Este trabajo es apoyado en parte por la Asociación Americana del Corazón GRA Primavera 09 Postdoctoral Fellowship (09POST2250268) y el NIH R01 CA097465. Apreciamos altamente Dr. Wen Li y el Dr. Vikas Gulani para la prueba de protocolo de la RM y la configuración, y la Sra. Yvonne Parker por su asistencia en la implantación del tumor.

Protocolo de adaptación de un estudio previo 11. 1. Conjugación de Gd-DOTA para CLT1 Péptido <ol><li> Uso de la síntesis de péptidos en fase sólida estándar, sintetizar CLT1 péptido (CGLIIQKNEC) a partir de aminoácidos protegidos con Fmoc en una resina de cloruro de 2-clorotritilo (1,0 mmol). </li><li> Después de la adición de aminoácido final, ciclar el péptido lineal en-resina con talio (III) trifluoroacetato (1,09 g, 2,0 mmoles, 2 equivalentes) en DMF (20 ml) a 0 º C durante 2 h. </li><li> Siguiente, conjugado de PEG y lisina secuencialmente a la N-terminal de CLT1 péptido (1,0 mmoles) en resina haciendo reaccionar secuencialmente con Fmoc-NH-PEG-COOH (3,0 mmoles), Fmoc-Lys (Fmoc)-OH (3,0 mmoles), y un segundo lote de Fmoc-Lys (Fmoc)-OH (6 mmol). </li><li> Eliminar Fmoc con piperidina. Añadir DOTA-tris (t-Bu) (4,58 g, 8,0 mmoles, 2 equivalentes a cada grupo amino) para reaccionar con los cuatro aminas libres de la dendrímero de lisina en la resina durante 2 horas. </li><li> Finalmente, escindir y desproteger el productode la resina por tratamiento con un cóctel de ácido trifluoroacético, agua, y triisobutylsilane (TIS) (20 ml, 95/2.5/2.5) durante 8 h a TA. Eliminar la resina por filtración seguido de lavado con ácido trifluoroacético (TFA). Añadir los filtrados combinados gota a gota a éter frío de etilo (200 ml), se centrífuga, y se lavan con éter etílico 4x. Secar al vacío para dar un sólido incoloro (1,99 g, rendimiento del 61%). </li><li> Se purifica el producto bruto por HPLC preparativa usando un gradiente de 0-40% de disolvente B (0,1% de TFA en acetonitrilo) en disolvente A (solución acuosa de TFA al 0,1%) durante 20 min y 40-90% de disolvente B en disolvente A durante 10 min . </li><li> Disolver CLT1-dl-(DOTA) 4 (250 mg, 0,077 mmol) en agua DI (15 ml) y ajustar el pH a 6 con 1 M de NaOH. Añadir Di-s (OAc) 3 0,4 H 2 O (472 mg, 0,462 mmol, 1,5 equivalentes a DOTA monoamida) en porciones a la solución, mientras se mantiene el pH a 6 con 1 M de NaOH. Se agita la solución de reacción a TA durante 48 h. </li><li> Complejo Gd residual (III) con el correoácido thylenediaminetetraacetic (EDTA) (90 mg, 0,31 mmoles), y purificar el producto crudo con una columna de exclusión por tamaño para dar el producto final CLT1-dl-(Gd-DOTA) 4 (169 mg, 57%). </li></ol> 2. Caracterización de los agentes de contraste <ol><li> Mida Gd (III) el contenido con la espectroscopia de emisión óptica con plasma acoplado inductivamente. </li><li> Adquirir láser de desorción / ionización espectros de masas asistida por matriz de tiempo de vuelo (MALDI-TOF) en el modo lineal con ácido 2,5-dihidroxibenzoico (2,5-DHB) como una matriz. </li><li> Medir los tiempos de relajación de la solución acuosa de los agentes de contraste de CLT1-dl-(Gd-DOTA) 4 y un agente de control de nontargeted sCLT1-dl-(Gd-DOTA) 4 con diferentes concentraciones a 60 MHz (1,5 T) con un relaxometer a 37 ° C. Utilice una secuencia de pulsos de inversión-recuperación para medir T 1 y una secuencia de Carr-Purcell-Meiboom-Gill con 500 ecos para medir T 2. Calcula T 1 y T 2 relaxividades de thagentes e de las laderas de las parcelas de 1 / T 1 y 1 / T 2 frente a las concentraciones de D-os. </li></ol> 3. Cultivo Celular y Desarrollo del Modelo Animal con ortotópico PC3 próstata Tumor <ol><li> PC3 Cultura células de cáncer de próstata con la expresión constitutiva de la proteína de fluorescencia verde (GFP) en medio RPMI suplementado con 5% de suero bovino fetal y penicilina / estreptomicina / fungizona, la cosecha por tripsinización y se resuspende a una densidad de 2,5 x 10 4 células / l de PBS. </li><li> Mantener los ratones machos NIH atímicos desnudos (4-5 semanas de edad) en la Instalación Athymic Animal Core, de la Universidad Case Western Reserve (CWRU) de acuerdo con un protocolo de los animales, aprobado por el Comité de Cuidado de Animales y el empleo Institucional CWRU. </li><li> Las superficies quirúrgicas se pulverizaron con una solución de clorhexidina al 2% y luego se cubre con cortinas absorbentes. Guantes quirúrgicos estériles fueron usados ​​durante el procedimiento. Antes de la cirugía los animales, IP inyectar Avertin (250 mg / kg) a anesthetize los ratones. Alternativamente, la ketamina / xilazina / acepromazina se puede utilizar a una concentración de 50/5/1 mg / kg. Pomada oftálmica se aplicó a la del ratón para mantener la humedad adecuada durante la anestesia. </li><li> Iniciar la cirugía mediante la reducción de una pequeña incisión a través de la piel y el peritoneo a lo largo de la línea media inferior a una longitud de aproximadamente 1 cm, utilizando un bisturí estéril. Un paño quirúrgico estéril se cubrió alrededor del sitio de la incisión durante las cirugías de supervivencia. </li><li> Exteriorizar suavemente y estabilizar los lóbulos dorsales próstata para exponer la próstata. Se inyecta la suspensión de células PC3-GFP en PBS (20 l) en la próstata con una aguja de