Nos gustaría dar las gracias a la Nikon Imaging Center de la Harvard Medical School, en concreto las aguas de Jennifer, Lara Petrak y Salmón Wendy, para la formación y el uso de microscopios sus timelapse. También nos gustaría dar las gracias a Rosa Ng y Besser Achim valiosa para los debates. Este trabajo fue apoyado por el NIH Grant 5695837 (a M. Iwanicki) y GM064346 de ACC, por una beca de la Dra. Miriam y Sheldon G. Adelson Fundación de Investigación Médica (con ACC).

1. Cáncer de ovario de la célula Esferoide Formación <ol><li> PP-expresando las células de cáncer de ovario se cultivan en medio de base 10% (un medio de cultivo celular personalizada que contiene una mezcla 50:50 de 199 y MCDB105, 10% suero bovino fetal inactivado y 1% de lápiz estreptococo). Para expresar RFP en las células de cáncer de ovario no etiquetados, transfectar las células con un plásmido que contiene solicitud de propuestas y seleccionar las células que expresan RFP. Alternativamente, los vectores virales pueden ser utilizados para expresar transitoriamente proteínas fluorescentes, o las células pueden ser pre-incuban con un color rojo fluorescente de células seguidor colorante (Invitrogen). </li><li> Antes de formar esferoides de cáncer de ovario, es necesario preparar baja adherencia 96 platos de fondo redondo de cultivo así. Para producir las placas de cultivo de baja adherencia, 30μl poli-HEMA (6mg polihidroxietilmetacrilato en 1 ml de EtOH al 95%) solución se añade a cada pocillo de una placa de cultivo de 96 pocillos Corning celular. Las placas de 96 pocillos se incuban en un 37 ° C no incubador humidificado para evaporar el etanol, leaving una película de poli-HEMA en cada pocillo. Esta película de poli-HEMA evita que las células se adhieran a la parte inferior del pozo, forzando a las células para crecer en suspensión 18. [Alternativamente, ultra-bajo placas de cultivo de fijación (Corning) puede utilizarse en lugar de poli-HEMA platos recubiertos.] </li><li> Después de las placas de baja adhesión de cultivo se preparan, trypsinize una placa de células de cáncer de ovario, pellet de las células en una centrífuga de mesa (Heraeus) a 900 RCF durante 3 minutos, Aspirar el sobrenadante y resuspender en medio de base 10%. </li><li> Cuenta las células utilizando un hemocitómetro. </li><li> Ajustar la concentración de tales células que hay 100 células por 50 l de medio de base 10%. </li><li> Añadir 50 l de la suspensión de células uniformemente suspendido diluido a cada pocillo de la placa 96 así poli-HEMA cultura revestido. </li><li> Se incuba la placa de 96 pocillos en un 37 ° C incubadora de cultivo celular durante 16 horas (esta cantidad de tiempo debe aumentarse o disminuirse dependiendo de la cantidad de tiempo que tardauna línea celular particular para formar esferoides multicelulares o deseadas condiciones experimentales) para permitir que las células de cáncer de ovario a agruparse entre sí, formando un esferoide sola multicelular en cada pocillo. Algunas células del tumor pueden someterse a la apoptosis durante este período, por lo que es importante elegir una hora antes de la inducción de la apoptosis. </li></ol> 2. Monocapa de células mesoteliales Formación <ol><li> En una campana de cultivo celular, pre-capa de los pocillos de un 6 bien de fondo de vidrio plato MatTek con fibronectina mediante la adición de 2 mL de una fibronectina 5μg / ml de solución de PBS a cada pocillo de la placa y la incubación a temperatura ambiente durante 30 minutos. La calidad óptica de los fondos de vidrio en los platos Mattek permiten imágenes de alta resolución microscópica. </li><li> GFP-células que expresan mesoteliales se cultivan en medio de base 10%. Trypsinize una placa de células mesoteliales, la desaceleración en una centrífuga de mesa (Heraeus) a 900 rpm durante 3 minutos, aspirar el sobrenadante, y vuelva a suspender en el medio de base del 10%. Lacélulas mesoteliales utilizados aquí fueron