Authors thank Jeffrey Sellers to assist orthotopic pancreatic cancer mouse modeling. This work was supported by Research Initiative Pilot Awards from the Department of Radiology at UAB and NIH grants 2P30CA013148 and P50CA101955.

Todos los procedimientos fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Alabama en Birmingham. Modelado 1. ortotópico tumor pancreático Ratón <ol><li> Cultura líneas estándar de células de páncreas-cáncer humano en medio de Eagle modificado por Dulbecco (DMEM) suplementado con suero bovino fetal al 10%. Mantener todas las culturas a 37 ° C en atmósfera húmeda con 5% de CO 2. </li><li> Utilice 8-10 ratones graves femeninos semanas de edad combinados inmunodeficientes. Coloque jaulas de los animales a las 12 hr luz y oscuridad ciclo de 12 horas a RT (21 ± 2 ° C) y 60% de humedad. </li><li> Anestesiar a todos los animales utilizando la ventilación con 2% de isoflurano mezclado con oxígeno (2 l / min) a lo largo de la cirugía. Confirme la profundidad de la anestesia por reflejo pizca dedo del pie. Coloque los animales en una almohadilla térmica (37 ° C) para mantener la temperatura corporal. Aplique un ungüento oftálmico veterinario en los ojos para evitar la sequedad mientras que bajo anestesia. Y# 160; </li><li> Quitar el pelo en el cuadrante superior izquierdo del abdomen de cada ratón, y dar un fármaco analgésico (carpofen, 5 mg / kg de peso corporal por vía subcutánea) en la zona. Aplique una solución de betadine a la piel expuesta. Preparar instrumentos quirúrgicos esterilizados en autoclave. </li><li> Hacer una incisión de 1 cm en la piel y el peritoneo usando iris tijeras rectas. Retire con cuidado el páncreas desde el abdomen con unas pinzas quirúrgicas. </li><li> Insertar 28 G aguja de una jeringa 0,5 ml de insulina en la cola del páncreas y luego infundir lentamente una solución de 2,5 millones de células de cáncer pancreático humano en 30 l de DMEM. Confirme que una pequeña ampolla se crea en la cabeza del páncreas por la solución. </li><li> Coloque suavemente el páncreas en el abdomen con unas pinzas quirúrgicas. Cierre el peritoneo y la piel en la capa 1 con 2 interrumpido 5-0 suturas de Prolene, y luego terminar la anestesia. No devuelva un animal que ha sido sometido a cirugía para la compañía de otros animales hasta que se recupere completamente.60; Retire las suturas en 7 ~ 10 días después de la cirugía. </li><li> Dale otra dosis del fármaco analgésico (carpofen, 5 mg / kg de peso corporal por vía subcutánea) a las 24 horas después de la cirugía. </li><li> Compruebe el tamaño del tumor mediante la palpación de la zona de la cirugía usando dos dedos. Los tumores suelen sentirse más denso y más baches de los tejidos y órganos circundantes. Normalmente se tarda ~ 1 - 2 semanas para empezar a sentirse un tumor. </li><li> Monitorear los animales diariamente para detectar signos de enfermedad. Cuando los animales parecen enfermos (conductas de aseo personal y de evitación falta normal), nosotros los terminamos por dislocación cervical, mientras que bajo anestesia. </li></ol> 2. Imagen de Resonancia Magnética <ol><li> Aplicar MRI cuando el tamaño del tumor es cerca de 5 - 7 mm de diámetro en generalmente 2 ~ 4 semanas después de la implantación de células. Utilice un escáner de RM dedicada a imágenes de pequeños animales o un escáner de RM clínica equipada con una bobina especializada para obtener imágenes de pequeños animales. NOTA: Se utilizó un animal pequeño escáner de RM 9.4T con una combinación de un volumen del resonador 1 H/ Transmisor y un receptor bobina de superficie (30 mm de diámetro) (Bruker BioSpin Corp., Billerica, MA). Una bobina de superficie proporciona una mejor relación señal-ruido (SNR) 14. </li><li> Preparar una MRI agente de contraste a base de gadolinio para inyectar ~ 0,1 a 0,2 mmol / kg a cada animal en ~ 0,1 hasta 0,2 ml de PBS (tampón fosfato salino). NOTA: Se utilizó gadoteridol, y se inyectaron 0,2 mmol / kg en 0,15 ml de PBS durante un período de 15 seg (0,1 ml / seg). </li><li> Prepare un tubo de micro-polietileno (longitud: 7,62 mm, diámetro interior: 0,28 mm, diámetro exterior: 0,64 mm). Inserte una aguja 30 G (longitud de 12,7 mm) en un extremo del tubo, y una aguja de 30 G romo de la punta (9,5 mm de longitud) en el otro extremo. Conectar una