This research was supported by research grants RO1CA109545 and RO1CA086984 to M.S.S. by the National Institutes of Health/National Cancer Institute and by an award from the Leo and Ann Albert Charitable Trust (to M.S.S.).

Todos los estudios in vivo fueron aprobados por la Universidad de Notre Dame Cuidado de Animales y el empleo Comisión y utilizaron ratones C57 / BL6J femeninos. 1. Cultivo de células murino cáncer de ovario <ol><li> Hacer que el medio de cultivo ID8 murino de ovario de células de cáncer de la siguiente manera: 1 L de de Eagle modificado por Dulbecco (DMEM) suplementado con 4% de suero bovino fetal (FBS), 1% penicilina / estreptomicina, 5 mg / ml de insulina, 5 mg / ml de transferrina y 5 ng / ml selenito de sodio. </li><li> Transducir ID8 células de cáncer de ovario murino 13 para expresar la proteína roja fluorescente (RFP) utilizando partículas lentiviral adquiridos comercialmente que expresan RFP y un marcador de selección (gen blasticidin). Tenga en cuenta que la proteína fluorescente verde se puede utilizar, pero proporciona una señal de fluorescencia más débiles. <ol><li> Inmediatamente antes de la transducción, las células de cultivo de 50 - 70% de confluencia y añadir medio fresco (sin penicilina / estreptomicina) que incluye 1 ml de polibreno (5 mg / ml finalconcentración). </li><li> Suavemente la pipeta RFP-lentivirus (20 l) gota a gota en el plato y se incuba a 37 ° C durante 72 horas. Retire medio y se incuban con medio fresco durante 24 horas. Comience la selección de células transducidas mediante la adición de blasticidin al medio de cultivo (5 g / ml) y monitorear de células rojas usando microscopía de fluorescencia (Figura 1). </li></ol></li><li> Cultivar las células RFP-etiquetados en medio ID8 por la siembra de aproximadamente 10 6 células en una placa de cultivo de tejidos y la visualización de todos los días usando un microscopio de luz hasta que la monocapa de células confluentes es. </li><li> Cuando confluentes, cosechar las células utilizando tripsina (0,25%, 3 min) y regresar el matraz al 37 ° C incubadora hasta se separan las células. </li><li> Neutralizar la tripsina con 6 ml de medio de cultivo que contiene suero ID8. Pipetear arriba y abajo contra la parte inferior del plato, a continuación, transferir a un tubo cónico de 15 ml. Centrifugar a 1200 rpm durante 2 min, a continuación, aspirar el medio utilizando un glpipeta culo. </li><li> Lavar las células con solución salina tamponada con fosfato (PBS) resuspendiendo suavemente en 6 ml PBS caliente y centrifugando como en 1.5 anteriormente. Vuelva a suspender en PBS hasta una concentración final de 5 x 10 6 células / ml. Mantenga las celdas calientes (37 ° C) hasta la inyección. </li></ol> 2. intra-peritoneal inyección de células ID8 imagen in vivo y <ol><li> Inyectar cada ratón con 2 ml de la solución caliente ID8-PBS durante un total de 10 millones de células a través de la inyección intra-peritoneal (ip). Permiten que las células tumorales que crecen in vivo durante aproximadamente 10 semanas, con imágenes en vivo semanalmente. </li><li> Inmediatamente después de la inyección y cada semana a partir de entonces, pesan los ratones y luego anestesiar cada ratón utilizando isofluorano (velocidad de flujo de 2,5% en / min O flujo de 0,5 L 2). Nota: Los ratones se pesaron, y no como una medida cuantitativa de la carga tumoral, sino más bien una medida más general de la progresión física ratón individual durante todo el estudio. Los ratones pesanTS se compararon con sus propios valores de los pesos anteriores. <ol><li> Anestesiar a los ratones mediante la colocación de una cámara de isofluorano. Mantener la anestesia constante, mientras que la exploración mediante la colocación de la cabeza del ratón en la ojiva de la exposición a isofluorano caudal 2,5% durante todo el procedimiento. </li></ol></li><li> Use crema depilatoria para quitar el pelo en el torso y el abdomen de cada ratón, como el cabello negro hará que la atenuación de la señal de fluorescencia. Los ratones se bañan con agua tibia después de la aplicación de la crema depilatoria y se secaron con toallas de papel RT. </li><li> Si bien anestesiado, escanear todo el cuerpo de cada ratón utilizando un pequeño sistema de