calibre 30. Finalmente cerrará la incisión con herida autoclip 12. </li><li> Para el monitoreo post-procedimiento, observar a los animales dos veces al día durante 1 semana. Los signos de la enfermedad son la falta de alimentación, la recogida de orina de color oscuro alrededor de la uretra y la marcha inestable, que podría indicar una infección u obstrucción de la vejiga en las grapas. </li><li> Después de 10 días, retire tél grapas utilizando un removedor de grapas. Monitorear los animales diariamente para evaluar el tamaño del tumor y cualquier evidencia de dolor, como la pérdida de peso, problemas de conducta, y los defectos de motor. La eutanasia a los animales que mostraron un tamaño del tumor superior al límite permitido éticamente o evidencia de dolor durante el crecimiento del tumor. </li></ol> 4. La confirmación de un tumor especificidad de unión de los péptidos con Fluorescencia de Imagen e Histología <ol><li> Después de la inoculación de células tumorales, permitir que aproximadamente 4 semanas de crecimiento del tumor antes de comenzar con la formación de imágenes. Antes de la inyección de las sondas de péptidos, adquirir imágenes de fluorescencia de GFP con ratones vivos en un generador de imágenes de fluorescencia para verificar la presencia de tumor. </li><li> Iv inyectar Rojo Texas péptidos marcados a la ratones portadores de tumor a una dosis de 10 nmol / ratón. </li><li> Sacrificar los ratones 2 horas más tarde por dislocación cervical, recoger el tumor y los órganos principales, y de inmediato la imagen de un generador de imágenes de fluorescencia. Utilice filtros de luz verde para GFP (excitación: 444-490 nm; emisiones: filtro de paso largo nm 515; ajustes de adquisición: 500 a 720 en pasos de 10 nm) y filtros de luz roja de Texas Red (excitación: 576-621 nm; emisiones: filtro de paso largo nm 635, la adquisición ajustes: 630-800 en 10 pasos nm). 10 ms tiempo de exposición de las buenas prácticas agrarias y 150 ms para Rojo Texas. </li><li> Inmediatamente después de la medición de la fluorescencia del tejido conjunto, recoger los tejidos tumorales, fijar con formalina, y criosección en rodajas de 5 micras. </li><li> Después de enjuagar con PBS, montar con 1 gota de medio de montaje que contiene 4 &#39;,6-diamino-2-fenilindol (DAPI), imagen inmediatamente en un microscopio láser confocal de barrido. </li></ol> 5. Imagen de Resonancia Magnética (MRI) <ol><li> Aproximadamente 4 semanas después de la inoculación de las células tumorales, permiten que los tumores crezcan hasta 0,3 hasta 0,6 cm de diámetro. Utilice un escáner 7 T MRI con un volumen de la radio frecuencia (RF) de la bobina para la RM estudio. </li><li> Anestesiar el ratón con una mezcla de isoflurano en oxígeno del 2% en una cámara de inducción con isoflurano. Ophthalmic ungüento se aplicó a la del ratón para mantener la humedad adecuada durante la anestesia. </li><li> Inserte una aguja de calibre 30 en un largo tubo de 1,0 m lleno de solución salina heparinizada. Coloque el catéter hecho a sí mismo en la vena de la cola del ratón. </li><li> Coloque el ratón en el imán y mantener bajo anestesia por inhalación con un 1,5% de isoflurano en oxígeno a través de un cono de la nariz. </li><li> Coloque un sensor respiratorio conectado a un sistema de monitoreo en el abdomen para controlar la frecuencia y profundidad de la respiración. Mantener la temperatura del cuerpo a 37 ° C por soplado de aire caliente en el imán a través de un sistema de control de realimentación. </li><li> Comience con la sección de imágenes sagital utilizando una secuencia de localización para identificar la ubicación del tumor (TR / TE = 200/3.7 ms, FOV = 3,0 cm, grosor de corte = 2,2 mm, rebanada número = 16, promedio = 2, flip = ángulo de 45 °, matriz = 128 x 128). </li><li> Utilice una secuencia de supresión de grasa gradiente potenciada en T1 2D para obtener imágenes axiales 2D para CE-MRI (TR / TE = 151.2/1.9 ms, FOV = 3,0 cm x 3,0 cm, sespesor piojos = 1,2 mm, número de segmento = 12, promedio = 1, flip = ángulo de 80 °, la matriz = 128 x 128). </li><li> Después de la adquisición de imágenes MR de línea de base previa a la inyección, comenzar a inyectar el agente vectorizado o el agente de control a una dosis de 0,03 mmol Gd / kg por lavado con 200 l de solución salina. </li><li> Seguir adquiriendo imágenes CE-MRI en diferentes momentos durante un máximo de 30 min. </li></ol> 6. Procesamiento y Análisis de Imágenes <ol><li> Utilice el software de imágenes para análisis de imágenes. </li><li> Dibuje las regiones de interés (ROI) en todo el tumor y los riñones en el plano de la imagen bidimensional y medir la intensidad media de la señal. </li><li> Para cuantificar los datos CE-MRI, medir tumor o aumento del contraste de los riñones (ΔSNR) mediante el cálculo del incremento de la post-contraste SNR sobre pre-contraste SNR usando la siguiente ecuación: ΔSNR = (S t / σ t) - (S 0 / σ 0), donde S 0 y S t denotan la señal en la tumo o los riñones antes y después de contraste, y σ 0 y σ t son la desviación estándar del ruido medido desde el fondo de aire antes y después de contraste. </li><li> Calcular los valores p usando la prueba t de dos colas del estudiante, asumiendo la significación estadística de p &lt;0.05. </li></ol>