jeringa de 1 ml que contiene agente de contraste MR a la aguja de punta roma, y ​​empujar lentamente la jeringa para llenar todo el tubo con el agente de contraste MR. </li><li> Anestesiar a los animales, utilizando ventilación con ~ 1 - 2% de isoflurano mezclado con oxígeno (2 L / min) durante la preparación y formación de imágenes. Confirme la profundidad de anesthesia por reflejo pizca dedo del pie. Aplique un ungüento oftálmico veterinario en los ojos para evitar la sequedad mientras que bajo anestesia. Dilatar la vena de la cola usando una lámpara de calor antes de la inserción de la aguja. Coge el medio de la aguja 30 G utilizando pinza de Kelly, y la inserta en la vena de la cola. Tape tanto la cola y el tubo en un pedazo de plástico o de papel cartón (10 mm de ancho x 100 mm de longitud) para mantener la recta cola. </li><li> Colocar el animal en posición supina en una cama de animales equipado con circulación de agua caliente (o aire caliente) para regular la temperatura corporal durante la exploración. Ajuste la temperatura en la cama a 37 ° C. Inserte una sonda de temperatura rectal para monitorear la temperatura del cuerpo durante la exploración. </li><li> Aplicar un tablero plástico ortogonalmente doblada en la zona abdominal. Asegúrese de que el tumor se localiza detrás del extremo superior de la placa, y luego tire hacia abajo el tablero ligeramente (~ 2 mm) para asegurarse de que el tumor se detecta por la junta. Tape el tablero a la cama de los animales con firmeza. </li><li> Pegue un transduce almohadilla respiraciónr (SA Instrument, Inc., Stony Brook, Nueva York) en el área del pecho para monitorear la respiración animal durante la exploración. Coloque una bobina de superficie en la parte superior de la región del tumor, y la cinta a la cama de los animales con firmeza. Empuje la cama de los animales en el escáner de RM para colocar la región del tumor en el centro de la bobina de volumen (diámetro interior: 72 mm). </li><li> La igualación y puesta a punto tanto para el receptor y el transmisor, seguido de calzas. </li><li> Comience con una secuencia de RM anatómica para localizar el tumor. Utilice una secuencia spin-echo (T2W) turbo T2 para obtener imágenes axiales con los siguientes parámetros de adquisición. Tiempo de repetición (TR) / tiempo de eco (TE) = 3000/34 mseg, 128 x 128 de matriz, 30 x 30 mm campo de visión, número de promedios = 1, longitud de los trenes de eco = 4, y 20 contiguos 1 mm en rodajas gruesas un modo entrelazado para cubrir la región entera del tumor (tiempo total de barrido: 1,6 min). NOTA: Dado que los tumores pancreáticos ortotópico son más difíciles que se encuentra que las subcutáneas, locali convencionalzer imágenes tienen una resolución más baja pueden no ser útiles. </li><li> Adquirir imágenes ponderadas en T1 (T1W) con varios ángulos de tirón para recuperar mapa T1. Para este propósito, usar un gradiente de eco enfoque ángulo MultiFlip con los siguientes parámetros: tiempo de repetición (TR) / tiempo de eco (TE) = 115/3 mseg, 128 x 128 de matriz, un campo de 30 x 30 mm de vista, el número de promedios = 4, ~ 5 - 7 1 mm de espesor cortes contiguos en un modo entrelazado para cubrir la región del tumor, y siete ángulos flip de 10, 20, 30, 40, 50, 60, y 70 (tiempo total de exploración por ángulo flip: 1 min). NOTA: Sin embargo, el enfoque ángulo MultiFlip es eficaz sólo cuando el campo B1 homogeneidad es alta. Si no es así, mapas de T1 se puede obtener con enfoque múltiple TR lugar 15. </li><li> Adquirir imágenes T1W antes y después de la inyección de contraste de gadolinio basada MR. Utilice los mismos parámetros de adquisición y la geometría para el mapeo T1 pero con el ángulo de rotación fija de 30. Usar codificación lineal para garantizar el estado de equilibrio cuando se obtiene el centro del espacio k, especially cuando se utilizan un corto TR y un ángulo bajo flip grado. Adquirir 5 imágenes de referencia antes de la inyección de contraste. Luego de adquirir 40 imágenes tras la inyección de contraste (tiempo total de barrido: 45 min). Utilice una bomba de jeringa para inyectar agente de contraste a una velocidad constante (0,01 ml / seg). </li><li> Controlar la respiración de los animales de forma continua, y ajustar la concentración de isoflurano para mantener la frecuencia