imagen óptica animal. Analiza todos los ratones en una cohorte en cada momento. Utilizar el protocolo siguiente paso dos (Figura 2): <ol><li> En el paso 1, para observar las células tumorales marcadas con fluorescencia (RFP), ejecutar la adquisición multiespectral para recoger un total de 5 imágenes utilizando filtros de excitación de 440 nm, 460 nm, 480 nm, 520 nm y 540 nm, y un filtro de emisión de 600 nm. Utilice los siguientes parámetros de adquisición: una exposición estándar de 15 segundos, 2 x 2 binning, FOV de 160 mm, y la abertura F de 1,1. </li><li> En el paso 2, para observar el animal entero, adquirir una imagen de reflectancia usando un filtro de excitación abierta (luz blanca), un filtro de emisión abierta, una exposición estándar de 0,2 segundos con 2 x 2 binning y un campo de visión de 16 cm. Nota: El tiempo de imagen real es ~ 1 min. </li></ol></li></ol> 3. Ratón La disección y Adquisición de imágenes <ol><li> Cuando la imagen en vivo muestra la presencia de la enfermedad intra-peritoneal o animales extensa acumulación de exposiciones de la ascitis (como se evidencia por la distensión abdominal), la pérdida o ganancia de peso corporal superior a 20%, letargo u otros signos de angustia, la eutanasia de cada ratón utilizando CO 2 anestesia seguido por dislocación cervical. </li><li> Cortar la piel anteriormente a lo largo de la línea media ventral del ratón, utilizando puntas dissection tijeras, desde la parte superior de los muslos de la parte inferior de la caja torácica. Tenga mucho cuidado al cortar a través de sólo la capa de la piel (Figura 2Bi). separar suavemente la piel del tejido peritoneal, asegurándose de no romper la pared peritoneal. </li><li> Con unas tijeras de disección puntiagudas, cortados lateralmente tanto a lo largo de la parte inferior de la caja torácica y la parte superior de los muslos para crear dos colgajos de piel (Figura 2B ii, iii). </li><li> Colocar cada ratón en su lado ventral (cavidad peritoneal mirando hacia abajo) con los colgajos de piel abrió y escanear cada ratón con sus órganos in situ utilizando el pequeño sistema de formación de imágenes ópticas de los animales (Figura 3A, B). <ol><li> Utilizar los mismos parámetros de adquisición de imágenes como se describe en 2.4.1.-2.4.2. </li></ol></li><li> Continuar la disección del ratón mediante la extracción de órganos peritoneales individuales incluyendo el hígado, epiplón / páncreas, estómago, diafragma, intestino delgado, intestino grueso, izquierda y derecha peritoneo, ovarios, kidneys, mesenterio y la almohadilla de grasa. <ol><li> Observar, enumerar y registrar los tumores visibles en cada órgano. Utilice un calibre para medir el diámetro de cada tumor. </li><li> Designar los tumores de menos de 2 mm de diámetro tan pequeño, en entre 2 y 5 mm como medio y superior a 5 mm de grande. </li></ol></li><li> Colocar los órganos en la plantilla de exploración de órganos (Figura 4) que identifica una posición predeterminada para cada órgano. Utilice PBS para mantener los órganos húmedo. </li><li> Analiza cada hoja de órganos utilizando el pequeño sistema de formación de imágenes ópticas de los animales (Figura 3 C, D). <ol><li> Utilizar los mismos parámetros de adquisición de imágenes como se describe en el paso 2.4. </li></ol></li><li> Después de la exploración, lugar órganos en formol al 10% para permitir el tratamiento posterior para la histología. </li></ol> 4. La cuantificación de la carga tumoral en THImágenes de fluorescencia de correo <ol><li> Con el fin de reducir la cantidad de auto-fluorescencia en cada exploración multiespectral, primero unmix los archivos usando software disponible con el sistema de imagen pequeña animal. <ol><li> Cargar en primer lugar la solicitud de ofertas: En Vivo ficha espectral, seguido por el Autofluor: En Vivo archivo de rojo en la ventana Imágenes sin mezclar y seleccione Deshacer mezcla. </li></ol></li><li> Exportar los archivos .bip como formato de 16 bits .tiff (sin escala). </li><li> Utilice el software ImageJ (ex vivo órganos y luego el cuerpo completo del ratón con órganos in situ). <ol><li> Utilice Archivo&gt; Abrir para abrir los archivos .tiff 16 bits de los archivos de plantilla de exploración de órganos en ImageJ. </li><li> Bajo