Apoyado por el NIH subvenciones CA051008 P30 y T32 CA009686 (ATR).

1. Generación de esferoides <ol><li> Preparar la suspensión de una sola célula para colgar culturas gota a separar los cultivos de células adherentes cáncer de ~ 70% de confluencia usando un lavado con PBS seguido de exposición a solución de tripsina-EDTA 0,05%. <ol><li> Neutralizar la solución de tripsina con medio de cultivo celular y contar las células usando una parte alícuota de la suspensión celular. Nota: El medio de cultivo celular específica dependerá de la línea celular se está probando. Siga ATCC recomendaciones de los medios, donde el medio de cultivo celular es típicamente DMEM + 10% FBS. Más información se puede encontrar en la Tabla de Materiales. </li><li> Realizar una dilución para permitir la siembra de 500-1.000 células por 20 l gota de medio de cultivo celular. </li></ol></li><li> Adquirir placa de 10 cm y añadir 5 ml de PBS estéril hasta el fondo. Nota: Este paso protege el ahorcamiento cae de evaporación. Bajo costo &quot;grado bacteriológicos&quot; platos puede ser usado en lugar de platos de grado de cultivo de células, como las células no lo hará contactuar de la superficie de cultivo. </li><li> Usar una pipeta multicanal para transferir 20 l gotitas de la suspensión celular diluida a la superficie interior de la tapa. <ol><li> Pipetear 40 gotas (5 filas de 8 gotas) en la tapa de la placa de 10 cm. </li></ol></li><li> Invierta la tapa y colocar en la placa de cultivo. Incubar los cultivos gota colgante a 37 ° C durante 72 horas para generar esferoides. <ol><li> Voltear la tapa de una manera segura, pero controlada. Nota: La inversión de la tapa demasiado rápido o demasiado lento puede hacer que las gotas se desplacen. Algunas líneas de células pueden formar esferoides en 48 horas o menos, mientras que otros pueden requerir más de 72 horas para producir agregados compactos. </li></ol></li></ol> 2. Incorporación de esferoides en Matrix 3D <ol><li> Descongelar una alícuota del sótano materiales de membrana del factor de crecimiento reducido a 4 ° C durante la noche antes de la incorporación de los esferoides. <ol><li> Opcional: pozos de capa con ECM de antelación para evitar posibles interacciones esferoide con la cultu tejidovolver a emerger. <ol><li> Pipeta 200 l de ECM por pocillo en una placa de 24 pocillos. </li><li> Inclinar la placa para asegurarse de que el ECM cubre toda la superficie del pocillo. </li><li> Eliminar cuidadosamente el exceso de ECM con una pipeta. </li><li> Incubar la placa hasta que los pocillos están secos: 3 hr a temperatura ambiente o durante la noche a 4 ° C. </li></ol></li></ol></li><li> Recoger los esferoides por la inclinación de la tapa de la placa de cultivo tisular y puesta en común de los medios de comunicación. La transferencia de los medios de comunicación con los esferoides en un tubo de microcentrífuga de 1,5 ml. </li><li> Permitir 10 min para los esferoides para ubicarse en la parte inferior del tubo de microcentrífuga. Esferoides deben ser visibles a simple vista. </li><li> Mezclar 100 l de los materiales de la membrana del sótano con 100 l de frío (4 ° C) de colágeno tipo I en un tubo de pre-enfriado por separado. Mantenga la mezcla a 4 ° C para prevenir ya sea en gel se solidifique antes de tiempo. <ol><li> Utilice puntas de pipeta pre-refrigerados si la transferencia de pequeños volúmenes. </li><li> Dar cuidado almezclar el material de la membrana basal y el colágeno de tipo I, que impiden la formación de burbujas de aire. Nota: Las burbujas de aire pueden obstaculizar las imágenes más tarde en el protocolo si se incrustan en el gel. </li></ol></li><li> Aspirar los esferoides de la porción inferior 40 l de la tubo de microcentrífuga, y se combinan con el material de la membrana basal / colágeno tipo I mezcla. Tenga cuidado para evitar la formación de burbujas de aire cuando la mezcla. Nota: La solución que contiene de 100 l material de la membrana basal, 100 colágeno tipo l I, y 40 l de los esferoides crearé material suficiente para 4 culturas 3D independientes. Esto se puede escalar hacia arriba o hacia abajo. </li><li> Pipetear 40 mu l gotas de la mezcla viscosa en los centros de los pozos en una placa de 24 pocillos. Mantenga el nivel de placa para evitar que la mezcla se ejecuta en el lado del pozo. Nota: Una sola columna de la placa de 24 pocillos - se recomienda para cada condición a ensayar - 4 pozos. </li><li> Coloque la placa de intOA 37 ° C incubadora y dejar en reposo durante 30 min. La cultura 3D polimeriza durante este período. </li><li> Lentamente sumergir las culturas 3D en 1 ml de medio de cultivo celular. <ol><li> Asegúrese de que los medios de comunicación es cálido al agregar que en los pocillos para promover aún más la polimerización del gel. </li><li> PASO CRÍTICO: añadir con cuidado los medios de comunicación a las culturas 3D. Pipetear los medios de comunicación demasiado rápido en los pocillos puede dar lugar a la separación de las culturas 3D a partir de la superficie de cultivo de tejidos. </li></ol></li></ol> 3. Seguimiento y análisis de esferoide Invasion <ol><li> Invasión de la imagen de los esferoides en la matriz 3D que rodea a los puntos de tiempo decidido por el investigador. Adquirir fotografías utilizando un microscopio invertido con la lente de objetivo de 