respiratoria a 50-100 respiraciones por min. Controle la temperatura corporal del animal a lo largo de la imagen. </li><li> Después de completar DCE-MRI, quítese la aguja y otras sondas, y colocar al animal en una jaula vacía camas con toallas de papel. Masajear suavemente la zona inferior del abdomen. La jaula debe ser colocado un medio bajo una lámpara de calor para permitir que el animal se mueva dentro y fuera del gradiente de calor a medida que se recupera. No deje a un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. </li></ol> 3. Procesamiento y Análisis de Imágenes <ol><li> Regi tumorales Segmento<html> en las imágenes T2W. En imágenes T2W, la intensidad de la señal en la región del tumor es más brillante que la de los tejidos circundantes, por lo que el límite del tumor se puede delinear manualmente. NOTA: técnicas de segmentación semi-automáticas tales como umbralización global o contorno activo se pueden utilizar 16,17, pero la intensidad de fondo desigual deben corregirse especialmente cuando se utiliza una bobina de superficie. </li><li> Crear mapas de T1 y densidad de protones. En las imágenes adquiridas T1W con una secuencia de eco de gradiente, suponiendo que el tiempo de eco (TE) es mucho menor que T2 * valor, el valor de píxel se determina por <img alt="Ecuación 1" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq1.jpg" /> donde S 0 es la densidad de protones, T es T 1 1 constante de tiempo de relajación, TR es tiempo de repetición, y θ es un ángulo flip. La ecuación (1) puede ser reescrita para 641eq2.jpg &quot;/&gt; cuando S (θ) / sinθ se sustituye con Y, y S (θ) / tanθ se sustituye por X. La ecuación (2) es una ecuación lineal, y su pendiente y la intersección se puede utilizar para recuperar T1 y S 0 valores, respectivamente. </li><li> Calcular la concentración de contraste MR en imágenes DCE-MR. Cuando se inyecta medio de contraste MR basado gadolinio, T Tiempo 1 relajación constante se cambia con el tiempo. Así, la ecuación (1) puede ser reescrita para <img alt="Ecuación 3" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq3.jpg" /> T 1 (t) está relacionada con la concentración de contraste MR, C (t), como sigue, <img alt="Ecuación 4" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq4.jpg" /> donde r 1 es la capacidad de relajación longitudinal del agente de contraste MR. Así, mediante la combinación de las ecuaciones (3) y (4), la concentración de contraste MR se determina por <img alt = &quot;Ecuación 5&quot; fo: contenido-width = src &quot;3in&quot; = &quot;/ files / ftp_upload / 52641 / 52641eq5.jpg&quot; /&gt; </li><li> Cuantificar los parámetros farmacocinéticos de MR agente de contraste. C p (t) presenta la concentración de contraste MR en el plasma sanguíneo en el tiempo t después de iniciar la inyección de contraste. C p (t) se denomina función de entrada arterial (AIF). Si AIF está disponible, los parámetros farmacocinéticos de agente de contraste MR pueden calcularse <img alt="Ecuación 6" fo:content-width="4in" src="/files/ftp_upload/52641/52641eq6.jpg" /> donde C t (t) es la concentración de contraste MR en un tejido diana, p v es el volumen de plasma de sangre fraccionada, v es el volumen extracelular e extravascular fraccionada, y K trans es la transferencia de volumen constante. Constante de velocidad de flujo, k ep, es igual a K trans dividido por v e. Si AIF no está disponible, entonces el modelo de región de referencia puede utilizarse en lugar 18,19. El modelo de región de referencia se basa en el modelo de Kety limitado de flujo 20 y utiliza la concentración de contraste en una región de referencia para eliminar la necesidad de AIF de la siguiente manera, <img alt="Ecuación 7" fo:content-width="5in" src="/files/ftp_upload/52641/52641eq7.jpg" /> donde C t, ROI (t), K trans, ROI, y v e, ROI son la concentración de contraste, la transferencia de volumen constante, y el volumen extravascular extracelular-fraccionada, respectivamente, en la región de interés (ROI), mientras que C t, RR (t), K trans, RR, y v e, RR son aquellos en la región de referencia. Muscular paravertebral se selecciona a menudo como la región de referencia, y v e, RR en pa murinomuscular ravertebral se supone que es constante a 0,08 21. Se utilizó el modelo de región de referencia. </li></ol>