Imagen&gt; Ajustar&gt; Brillo / Contraste, optar por establecer el mínimo valor visualizado a 0 y el máximo valor visualizado a 35.353 y luego bajoImagen&gt; tablas de búsqueda, seleccionar candente. Nota: Los distintos modelos animales se requieren diferentes niveles de brillo, pero es importante para mantener el brillo constante a través de un estudio determinado. </li><li> Con la herramienta Selección a mano alzada, dibujar una región de forma libre de la selección interés (ROI) alrededor de cada órgano y utilizar la herramienta Medición (CTRL + M) para calcular la superficie de cada órgano; grabar la superficie de cada órgano en una hoja de cálculo. </li><li> Con el retorno de la inversión dibujado alrededor de cada órgano, haga clic derecho en el ROI y seleccione Duplicar para incluir sólo el órgano. </li><li> Mientras observa el órgano individual, en virtud de Imagen&gt; Ajustar&gt; Umbral, optar por establecer el umbral de la imagen a un nivel inferior del umbral de + 1200 y un umbral de nivel superior de + 1700 para seleccionar ONLy las regiones más brillantes y fluorescentes de verificación para seleccionar el fondo oscuro; seleccione Aceptar. Nota: Las imágenes pueden requerir la manipulación manual de los controles deslizantes de umbral en ImageJ manera que las áreas más brillantes son seleccionados previamente para la etapa de análisis, como se muestra arriba. Diferentes modelos de enfermedad se requieren diferentes limitaciones de umbral, pero es importante para mantener los niveles de umbral consistentes a lo largo de un determinado estudio. </li><li> Bajo Análisis&gt; Análisis de Partículas, cambiar el tamaño (píxeles ^ 2) a 10-Infinity, optar por mostrar: contornos, comprobar para seleccionar sólo &quot;Mostrar resultados&quot; y haga clic en OK para medir tanto las áreas y densidades integradas primas de estas regiones brillantes, Los tumores que se consideren. </li><li> Registre los valores de la zona de superficie y la densidad aparente Integrado de cada selección del tumor que aparece en el <strong&gt; tabla de resultados en la misma hoja de cálculo con las superficies de órganos. </li><li> Bajo Análisis&gt; Herramientas&gt; Calibración de bar, añadir una barra de escala de calibración de las imágenes de los órganos. Nota: En este ejemplo, esto se hace cambiando la ubicación de Arriba a la derecha, el color de relleno de Negro, la etiqueta de color al blanco, el número de etiquetas a 5, el número de decimales a 0, el tamaño de fuente a 12 y el zoom factor a 4.0 y que permite el texto en negrita. </li><li> En Archivo&gt; Guardar como ..., salvo el montaje como .tiff y .jpeg. </li><li> Utilice Archivo&gt; Abrir para abrir el archivo .jpeg de los órganos y luego en Insertar&gt; Cuadro de texto </strong&gt;, añadir etiquetas de órganos mediante la creación de un cuadro de texto por debajo de cada una de las tres filas de órganos. </li><li> Utilice Archivo&gt; Abrir para abrir cada uno de los archivos .tiff 16 bits de los escáneres de cuerpo completo en ImageJ en orden de número de ratón. </li><li> Bajo Imagen&gt; Ajustar&gt; Brillo / Contraste, optar por establecer el valor mínimo que se muestra a 0 y el máximo valor visualizado a + 35353 a continuación, en Imagen&gt; tablas de búsqueda, seleccionar candente. </li><li> En Archivo&gt; Guardar como ..., guardar el montaje como un. TIFF y .jpeg. </li></ol></li></ol> Análisis 5. Datos <ol><li> Añadir las áreas de selección de tumores y densidades integradas primas, respectivamente, para cada órgano de cada ratón y registrar el área total del tumor de órganos para cada ratón. </li><li> Normalizar el área de grabado y integrat primalos valores de densidad ed para el tamaño de cada órgano dividiendo el área total del tumor y los valores de densidad integradas primas totales por el área de superficie del órgano correspondiente (Tabla 1). </li><li> Calcular y representar gráficamente la media de ambas las áreas de superficie tumor normalizados y los valores de densidad integrados bruto normalizado (Figura 5A, B). </li><li> Compare estos valores medios a los obtenidos mediante el recuento visual de los tumores que eran visibles a simple vista durante la inspección de cada órgano para lesiones metastásicas. </li></ol>

