido para la progresión. Metástasis a menudo se producen sin ninguna indicación previa de la invasividad tumoral 4. Esta heterogeneidad clínica se atribuye a la diversidad molecular de cáncer de próstata. Comprender la composición molecular de estos tumores letales es la clave para diseñar mejores estrategias diagnósticas y terapéuticas para esta enfermedad. En consecuencia, la investigación del cáncer de próstata se centra actualmente en la comprensión y la prevención de la metástasis. Preclínicos modelos de ratón in vivo ofrecen una variedad de opciones para comprender los mecanismos moleculares de la progresión del cáncer de próstata metastásico avanzado a la enfermedad. Además, estos modelos son importantes para las evaluaciones preclínicas de nuevas estrategias terapéuticas contra esta enfermedad. Los modelos animales más comúnmente usados ​​incluyen modelos de ratones transgénicos, la cola de inyección venosa, implantación intra-cardiaca y modelos de ratón ortotópico humanos. estudios transgénicos son consumi tiempong y correlación de desarrollo del cáncer de próstata en ratones con la de los seres humanos han demostrado la variabilidad 11. En modelos de ratón de metástasis espontáneas, las células se inyectan directamente en la circulación y, sin embargo, de que tengan tiempo de respuesta rápido, no se pueden utilizar para estudiar el tumor primario o los pasos iniciales de la cascada metastásica 5. modelos de xenoinjerto ortotópico tienen la limitación del desarrollo de lesiones metastásicas óseas, el sitio común de metástasis del cáncer de próstata. Sin embargo, el cáncer de próstata modelo de ratón con xenoinjerto ortotópico humano está bien caracterizado y ampliamente usado para estudiar los eventos moleculares del desarrollo del tumor primario, la diafonía entre tumor y microambiente de órganos, la fase inicial de la enfermedad metastásica y el uso de fármacos experimentales para la intervención terapéutica 6 , 7,8-11.

<table border="0" cellpadding="0" cellspacing="0" width="732">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PC3 prostate cancer cell line&#160;</td>
			<td style="width:188px;">ATCC</td>
			<td style="width:111px;">CRL-1435</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Medium (MEM)&#160;</td>
			<td>GIBCO,Life Technology</td>
			<td>11095-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>GIBCO,Life Technology</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO,Life Technology</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeocin</td>
			<td>Invitrogen,Life Technology</td>
			<td>R250-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin&#160;</td>
			<td>GIBCO,Life Technology</td>
			<td>25300-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS&#160;</td>
			<td>Xenogen-Caliper</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin Syringes (300ul, 28.5g)</td>
			<td>Becton Dickinson</td>
			<td>309300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mice</td>
			<td>Charles River Laboratories, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol Swabs</td>
			<td>MEDEquip Depot</td>
			<td>326895 BD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVP Iodine Prep Pad</td>
			<td>MEDEquip Depot</td>
			<td>C12400PDI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical CatGut Chromic Suture</td>
			<td>Demetech</td>
			<td>CC224017F0P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>Corning</td>
			<td>354248</td>
			<td></td>
		</tr>
	</tbody>
</table>

pre-clinical orthotopic murine model of human prostate cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Después de la implantación de las células ortotópico PC3M-Luc-C6 en el lóbulo de la próstata posterior, los ratones fueron imágenes semanal mediante el uso de un sistema de imágenes de bioluminiscencia animal vivo para controlar la colonización de células y el crecimiento del tumor durante el transcurso de experimento (Figura 5A - B). La cuantificación de la señal bioluminiscente indicó que las células PC3M-Luc-C6 colonizados con éxito los...

Watch this Scientific Journal Video about Pre-clinical Orthotopic Murine Model of Human Prostate Cancer at JoVE.com

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Preclínica modelo murino ortotópico de cáncer de próstata humano

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

pre-clinical-orthotopic-murine-model-of-human-prostate-cancer

Research

JoVE Journal

Medicine

14.1K vistas.  VA Medical Center and UCSF. Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Video: Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

An Orthotopic Model of Murine Bladder Cancer

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

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

Un modelo ortotópico de cáncer de vejiga murino

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

Investigación

Medicina

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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

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

Combinación de radioterapia en un modelo ortotópico de tumor cerebral del ratón

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

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

El desarrollo de bronquiolitis obliterante en un modelo murino de trasplante de pulmón ortotópico

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Un modelo ortotópico de cáncer de vejiga de Estudios de liberación de genes

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

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

La formación del epitelio prostático humano utilizando tejido recombinación de roedores Urogenital mesénquima seno y células madre humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

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

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

Modelo ortotópico bioluminiscente de la progresión del cáncer de páncreas

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

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Evaluación previa a la clínica de inhibidores de tirosina cinasa para el tratamiento de la leucemia aguda

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

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

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

Un modelo murino de cáncer de próstata humano Metástasis ortotópico

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

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

El aislamiento de las células madre del cáncer de muestras de cáncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.