Este trabajo fue apoyado en parte por los Institutos Nacionales de Salud R01 CA131990 (BFS y MRR). En vivo de células de imágenes se realizó en el Núcleo de Microscopía, imágenes y Citometría de Recursos apoyada, en parte, por el NIH Centro de concesión P30 CA22453 del Instituto Karmanos del Cáncer, Universidad Estatal de Wayne y por la Sección de Investigación de Perinatología del Instituto Nacional de Salud Infantil y Desarrollo , Wayne State University.

1. Preparar DQ-sustratos <ol><li> Permitir liofilizó DQ-sustratos calentar a temperatura ambiente antes de la apertura de viales, preparar la solución madre de 1 mg / ml de DQ-sustrato en agua desionizada, se dividen en 50 ml de alícuotas y se almacena a 4 ° C. </li></ol> Puede ser necesario agitar DQ-sustrato en un baño de agua ultrasónico durante ~ 5 minutos y se calienta a 50 ° C para facilitar la dispersión. <ol start="2"><li> RBM Descongelar en hielo durante la noche a 4 ° C; RBM debe ser manejado en hielo en todo momento. </li><li> Para hacer fosfato 10X tamponado salino (PBS), se disuelven 80 g de NaCl (1,37 M), 2 g de KCl (0,027 M), 14,4 g de Na 2 HPO 4 (1 M) y 2,4 g de KH 2 PO 4 (0,02 M) en 800 ml de agua ultrapura. Ajustar el pH de la solución tampón PBS a 7,4 con 1,0 M de HCl. Aumentar el volumen hasta 1 litro, esterilizar por filtración. </li><li> Preparar colágeno solución I sobre hielo: 8 partes de colágeno refrigerados I de 1 parte de refrigerados 10X PBS. Ajustar el pHde mezcla a 7.2-7.6 usando estéril 0,1 M de NaOH. Verificar el pH con tiras de pH y ajustar el volumen final a 10 partes con agua estéril. </li><li> Preparar DQ-colágeno I: colágeno I matriz mediante la dilución de DQ-colágeno I en colágeno I solución en un tubo previamente enfriado a una concentración final de 25 mg / ml y 2,4 mg / ml, respectivamente. </li><li> Preparar DQ-colágeno IV: membrana basal reconstituida (GBR) matriz mediante la dilución de DQ-colágeno IV en la GBR (Cultrex o Matrigel) en un tubo previamente enfriado a una concentración final de 25 mg / ml y 12-15 mg / ml, respectivamente. Mezcle el hielo con pipeteo suave para evitar la creación de burbujas. </li></ol> 2. Preparar cocultivos MAME Véase la Figura 1 para un diagrama esquemático de cocultivos. <ol><li> Coloque dos cubreobjetos de plástico rectangulares (esterilizada en alcohol al 70% durante 10 minutos y se seca al aire) en una placa de cultivo de 35 mm o utilizar un ibiTreat de 35 mm μ-Dish. Llegar aquí son para los dos cubreobjetos y u-Dishes, que utilizamos para estudiantesmuere en un microscopio vertical. Para los estudios en un microscopio invertido, sólo usamos μ de platos. </li><li> Mezclar el número deseado de fibroblastos con DQ-colágeno I: colágeno I matriz. Para los cocultivos largo plazo ilustradas en las Figuras 2 y 3, se utiliza 500 fibroblastos en 10 l de medios más 60 l de DQ-colágeno I: colágeno I de la matriz. </li><li> Usando una micropipeta 100-l, y se extendió cuidadosamente pipeta 70 l de fibroblastos: DQ-colágeno I: colágeno I matriz sobre la superficie entera de cada cubreobjetos y dejar a 37 ° C en un incubador humidificado sin CO 2 durante 30 minutos para solidificar. </li><li> Transferencia de 35-mm platos que contienen cubreobjetos a 37 ° C incubadora con 5% de CO 2 durante 10 min para equilibrar. </li><li> Eliminar de la incubadora y dejar los platos de 35 mm bajo el capó hasta que llegue a temperatura ambiente. </li><li> Añadir 60 l de DQ-colágeno IV: GBR matriz en la parte superior de la solidificado DQ-colágeno I: colágeno I matriz con fibroblastos embebidos. Con punta de la pipeta, carefully extendió DQ-colágeno IV: GBR matriz de manera uniforme, evitando rayar los fibroblastos: DQ-colágeno I: colágeno I de la matriz. </li><li> Transferencia de 35-mm platos que contienen cubreobjetos a 37 ° C incubadora con 5% de CO 2 durante 10 minutos para solidificar. Mientras que el IV DQ-colágeno: GBR matriz se solidifica, trypsinize y contar las células epiteliales. </li><li> Colocar 50 l de una suspensión de células epiteliales / tumoral sobre