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Este artículo es parte de una edición especial sobre Imaging Multimodal Preclínica, patrocinado por Bruker Biospin.

En contraste con los estudios que utilizan las células de cáncer de ovario humanos que deben ser realizados en ratones inmunodeficientes, el protocolo descrito anteriormente utiliza los ratones inmunocompetentes C57 / Bl6 y las células de cáncer de ovario murinos singénicos. Si bien esto permite la evaluación de la posible función de los infiltrados inmunes en la progresión tumoral y la metástasis, la presencia de pelo oscuro en la superficie abdominal hace menos sensible de formación de imágenes. El uso de u...

Cáncer de ovario epitelial (EOC) es la causa más común de muerte por neoplasia ginecológica, con un estimado de 21.290 nuevos diagnósticos en los EE.UU. en 2015 y un estimado de 14.180 muertes 1. La gran mayoría (&gt; 75%) de las mujeres son diagnosticadas con enfermedad en etapa tardía (estadio III o IV) caracteriza por metástasis difusa intra-peritoneal y mal pronóstico. Recurrencia de la enfermedad en la cavidad peritoneal después de la quimioterapia de primera línea también es común y representa una causa importante de mortalidad 2,3. EOC metástasis por un mecanismo único que implica tanto la extensión directa del tumor primario a los órganos peritoneales vecinos, así como por la disociación o desprendimiento de las células de la superficie del tumor primario como células individuales o agregados multicelulares. Las células se desprenden en la cavidad peritoneal, en el que se resisten a la apoptosis inducida por el desprendimiento-4. La acumulación de ascitis peritoneal es común, como las células tumorales arrojar bloquean el drenaje linfático peritoneal y tumors producen factores de crecimiento que alteran la permeabilidad vascular. Una parte de las células tumorales cobertizo se adhieren a la superficie de los órganos peritoneales y estructuras, incluyendo el intestino, el hígado, epiplón y mesenterio, después de lo cual se anclan y proliferan para producir múltiples lesiones secundarias ampliamente difundidos 3,5. metástasis hematógena es infrecuente. Por lo tanto, el manejo clínico consiste comúnmente de la cirugía citorreductora incluyendo &quot;citorreducción óptima&quot;, definida como la resección de todo el tumor visible (no importa cuán pequeño). Citorreducción completa se asocia con un aumento significativo en la supervivencia global 6,7 y se asocia con el reto de la identificación y eliminación de las lesiones &lt;0,5 cm. los pequeños modelos animales han demostrado utilidad en la investigación del cáncer de ovario en la mejora de nuestra comprensión de la evolución de la enfermedad, así como en la identificación de biomarcadores pronósticos y ensayo de nuevas quimioterapias o aproximaciones de la terapia de combinación. A medida que la primariasitio de la incidencia de cáncer de ovario y de la metástasis es la cavidad peritoneal, modelos ortotópico de EOC metástasis implica el análisis y caracterización de la enfermedad intraperitoneal. Aunque ha habido recientes mejoras en la capacidad de las células tumorales de imagen, incluso a nivel de células individuales, aún existen dificultades significativas en la cuantificación de la carga tumoral metastásica del COE. Estos retos surgen debido a la cantidad, el tamaño y la localización anatómica de las lesiones metastásicas. Además existe una necesidad de células de cáncer de etiqueta para distinguirlas de las células huésped normales. Estudios anteriores han utilizado protocolos de etiquetado a base de anticuerpos o transfección de células tumorales con 8,9 luciferasa. El marcaje fluorescente directa de las células cancerosas se informó por primera vez por Chishima y compañeros de trabajo en 1997 10. Los marcadores fluorescentes no requieren la adición de sustrato exógeno y proporcionan exquisita especificidad de las células tumorales, proporcionando un medio más eficaz para rastrear la metástasis del cáncer 11,12 </sup&gt;. Aquí se describe un método de formación de imágenes ópticas para el análisis cuantitativo de la enfermedad metastásica utilizando un modelo de xenoinjerto ortotópico singénico compuesto por proteína fluorescente roja (RFP)-etiquetados murino ID8 células de cáncer de ovario 13 y C57 / BL6 ratones inmunocompetentes. Se demuestra un nuevo método de cuantificación relativa de la carga tumoral combinando in vivo y ex vivo de formación de imágenes con la eliminación de tejido auto-fluorescencia. Este enfoque tiene utilidad potencial en estudios diseñados para evaluar el efecto de específico genética, epigenéticos o micro-ambientales modificaciones y / o modalidades de tratamiento en la metástasis de órganos específicos de cáncer de ovario.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:183px;">Corning</td>
			<td style="width:117px;">10-014-CM</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin/streptomycin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Insulin-transferrin-sodium selenite media supplement</td>
			<td>Sigma</td>
			<td>I-1884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Xtreme small animal imaging system</td>
			<td>Bruker Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Multispectral software</td>
			<td>Bruker Corp</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">lentiviral particles with Red fluorescent protein</td>
			<td>GenTarget, Inc.</td>
			<td>LVP023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin for cell culture</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">depilatory cream (such as Nair Hair Remover Lotion)</td>
			<td>purchases from drugstore&#160;</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software&#160;</td>
			<td>http://imagej.nih.gov/ij/&#160;</td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (forceps)</td>
			<td>Roboz Surgical Instrument</td>
			<td>&#160;RS 5130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (Scissors)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS 5910</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitation of intra-peritoneal ovarian cancer metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