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No hay conflictos de interés declarado.

Pasos críticos La selección de biomarcadores adecuada y focalizada en los pequeños Péptido Para desarrollar con éxito un agente de contraste objetivo con el tamaño pequeño, dos puntos clave deben ser considerados. En primer lugar, es importante seleccionar biomarcadores moleculares adecuados que son abundantemente presente en los tejidos enfermos con poca presencia en los tejidos normales. Nuestro biomarcador relacionado con el cáncer se sel...

De formación de imágenes eficaz de dianas moleculares relacionados con el cáncer es de gran importancia para mejorar la precisión de la detección del cáncer antes y diagnóstico. La resonancia magnética (MRI) es una poderosa técnica de imagen clínica con alta resolución espacial y no la radiación de ionización 1. Sin embargo, ningún agente de contraste específico está disponible para la imagen molecular del cáncer MR clínica. El diseño innovador y el desarrollo de agentes de contraste de resonancia magnética dirigidos avanzarían en gran medida la aplicación de la imagen molecular del cáncer de MR. Se han hecho esfuerzos significativos para desarrollar agentes de contraste dirigidos para formación de imágenes MR de los biomarcadores expresados ​​en la superficie de las células cancerosas. Debido a la relativamente baja sensibilidad de la RM y la baja concentración de estos biomarcadores, es un reto para generar la mejora del contraste suficiente para MR efectiva de imagen molecular usando pequeños agentes de contraste diana molecular 2,3. Con el fin de obtener la mejora suficiente, varios sistemas de administración de such como liposomas, nanopartículas y conjugados de polímeros con una alta carga útil de paramagnéticas Gd (III) quelatos se han preparado para aumentar la concentración local de agentes de contraste en los sitios diana de 4,5. Aunque estos sistemas de suministro fueron capaces de generar mejora significativa del tumor en modelos animales, sus grandes tamaños resultaron en eliminación lenta e incompleta desde el cuerpo, resultando en la acumulación prolongada de Gd (III) iones tóxicos, que pueden causar graves problemas de seguridad 6. Recientemente, algunos estudios han demostrado que las limitaciones de la RMN para imágenes moleculares se pueden superar mediante la selección de biomarcadores moleculares adecuados con alta expresión local en las lesiones y el uso de pequeños agentes moleculares que pueden ser excretados fácilmente 7,8. La característica clave de estos agentes es que se dirigen a los marcadores moleculares abundantemente presentes en los tejidos enfermos con poca presencia en los tejidos normales. Una alta concentración de agentes de contraste se puede unir a estos objetivos, lo que resulta en sufiaumento del contraste ciente para imagen molecular MR eficaz. Dado que su tamaño es menor que el umbral filtración renal, agentes de contraste no unidos pueden ser fácilmente excretados del cuerpo con un menor ruido de fondo. Hemos seleccionado un biomarcador relacionado con el cáncer universales, las proteínas del plasma coagulado, que existen en abundancia en el estroma del tumor, y no suelen estar presentes en los tejidos normales 9. Se sintetizó un agente de contraste diana que contiene un pequeño péptido de direccionamiento CGLIIQKNEC (CLT1), que mostró una fuerte unión específica para el modelo de tumor de próstata PC3 10, y cuatro quelatos de monoamida Gd-DOTA. En este caso, se propone una metodología para el cáncer de RM molecular para detectar tumores en ratones.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Fmoc protected amino acids</td>
			<td>EMD Chemicals Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DOTA-tris(t-Bu)</td>
			<td>TCI America</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PyBOP, HOBt, HBTU</td>
			<td>Nova Biochem</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DIPEA, Thallium(III) trifluoroacetate, TIS</td>
			<td>Sigma-Aldrich Corp.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Texas Red, succinimidyl ester, single isomer</td>
			<td>Invitrogen</td>
			<td>T20175</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENTS</td>
		 </tr>
		 <tr>
			<td>Agilent 1100 HPLC system</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ZORBAX 300SB-C18 PrepHT column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ICP-OES Optima 3100XL</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>AutoflexTM Speed</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maestro FLEX In Vivo Imaging System</td>
			<td>Cambridge Research &amp; Instrumentation, Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biospec 7T MRI scanner</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