cubreobjetos revestidos con DQ-colágeno IV: GBR matriz. Coloque de 35 mm platos que contienen los cubreobjetos en una incubadora a 37 ° C. Permitir 40 a 60 minutos para que las células se adhieren a DQ-colágeno IV: GBR matriz. Para los cocultivos largo plazo ilustradas en las Figuras 2 y 3, se utiliza 2500 células epiteliales o tumor. </li><li> Añadir 2 ml de medio de cultivo que contiene 2% RBM a cada plato de 35 mm. Si al hacer los tratamientos de drogas, los medicamentos se debe agregar en este momento. </li><li> Incubar durante período de tiempo deseado antes de imágenes. Para cultivos a largo plazo, los medios de comunicación debe ser cambiado cada 3-4 días. Las células se pueden manteneren su medio de cultivo normal. Para cocultivos MAME de mama líneas celulares, se utiliza medio basal del epitelio (MEBM-PRF), complementado con SingleQuots MEGM. </li></ol> 3. Realizar 4D (3D + tiempo) de la imagen en vivo cocultivos MAME por microscopía confocal <ol><li> Para los estudios de prueba de principio ilustrado, cocultivos MAME fueron marcadas con un colorante fluorescente de la célula de seguimiento de acuerdo a nuestro procedimiento publicado 1. Después de lavar con PBS, se incubaron con cocultivos 5 Naranja CellTracker mM en el medio MEGM durante 45 minutos en una incubadora a 37 ° C, se lavó de nuevo con PBS y se incubaron con MEGMmedium precalentado durante 30 minutos en 37 ° C con 5% de CO 2 incubadora antes de imágenes . </li><li> Imagen cocultivos MAME en vivo en un bajo aumento (10X, inmersión en agua) en un microscopio Zeiss LSM 510 META confocal. </li><li> Secciones ópticas son capturados en intervalos a lo largo de toda la profundidad de las estructuras. Para nuestros estudios, podemos capturar secciones ópticas a las 10intervalos m. </li><li> Utilice las secciones ópticas para la reconstrucción de imágenes en 3D. Utilizamos el software Volocity para generar reconstrucciones en 3D. </li><li> Hacer películas de las reconstrucciones en 3D de manera que las estructuras, de células: las interacciones celulares y las interacciones con las matrices de los alrededores se pueden visualizar. Hemos hecho películas con el Reproductor de Quiktime 7. </li></ol> 4. Los resultados representativos El co-cultivos de MAME que hemos desarrollado son un modelo manejable experimental para imágenes en vivo de las células en tiempo real de las interacciones entre los diversos componentes celulares que forman un tumor de mama y su microambiente. Aquí se demuestra la capacidad de los modelos de MAME para recapitular la célula: las interacciones celulares durante la progresión del cáncer de mama y de células vivas de imágenes para seguir las interacciones a través del tiempo. Se ilustra la capacidad de las interacciones de imagen entre una línea de preinvasiva MCF10.DCIS de células humanas de mama y un cáncer de mama humano asociado a la línea de fibroblastos, WS-12Ti, a lo largoun período de más de tres semanas en cultivo. El co-cultivos fueron visualizados a los 3, 16 y 23 días. Secciones ópticas a través de todo el volumen de la cocultivos fueron reconstruidas en 3D y ejemplos representativos se muestran para cada uno de los tres puntos de tiempo en la Figura 2. La fluorescencia roja en la Figura 2 representa el MCF10.DCIS y células de WS-12TI. Para discriminar entre los dos tipos de células y de largo plazo co-cultivos, se puede transfectar o transducción de los distintos tipos de células con proteínas fluorescentes previo al establecimiento de los cultivos. Un cocultivo de las células MAME MCF10.DCIS y WS-12Ti que están diferencialmente marcadas con proteínas fluorescentes para la identificación se ilustra en la Figura 3. Por incorporación de sustratos, en este caso DQ-colágeno, en la co-cultivos de MAME, podemos evaluar y cuantificar los cambios en el tiempo en la proteólisis asociados con la transición a un fenotipo invasivo. Hemos establecido los métodos para la cuantificación de los productos de su desdoblamiento fluorescentes que dan como resultadode DQ-colágeno degradación 2,3. Nos