ya expresando GFP cuando se obtuvieron, pero sin marcar las células mesoteliales pueden ser producidos por transfección con un plásmido GFP ADNc que contiene, o preincubating las células en un verde fluorescente de células seguidor colorante (Invitrogen). </li><li> Después de la incubación de fibronectina de 30 minutos (en el paso 2.1), lavar los pocillos de la placa MatTek con 2 mL de PBS. </li><li> Aspirar el PBS y placa de 6 x 10 5 células mesoteliales por pocillo en cada pocillo de la placa de 6 pocillos MatTek. Incubar el plato MatTek en un 37 ° C de cultivo celular durante la noche para permitir que las células mesoteliales para insertarse en el plato y forman una monocapa incubadora. </li></ol> 3. Ensayo de células mesoteliales Liquidación <ol><li> Utilizar una pipeta para recoger los esferoides de cáncer de ovario del 96 bien poli-HEMA placa revestida. </li><li> Aspirar el medio de un pozo de la placa de MatTek 6 y que contiene una monocapa de células mesoteliales. Lavar una vez con 2 ml de PBS. Agregue todos los esferoides de la 96 y ptarde a un pocillo de la placa MatTek (~ 3 veces el número de esferoides que van a ser fotografiada para dar cuenta de aterrizaje esferoides en la parte del plato que no se pueden obtener imágenes). </li><li> Coloque el plato MatTek en la etapa de un microscopio invertido campo amplio de fluorescencia capaz de realizar de lapso de tiempo de imágenes para la duración de al menos 8 horas. Utilice una platina motorizada de posiciones de la imagen múltiple en el plato, con múltiples eventos de intercalación esferoide, en un solo experimento. Usamos una cámara Nikon Ti-E invertido motorizado de fluorescencia de campo amplio lapso de tiempo microscopio con sistema integrado de enfoque perfecto y baja [20x-0.75 apertura numérica (NA)] Aumentos / NA de contraste de interferencia diferencial (DIC), la óptica, un transiluminador de halógeno Nikon con 0.52 NA de larga distancia de trabajo (LWD) condensador, Nikon rápida (&lt;100 milisegundos de tiempo de conmutación) filtros de excitación y emisión de las buenas prácticas agrarias (ex 480/40, Em 525/50, PP-mCherry Ex 575/50 Em 640/50), Sutter rápida transmisión Apagar la luz y de epifluorescencia ruta inteligentetros, una Nikon lineal codificado etapa motorizada, un Hamamatsu ORCA-AG se enfrió dispositivo de carga acoplada (CCD), una cámara microscopio hecha a la medida de la incubación con temperatura y el CO 2 de control, Nikon NIS-Elements AR software de la versión 3, y un TMC aislamiento de vibraciones mesa. </li><li> Los esferoides de cáncer de ovario de células se depositan en el fondo de un molde y se unen a la monocapa de células mesoteliales. Recopilar las buenas prácticas agrarias, RFP y las imágenes de fase de 20 + esferoide / monocapa interacciones, cada 10 minutos, durante 8 horas. </li><li> En el PP-que expresan las células de cáncer de ovario esferoides va a invadir a la monocapa de células que expresan GFP mesotelial la creación de un agujero en la monocapa. Después de 8 horas, medir las dimensiones de los orificios mediante el trazado de los agujeros negros en las imágenes GFP utilizando software de Elementos (u otro software adecuado tal como J imagen). Normalizar el tamaño del agujero al tamaño esferoide inicial dividiendo el tamaño del agujero de 8 horas por el tamaño del esferoide en la correspondiente imagen RFP en el tiempo cero. En este exejemplo, el tamaño del agujero se midió una sola vez, pero se puede medir varias veces durante todo el experimento de ocho horas para comprender mejor la dinámica de la intercalación. </li></ol> 4. Los resultados representativos En este ejemplo, se comparó la capacidad de depuración mesotelial de esferoides celulares OVCA433 de cáncer de ovario que tienen expresión atenuada de talina-1 para controlar OVCA433 esferoides. OVCA433 esferoides de cada grupo se añadieron a un plato MatTek contiene ZT monocapas de células mesoteliales. Seis