mr molecular imaging of prostate cancer with a small molecular clt1 peptide targeted contrast agent

Matriz extracelular del tumor tiene abundancia de proteínas relacionadas con el cáncer que se pueden utilizar como biomarcadores para el diagnóstico por imagen molecular del cáncer. En este trabajo, hemos demostrado el cáncer de RM molecular eficaz con un pequeño péptido molecular dirigida Gd-DOTA complejo monoamida como objetivo MRI agente de contraste específico a las proteínas plasmáticas coagulada en el estroma tumoral. Se realizó el experimento de evaluación de la eficacia del agente para la detección no invasiva de tumor de próstata con la RM en un modelo ortotópico de cáncer de próstata PC-3 de ratón. El agente de contraste diana era eficaz para producir la mejora del contraste significativa del tumor a una dosis baja de 0,03 mmol Gd / kg. El péptido agente de contraste de resonancia magnética específica es prometedor para la RM molecular del tumor de próstata.

La Figura 1 muestra la síntesis del agente de contraste dirigidos CLT1-dl-(Gd-DOTA) 4 y el esquema general del experimento. CLT1-dl-(Gd-DOTA) 4 muestra la capacidad de relajación mucho más alto que el Gd-DOTA clínica (Tabla 1). A 1,5 T, la capacidad de relajación T 1 por gadolinio de CLT1-dl-(Gd-DOTA) 4 en PBS (pH 7,4) es de aproximadamente 3 veces mayor que la de Gd-DOTA 10. Maestro de imagen confirma la fuerte unión específica de Rojo Texas...

Watch this Scientific Journal Video about MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent at JoVE.com

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Para demostrar la imagen molecular del cáncer de MR con un pequeño péptido agente de contraste de MRI específica a las proteínas plasmáticas coagulados en estroma tumoral en un modelo de cáncer de próstata de ratón dirigido.

MR Imagen Molecular del cáncer de próstata con un agente de contraste dirigidos Pequeño Molecular CLT1 Péptido

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

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JoVE Journal

Medicine

11.1K vistas.  Case Western Reserve University.  Para demostrar la imagen molecular del cáncer de MR con un pequeño péptido agente de contraste de MRI específica a las proteínas plasmáticas coagulados en estroma tumoral en un modelo de cáncer de próstata de ratón dirigido. 

Video: MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

En vivo en el infrarrojo cercano fluorescencia (NIRF) imagen intravascular molecular de placa inflamatoria, un enfoque multimodal de imágenes de la aterosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Investigación

Medicina

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 ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluación de la absorción de nanopartículas en los tumores en tiempo real utilizando intravital imagen

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Imagen Molecular a Target trasplantó células progenitoras musculares

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

En vivo los macrófagos de imágenes utilizando MR Agente de contraste dirigida para la Evaluación Longitudinal de Artritis séptica

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages 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 ...

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.

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 ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generación del paciente con cáncer de próstata Derivado modelos de xenoinjertos de células tumorales circulantes

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Un tumor cerebral / organotípicos rebanada Co-cultura del sistema para estudiar microambiente tumoral y dirigida Medicamentos Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...