normalizar los productos de degradación a lo largo de todo el volumen 3D de la cocultivos MAME en una base por célula mediante el etiquetado y contando los núcleos celulares. Además, se puede localizar la degradación de los compartimentos intracelulares y extracelulares y cuantificar el total de productos, la degradación intracelular y extracelular, como hemos descrito anteriormente 2. La fluorescencia verde en las figuras 2 y 3 representa productos de escisión de los dos sustratos de colágeno-DQ. En este momento, colágenos diferencialmente marcados no están disponibles que nos permiten distinguir entre la degradación del colágeno tipo IV y la degradación del colágeno tipo I. Las interacciones en el cocultivos MAME son dinámicos y pueden cambiar con el tiempo. Para obtener información temporal y dinámico, el mismo campo de visión puede ser secciones con imagen y ópticas tomadas a través de la muestra completa del volumen repetidamente durante un período de tiempo de minutos a semanas,por lo tanto la recolección de datos en 4-D. En la figura 2, que ilustran los cambios más dramáticos que se producen más de 23 días: los cambios en el número de células, la migración celular y las interacciones celulares, los volúmenes de la estructura, las formas de la estructura, las desviaciones de las estructuras de esfericidad, excrecencias invasivos y proteólisis. Por otra parte, también demuestran un cambio general en el volumen debido a la co-cultivos MAME degradantes de la matriz extracelular que rodea a su durante este período. A los tres días como se ilustra en la Figura 2A, el volumen es 110.000 m 3. En contraste, el volumen de la cocultivos MAME menos 16 y 23 días es sólo 46.250 m 3, como se ilustra en las figuras 2B y C. <img alt="Figura 1" src="/files/ftp_upload/3661/3661fig1.jpg" /> Figura 1. Diagrama esquemático de cocultivos MAME. Cubreobjetos de plástico se recubre con colágeno I, que contiene DQ-colágeno I y fibroblastos (Magenta). Una 2 ª capa de membrana basal reconstituida (RBM), que contiene DQ-collagen IV se añade. Las células tumorales (rojo) se colocan en la parte superior y el 2% en la cultura de gestión basada en los medios de comunicación se superpone (puntos blancos) con cada cambio de los medios de comunicación. El verde representa los productos de desdoblamiento fluorescentes de DQ-colágeno I y IV. <img alt="Figura 2" src="/files/ftp_upload/3661/3661fig2.jpg" /> Figura 2. MAME cocultivos de células MCF10.DCIS de mama humanos y WS-12TI fibroblastos humanos de mama. Los fibroblastos fueron incorporados en el colágeno I / DQ-colágeno I y cubierta con GBR contiene DQ-colágeno IV. Las células de DCIS fueron sembradas en la gestión por resultados y recubierta con un medio que contiene 2% de la RBM. Durante los 23 días en cocultivo ilustrados aquí, hubo proliferación celular, la degradación de DQ-colágeno IV y I (verde) y las matrices de colágeno, como se indica por la reducción en el volumen de la cocultivos. Las células fueron localizados por el etiquetado con Orange CellTracker in situ antes de la imagen (rojo). Los co-cultivos vivos mismas se observaron durante los 23 días con las imágenes captadas por cmicroscopia onfocal a los 3, 16 y 23 días de cocultivo. Los cortes resultantes ópticos fueron reconstruidas en 3D con el software de Volocity. El aumento, 10 veces más. <img alt="Figura 3" src="/files/ftp_upload/3661/3661fig3.jpg" /> Figura 3. MAME 16 días cocultivos de células MCF10.DCIS de mama humanos y WS-12TI fibroblastos humanos de mama que expresan RFP (rojo) y YFP (blanco), respectivamente. Cocultivos se establecieron y se analizó como en la Figura 2. Los dos tipos de células que se muestran aquí había, sin embargo, ha diferencialmente etiquetados de modo que pudieran ser distinguidos unos de otros. Las imágenes de aumento mayor en esta figura, en comparación con las de la figura 2, ilustran la proteolisis de DQ-colágeno IV en la superficie de las células DCIS y proteolisis difusa de DQ-colágeno I en las zonas cercanas a los fibroblastos. Ampliación, 20X.