esferoides de cada grupo se obtuvieron imágenes cada 10 minutos durante ocho horas (Figura 4, 3 Película, Película 4). Los agujeros producidos en la monocapa por los esferoides de propagación se midieron y seis posiciones de cada grupo se promediaron. La Figura 4 muestra que la zona de aclaramiento promedio creado por esferoides Talin desmontables 1 fue significativamente menor que el área media creado por esferoides de control, lo que sugiere que se requiere para talina aclaramiento mesotelial por OVCA433 esferoides de cáncer de ovario. <img alt="Figura 1" src="/files/ftp_upload/3888/3888fig1.jpg" /> Figura 1. La metástasis del cáncer del ovario. Los tumores primarios de ovario desarrollan ya sea desde el epitelio superficial del ovario o trompas de Falopio. Las células tumorales o grupos se desprenden del tumor primario y se acumulan en la cavidad peritoneal. Las células tumorales a continuación, pueden agregarse para formar esferoides multicelulares. Esferoides continuación, se adhieren a las monocapas de células mesoteliales que recubren la cavidad peritoneal. Las células mesoteliales están excluidos de debajo del esferoide de cáncer de ovario adjunto, permitiendo que los esferoides para obtener acceso a la membrana basal subyacente. Película 1. La metástasis del cáncer de ovario. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">Haga clic aquí para ver la película</a> . <img alt="Figura 2" src="/files/ftp_upload/3888/3888fig2.jpg" /> Figura 2. </strong&gt; Línea células mesoteliales de la superficie del tejido peritoneal humano y se excluyen de debajo de los implantes de células cancerígenas de ovario. <img alt="Figura 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> Figura 3. Ensayo de Liquidación mesoteliales. Esferoides de cáncer de ovario se forman mediante la incubación de 100 RFP-células que expresan el cáncer de ovario por pocillo en un poli-HEMA revestida 96 placa inferior la cultura y todo a 37 ° C durante 16 horas. Poli-HEMA evita que las células se adhieran a la placa de cultivo, permitiendo que las células permanezcan en suspensión y se adhieren entre sí para formar un solo grupo por pocillo. Monocapas de células mesoteliales se preparan mediante siembra de 6x10 5 células mesoteliales por pocillo en una fibronectina recubierto 6 así plato MatTek e incubando la placa a 37 ° C durante 16 horas. Los esferoides se transfieren luego al plato MatTek con la monocapa mesotelial y las dos poblaciones de células se visualizan cada 10 minutos durante 8 horas utilizando una Nikon Ti-EX Inverted motorizado de fluorescencia de campo amplio lapso de tiempo microscopio y software de los elementos. Película 2. Ensayo de Liquidación mesoteliales. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">Haga clic aquí para ver la película</a> . <img alt="Figura 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> Atenuación de la Figura 4. Talina de una expresión en OVCA433 esferoides disminuye la capacidad de depuración mesotelial. OVCA433 esferoides (rojo) con y sin expresión atenuada de una talina se permite adjuntar a e invadir en una monocapa mesotelial ZT (verde). Las dos poblaciones de células se obtuvieron imágenes cada 10 minutos durante 8 horas utilizando una Nikon Ti-E invertido motorizado de fluorescencia de campo amplio lapso de tiempo microscopio y software de los elementos. El gráfico muestra que talina una atenuación dedisminuye significativamente el aclaramiento de células mesoteliales (parcela Quantile con barras verdes en el medio). Película 3. Control de OVCA433 esferoides (rojo) invadiendo en una monocapa mesotelial (verde). <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">Haga clic aquí para ver la película</a> . Atenuación de la película 4. De talina una expresión en OVCA433 esferoides (rojo) disminuye mesotelial (verde) la capacidad de depuración. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">Haga clic aquí para ver la película</a> .