<ol>
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Authors do not have any conflicts of interest to declare.

Hemos introducido los métodos detallados de modelado tumor pancreático ortotópico utilizando ratones inmunodeficientes, DCE-MRI de los tumores abdominales en ratones, y la cuantificación de sus parámetros cinéticos. En el modelado tumor pancreático ortotópico, se debe tener cuidado cuando se inserta una aguja en la cola del páncreas. Si tiene éxito, las células se transfieren a la cabeza de páncreas creando una pequeña ampolla. Cuando se aplica una tabla de plástico ortogonalmente doblada, es fundamental p...

El objetivo general de este método consiste en aplicar un mayor contraste de imagen dinámica de resonancia magnética (RM-RT) para xenoinjertos de tumores pancreáticos ortotópico en ratones. DCE-MRI es un método no invasivo para evaluar la microvasculatura en un tejido diana mediante la supervisión del cambio de contraste MR durante un cierto período de tiempo después de la inyección. DCE-MRI se ha utilizado para diagnosticar los tumores malignos y para evaluar la respuesta del tumor a diversas terapias 1-4. Cuantitativa DCE-MRI ha presentado una alta reproducibilidad 5. Para cuantificar los parámetros farmacocinéticos de un agente de contraste MR en un tejido diana, todas las imágenes DCE-MR adquiridos en diferentes puntos temporales y mapa T1 obtenidas antes de la inyección de contraste deben ser corregistrados 6. Sin embargo, debido a los movimientos respiratorios y peristálticas en la zona abdominal, cuantitativa DCE-MRI ha tenido una aplicación limitada para los tumores gastrointestinales. Se han utilizado modelos de tumores de páncreas ortotópico para evaluarrespuesta de páncreas-tumor siguientes terapias biológicas y las quimioterapias 7,8. Modelos de tumores ortotópicos se consideran superiores a los modelos subcutáneos convencionales, puesto que el microambiente en el sitio del tumor original se refleja y de ese modo la respuesta del tumor a la terapia humana se puede predecir con más precisión. Sin embargo, el páncreas del ratón se encuentra en el cuadrante superior izquierdo del abdomen, por lo cuantitativo DCE-MRI de ortotópico xenoinjertos de tumores pancreáticos en ratones no se ha aplicado fácilmente. Hemos establecido un protocolo de DCE-MRI de los tumores abdominales en ratones mediante la fijación de los tumores usando un tablero plástico ortogonalmente doblada para evitar la transferencia de movimiento de la región del pecho 9. La presión aplicada por este tablero fue localizado en el área abdominal, y no ha dado lugar a dificultad respiratoria. Una técnica automatizada imagen coregistration ha sido validado para DCE-MRI de los órganos abdominales en un modo sin respiración, pero realiza effectively sólo cuando las regiones objetivo se mueve lenta y regularmente 10. La frecuencia respiratoria de los animales es variable durante la exploración, la restricción de manera física en la zona abdominal será necesario recuperar los parámetros farmacocinéticos fiables en modelos de ratón de tumores pancreáticos ortotópico. Hemos cuantificado con éxito los parámetros farmacocinéticos de un agente de contraste MR en ortotópico xenoinjertos tumorales pancreáticas utilizando el tablero plástico ortogonalmente doblada en DCE-MRI 11-13. Aquí presentamos el procedimiento detallado de modelado tumor pancreático ortotópico, DCE-MRI de los xenoinjertos de tumores en ratones, y la cuantificación de los parámetros farmacocinéticos.