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	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

No tenemos nada que revelar.

Como se ha demostrado, la cocultivos MAME puede ser utilizado para vivo de células de imágenes en tiempo real de las interacciones entre los diferentes constituyentes celulares que comprenden un tumor de mama y su microambiente. En los estudios en curso en nuestro laboratorio, hemos utilizado cocultivos MAME para identificar las vías proteolíticas asociadas con la transición de carcinoma ductal in situ de carcinoma ductal invasivo, así como las interacciones entre las vías proteolíticas y otras vías que partici...

<table border="1"><tbody><tr><td> Reactivo / Equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Membrana basal reconstituida (Cultrex) </td><td> Trevigen </td><td> 3445-005-01 </td><td> Comparable a Matrigel (BD Biosciences) </td></tr><tr><td> Colágeno I </td><td> Laboratorios de cohesión </td><td> 5005-B </td><td></td></tr><tr><td> DQ-sustratos (Colágeno I, IV) </td><td> Invitrogen </td><td> DQ-Col-D12060 DQ-col IV D12052 </td><td></td></tr><tr><td> 22-mm cubreobjetos de plástico </td><td> Fisher Scientific Co. </td><td> 12-547 </td><td> Cortar por la mitad, dejar en el 70% de etanol durante 10 minutos y secar al aire antes de su uso. </td></tr><tr><td> Medio de cultivo de la célula </td><td> Lonza </td><td> MEBM CC-PRF-3153 MEGM CC-4136 </td><td> Rojo fenol libre </td></tr><tr><td> ibiTreat μ-Dish </td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Volocity de software </td><td> Perkin-Elmer </td><td> Versión 5.5 </td><td> Otras marcas de software de imagen se puede utilizar para generar reconstrucciones en 3D y las películas. </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