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M., You, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+and+micromechanics+of+tumor+cell+chemotactic+protrusion,+locomotion,+and+extravasation.">In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation.</a> Ann. Biomed. Eng. 30, 344-355 (2002).</li></ol>

No tenemos nada que revelar.

El &quot;Ensayo de Liquidación mesoteliales&quot; que aquí se presenta utiliza lapso de tiempo de microscopía para controlar las interacciones de los esferoides multicelulares de cáncer de ovario y monocapas de células mesoteliales, con gran detalle espacial y temporal. Anteriormente, varios grupos de 8-14 había utilizado los ensayos de punto final para demostrar que las células de cáncer de ovario y de adherirse a invadir en monocapas de células mesoteliales. Este ensayo es único en que utiliza cé...

<table border="1"><tbody><tr><td> Reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> OVCA433 células de cáncer ovárico </td><td></td><td></td><td> Regalo del Dr. Dennis Slamon </td></tr><tr><td> ZT células mesoteliales </td><td></td><td></td><td> Regalo del Dr. Tan Ince </td></tr><tr><td> Medio 199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> Aplicaciones de la célula Inc. </td><td> 117-500 </td><td></td></tr><tr><td> FBS-inactivado por calor </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> Pen-Strep </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Placas de 96 pocillos </td><td> Corning Costar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polihidroxietilmetacrilato (poli-HEMA) </td><td> Sigma Aldrich </td><td> 192066-25G </td><td> Para poli-HEMA solución disolver 6mg poli-HEMA polvo en 1 ml de EtOH al 95% </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> Diluir al 95% en dH 2 0 </td></tr><tr><td> Celular campana de la cultura </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> Cultivo de tejidos incubadora </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> incubadora de poli-HEMA placas </td><td> Labline Instruments </td><td> Imperial III 305 </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 y plato de fondo de vidrio </td><td> MatTek corp. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> La fibronectina </td><td> Sigma </td><td> F1141-1mg </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21 a 040 CV </td><td></td></tr><tr><td> Timelapse microscopio: </td><td></td><td></td><td></td></tr><tr><td> Microscopio </td><td> Nikon </td><td></td><td> Ti-E invertida motorizada de fluorescencia microscopio de lapso de tiempo con el sistema integrado de enfoque perfecto </td></tr><tr><td> Lente </td><td> Nikon </td><td></td><td> 20X-0.75 apeture numérica </td></tr><tr><td> Halógeno transiluminador </td><td> Nikon </td><td></td><td> 0.52 NA larga jornada de trabajo del condensador a distancia </td></tr><tr><td> Filtros de excitación y emisión </td><td> Chroma filtros de una sola pasada en la vivienda de Nikon </td><td></td><td> Las buenas prácticas agrarias Ex 480/40, Em 525/50 SP-mCherry Ex 575/50 Em 640/50 </td></tr><tr><td> Transmitido y Epifluoresce trayectoria de la luz </td><td> Sutter </td&gt; <td></td><td> Persianas inteligentes </td></tr><tr><td> Lineal codificado platina motorizada </td><td> Nikon </td><td></td><td></td></tr><tr><td> Refrigerado de carga acoplada dispositivo de cámara </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Microscopio cámara de incubación con 2 temperatura y el control de CO </td><td> hecha a la medida </td><td></td><td></td></tr><tr><td> Vibración mesa de aislamiento </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elements </td><td> Nikon </td><td></td><td> La versión 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