<table border="0" cellpadding="0" cellspacing="0" width="1275">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:712px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Harlan Laboratories</td>
			<td>BT-9501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td style="width:199px;">Purdue products</td>
			<td style="width:119px;">67618-153-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Prolene sutures</td>
			<td style="width:199px;">Ethicon</td>
			<td style="width:119px;">8720H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">9.4T MR scanner</td>
			<td>Bruker Biospin Corporation</td>
			<td style="width:119px;">BioSpec 94/20 USR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Gadoteridol</td>
			<td>Bracco Diagnostics Inc</td>
			<td style="width:119px;">NDC 0270-1111-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-polyethelene tube</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">#PE-10-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G blunt tip needle</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">89134-194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring and gating system</td>
			<td style="width:199px;">SA instruments, Inc</td>
			<td style="width:119px;">Model 1030</td>
			<td>This is an MR compatiable system to measure resiratory rating and body temperature of small animals at the same time.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump</td>
			<td>New Era Pump Systems, Inc.</td>
			<td>NE-1600</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic contrast enhanced magnetic resonance imaging of an orthotopic pancreatic cancer mouse model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

Células tumorales pancreáticas humanas crecen con éxito en páncreas de ratón creando un tumor sólido. La figura 1 muestra fotografías de (A) un páncreas normal donde se inyecta solución de células de tumor, y (B) un ratón representativo que lleva un xenoinjerto de tumor de páncreas ortotópico (MIA PaCa-2 ). Tumor está localizado en el cuadrante superior izquierdo del abdomen, al lado del bazo. Por lo general toma 2-4 semanas para que los tumores crezcan hasta 5-7 mm de diámetro después d...

Watch this Scientific Journal Video about Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model at JoVE.com

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic Contrast mejorada de imagen de resonancia magnética de un modelo ortotópico de cáncer pancreático del ratón

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

dynamic-contrast-enhanced-magnetic-resonance-imaging-an-orthotopic

Research

JoVE Journal

Medicine

15.0K vistas.  University of Alabama at Birmingham. The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy. 

Video: Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Contraste de imagen mejorada del buque utilizando MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Investigación

Medicina

An Orthotopic Model of Murine Bladder Cancer

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

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

Un 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 ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotón imágenes de invasión de células tumorales en un modelo de ratón ortotópico de carcinoma oral de células escamosas

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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

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

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodal de imágenes de la angiogénesis en un modelo de rata desnuda de las metástasis óseas del cáncer de pecho utilizando imágenes de resonancia magnética, tomografía computarizada y la ecografía volumétrica

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modelo de ratón ortotópico de carcinoma de tiroides anaplásico

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

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 ...

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 ...

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  ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Simultánea de imágenes PET / RM Durante cerebral del ratón hipoxia-isquemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...