El mecanismo metastásica de cáncer de ovario se caracteriza por metástasis intra-peritoneal altamente difusa compuesta de numerosas lesiones de tamaño variable, incluyendo múltiples (&lt;2 mm) lesiones pequeñas. Por lo tanto, el uso de células tumorales RFP marcado (Figura 1) y de formación de imágenes óptico proporciona un método alternativo para el conteo manual y la medición de tamaño de la lesión. El desarrollo de la carga tumoral con el tiempo se puede...

Watch this Scientific Journal Video about Quantitation of Intra-peritoneal Ovarian Cancer Metastasis at JoVE.com

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

La cuantificación de la intra-peritoneal del cáncer ovárico Metástasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

quantitation-of-intra-peritoneal-ovarian-cancer-metastasis

Research

JoVE Journal

Medicine

10.8K vistas.  University of Notre Dame. Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Video: Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Ensayo experimental de metástasis

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Investigación

Medicina

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Análisis cuantitativo de la metástasis del cáncer utilizando un modelo de embrión aviar

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ensayo in vitro Liquidación mesoteliales que modela los pasos iniciales de la metástasis del cáncer ovárico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Los modelos de metástasis en los huesos

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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  ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para la obtención de las células del cáncer de ovario primarios a partir de muestras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Evaluación de Cáncer de Ovario esferoide El apego y la invasión de células mesoteliales en Tiempo real

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modelo murino de imagen no invasivas para detectar y vigilar cáncer ovárico de Recurrencia

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

El enriquecimiento de las células madre quimiorresistente cáncer ovárico de líneas celulares humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

En Enriquecimiento vitro de células iniciadoras del tumor del cáncer de ovario

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...