El cáncer de ovario es la quinta causa principal de muertes relacionadas con el cáncer en los Estados Unidos 1. A pesar de una respuesta inicial positiva a las terapias, del 70 al 90 por ciento de las mujeres con cáncer de ovario desarrollan metástasis a los nuevos, y la recurrencia es a menudo fatal 2. Es, por tanto, necesaria para entender cómo surgen metástasis secundarias con el fin de desarrollar mejores tratamientos para el cáncer de ovario en etapa intermedia y tardía. La metástasis del cáncer de ovario ocurre cuando las células malignas se desprenden de la localización del tumor primario y difundir toda la cavidad peritoneal. Las células diseminadas pueden formar grupos multicelulares, o esferoides, que o bien permanecerá sin conectar, o implantes en órganos dentro de la cavidad peritoneal 3 (Figura 1, la película 1). Todos los órganos dentro de la cavidad peritoneal se revisten con una sola capa continua, de las células mesoteliales 4-6 (Figura 2). Sin embargo, las células mesoteliales están ausentes de debajolas masas tumorales peritoneales, como lo revelan los estudios de microscopio electrónico de secciones de tejidos humanos extirpados tumores 3,5-7 (Figura 2). Esto sugiere que las células mesoteliales se excluyen de debajo de la masa tumoral por un proceso desconocido. Anterior en experimentos in vitro han demostrado que las células primarias de cáncer de ovario adjuntar más eficientemente a la matriz extracelular que a las células mesoteliales 8, y los estudios más recientes demostraron que las células primarias mesoteliales peritoneales realmente proporcionar una barrera para la adhesión celular de cáncer de ovario y la invasión (en comparación con la adhesión y la invasión sobre soportes que no fueron cubiertas con células mesoteliales) 9,10. Esto sugeriría que las células mesoteliales actuar como una barrera contra la metástasis de cáncer de ovario. Los mecanismos celulares y moleculares por los cuales las células de cáncer de ovario incumplan esta barrera, y no incluyen el mesotelio, hasta hace poco, sigue siendo desconocido. A continuación se describe ºmetodología e para un ensayo in vitro que los modelos de la interacción entre los esferoides de ovario de células cancerosas y las células mesoteliales in vivo (Figura 3, la película 2). Nuestro protocolo es una adaptación de los métodos descritos anteriormente para el análisis de las interacciones de células de tumor de ovario con monocapas mesoteliales 8-16, y fue descrito por primera vez en un informe que muestra que las células tumorales de ovario utilizar una activación de la integrina-dependiente de la miosina y la fuerza de tracción para promover la exclusión de la las células mesoteliales de debajo de un esferoide del tumor 17. Este modelo toma ventaja de la microscopia de fluorescencia de lapso de tiempo para controlar las poblaciones de células de dos en tiempo real, proporcionando la información espacial y temporal de la interacción. Las células de cáncer de ovario expresan la proteína fluorescente roja (RFP), mientras que las células mesoteliales expresan la proteína verde fluorescente (GFP). RFP-expresando ovario esferoides celulares de cáncer de adjuntar a la monocapa mesotelial que expresan GFP. La difusión esferoides, invadir yforzar a las células mesoteliales lado creando un agujero en la monocapa. Este agujero se visualiza como el espacio negativo (negro) en la imagen de las buenas prácticas agrarias. El área del agujero se puede medir a analizar cuantitativamente diferencias en la actividad holgura entre el control y las poblaciones experimentales de cáncer de ovario y / o células mesoteliales. Este ensayo requiere sólo un pequeño número de células de cáncer de ovario (100 células por esferoides X 20-30 esferoides por condición), por lo que es factible realizar este ensayo utilizando preciosas muestras primarios de células tumorales. Además, este ensayo se puede adaptar fácilmente para el cribado de alto rendimiento.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

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

El ensayo de depuración mesotelial se describe aquí se aprovecha de las células de la etiqueta fluorescente y time-lapse microscopía de vídeo para visualizar y medir cuantitativamente las interacciones de los esferoides multicelulares de cáncer de ovario y monocapas de células mesoteliales. Este ensayo permite simular las primeras etapas de la metástasis del cáncer de ovario.

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 ...

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Research

JoVE Journal

Medicine

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

14.4K vistas.  Harvard Medical School. El ensayo de depuración mesotelial se describe aquí se aprovecha de las células de la etiqueta fluorescente y time-lapse microscopía de vídeo para visualizar y medir cuantitativamente las interacciones de los esferoides multicelulares de cáncer de ovario y monocapas de células mesoteliales. Este ensayo permite simular las primeras etapas de la metástasis del cáncer de ovario.

Video: In vitro Mesothelial Clearance Assay that Models the Early Steps of 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

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME Modelos de 4D Live de células de imagen de un tumor: Interacciones microambiente que la progresión maligna del impacto

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotípicos tridimensional<em&gt; In Vitro</em&gt; Modelado de estromal-epitelial interacciones durante la iniciación y progresión del cáncer de ovario

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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  ...

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 ...

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 ...

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.

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 ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modelado de los primeros pasos de cáncer de ovario Difusión en una cultura organotípicos del Peritoneal Cavity Humano

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...