Hemos desarrollado modelos 3D co-cultivo, que llamamos MAME (m ammary un RQUITECTURA m NGENIERÍA icroenvironment e), y los utiliza para imágenes en vivo de las células en tiempo real de células: las interacciones celulares. Nuestro objetivo general fue desarrollar modelos que recapitular la arquitectura de las lesiones preinvasoras del seno para estudiar su progresión a un fenotipo invasivo. Específicamente, hemos desarrollado modelos para analizar las interacciones entre premalignas variantes de células epiteliales de mama y otros tipos de células del microambiente del tumor que han sido implicados en la mejora o reducción de la progresión de las células epiteliales de mama preinvasoras a carcinomas ductales invasivos. Otros tipos de células estudiadas hasta la fecha son las células mioepiteliales, fibroblastos, macrófagos y la sangre y las células endoteliales microvasculares linfáticos. Además de los modelos de MAME, que están diseñados para recapitular las interacciones celulares en el pecho durantela progresión del cáncer, hemos desarrollado modelos comparables de la progresión de los cánceres de próstata. Aquí se ilustran los procedimientos para establecer los cocultivos 3D junto con el uso de imágenes en vivo de células y un ensayo de proteolisis funcional para seguir la transición de cocultivos de carcinoma ductal in situ (CDIS) y fibroblastos células a un fenotipo invasivo en el tiempo, en este caso más de veinte y tres días de cultivo. El co-cultivos MAME consisten en varias capas. Los fibroblastos están incrustadas en la capa inferior de colágeno de tipo I. En que se coloca una capa de membrana basal reconstituida (RBM) en el cual las células se siembran DCIS. Una capa final de la parte superior del 2% RBM se incluye y se repone con cada cambio de los medios de comunicación. Para la proteólisis de la imagen asociada con la progresión a un fenotipo invasivo, se utiliza proteínas fluorescentes de tinte enfriadas (DQ) de la matriz (DQ-colágeno I mezclado con la capa de colágeno I y IV DQ-colágeno mezclado con la capa media de rBM) y observar a las culturas vivas mediante microscopía confocal. Secciones ópticas son capturados, procesados ​​y reconstruido en 3D con el software de visualización de Volocity. En el transcurso de 23 días en cocultivos MAME, las células proliferan y DCIS se unen en grandes estructuras invasivas. Los fibroblastos migran y se incorporan a estas estructuras invasivas. Fluorescentes fragmentos proteolíticos de los colágenos se encuentran en asociación con la superficie de las estructuras DCIS, intracelularmente, y también dispersa por toda la matriz circundante. Los medicamentos que las vías proteolíticas objetivo, quimiocinas / citocinas y la quinasa o modificaciones en la composición celular de la co-cultivos pueden reducir la invasividad, lo que sugiere que los modelos de MAME se puede utilizar como pantallas preclínicos de nuevos enfoques terapéuticos.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

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

Hemos desarrollado modelos 3D para el cocultivo de células vivas imágenes en tiempo real de las interacciones entre las células tumorales de mama y otras células en el microambiente que afectan la progresión a un fenotipo invasivo. Estos modelos pueden servir como pantallas preclínicos de los medicamentos que se dirigen a paracrinos inducidos por las vías proteolíticas, quimiocinas / citocinas y la quinasa implicados en la invasión.

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 ...

mame-models-for-4d-live-cell-imaging-tumor-microenvironment

Research

JoVE Journal

Medicine

14.3K vistas.  Wayne State University. Hemos desarrollado modelos 3D para el cocultivo de células vivas imágenes en tiempo real de las interacciones entre las células tumorales de mama y otras células en el microambiente que afectan la progresión a un fenotipo invasivo. Estos modelos pueden servir como pantallas preclínicos de los medicamentos que se dirigen a paracrinos inducidos por las vías proteolíticas, quimiocinas / citocinas y la quinasa implicados en la invasión.

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

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Xenotrasplante ortotópico de los Derechos Humanos-luciferasa Tagged células malignas del tumor vaina de nervio periférico para In vivo Pruebas de candidatos a agentes terapéuticos

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Investigación

Medicina

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Evaluación de la migración de las células madre del cáncer compartimentar Utilizando dispositivos de microfluidos y imágenes de células vivas

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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 ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Imágenes en directo de las respuestas de drogas en el microambiente tumoral en modelos de ratón de cáncer de mama

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Una novela de alta resolución<em&gt; En vivo</em&gt; Técnica de escaneo para el Estudio de la respuesta dinámica de estructuras intracraneales al crecimiento del tumor y Terapéutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

Alta frecuencia de imágenes por ultrasonido del mouse ganglios linfáticos cervicales

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

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

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

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

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

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

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Método de cuantificación del tumor bioluminiscencia-base para la progresión tumoral Monitoreo y efectos del tratamiento en modelos de ratón linfoma

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Ex vivo de imágenes en vivo de metástasis pulmonar y su microambiente

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...