20x. Nota: Los puntos de tiempo Ideal serán diferentes dependiendo de la línea celular que se está probando. Líneas celulares más invasivos comenzarán su egreso de los esferoides, poco después de la siembra y, por tanto, tomar fotografía inicials dentro de un par de horas después de la siembra. Generalmente, las fotografías se toman a 0 h (después de la siembra), las 24 horas, y 48 horas. Invasión normalmente concluye 24 a 48 horas después de la siembra. </li><li> Cuantificar la capacidad invasiva utilizando el software de análisis de imagen como imagen J. <ol><li> Analizar la invasión como la distancia celular desde el borde del esferoide. <ol><li> Utilice la herramienta Draw recta para marcar el radio y / o la distancia invasivo máxima. </li><li> Haga clic en &quot;Analizar&quot; en el menú superior y haga clic en &quot;Medir&quot; para mostrar la medición de longitud. </li></ol></li><li> Analizar la invasión como la zona invasivo totales fuera del esferoide. <ol><li> Utilice la herramienta de dibujo a mano alzada para trazar la frontera del esferoide y / o área invasivo totales. </li><li> Haga clic en &quot;Analizar&quot; en el menú superior y haga clic en &quot;Medir&quot; para mostrar la medición de área. </li></ol></li><li> Use un plugin como herramientas del Administrador de retorno de la inversión para crear mediciones para una pila de imágenes. </li></ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Este estudio evaluó el rendimiento de un panel de líneas celulares de cáncer en un ensayo de invasión esferoide (Tabla 1). En general, nos encontramos con que la formación de esferoides es mayor en los más líneas de células epiteliales de aspecto, donde la presencia de uniones célula-célula promueve la formación de una arquitectura esferoide similar. Líneas celulares conocidos que se han sometido a una transición epitelial-mesenquimal, como MDA-MB-231 células no forman esferoides en el cul...

Se establece que la motilidad de células de cáncer es predictivo de potencial metastásico, dado que tal comportamiento facilita la invasión a través de la membrana basal y la entrada en el sistema circulatorio 1. Mayoría de las investigaciones sobre la motilidad se ha centrado en cómo las células se comportan en un entorno de dos dimensiones (2D), a pesar de que se está ampliamente reconocido que el movimiento de las células en una matriz (3D) tridimensional es más representativo de cómo estas mismas células en realidad comportarse 2 in vivo. Sistemas de cultivo en 3D se utilizan cada vez más para estudiar comportamientos celulares que van desde la morfología celular, a la cinética de crecimiento y la sensibilidad de drogas 3. El deseo de supervisar la invasión de células de cáncer dentro del contexto de un entorno 3D ha llevado a la síntesis de las técnicas previamente establecidas que implican la generación de agregados de células 3D (esferoides) a través del método de cultivo gota colgante 4, seguido por la incorporación de estos esferoides en un extracelular 3D matriz (ECM) compuesto de colágeno y de la membrana basal materiales 5. Este método tiene por objeto mejorar en estas técnicas establecidas anteriormente proporcionando un método simplificado que se puede utilizar fácilmente para comparar invasión bajo una variedad de condiciones experimentales. Más formas tradicionales para evaluar la motilidad celular in vitro son el ensayo de rayado de la herida y el ensayo transwell 6. El primero representa ensayo de la motilidad celular en un entorno 2D, y es por lo tanto independiente de una variedad de características críticas para la invasión in vivo, por ejemplo, actividad de la proteasa 7. El ensayo transwell puede invasión de células modelo mejor cuando los insertos así se recubren con sustratos de ECM, pero sólo un único parámetro, es decir, la aparición de células en la superficie de la membrana opuesta se mide, y muchos matices de la invasión de células por lo tanto no son fácilmente observables. En contraste con estas técnicas, el ensayo de invasión de esferoides de células del cáncer (Figura 1 &lt;/ strong&gt;) permite la monitorización en tiempo real de la invasión de células en un entorno que no sólo es fisiológicamente relevante, sino que también permite que las características de la línea específica de células importantes para ser visualizados, como la migración de células individuales vs. colectiva 8. Este método también ofrece ventajas sobre los ensayos de crecimiento de cultivo 3D estándar. La generación de agregados celulares a través del método de la gota colgando limita inicialmente el movimiento celular, por lo que las células serán incentivados para invadir después de que se levantó esta restricción. Además, una vez que se levanta restricción, la salida de células procederá en una dirección uniforme que luego pueden ser convenientemente cuantificó. Los materiales de ECM más populares utilizados para esferoides ensayos de células del cáncer son Matrigel y colágeno tipo I, en donde cada uno de estos componentes tiene un papel importante y distintas en influir en el comportamiento metastásico. Matrigel es una mezcla secreta de las proteínas producidas por las células del sarcoma Engelbreth-Holm Swarm ratón, y se enriquece en sótano ocupandoproteínas de membrana nt como laminina, entactina, y el colágeno tipo IV 9. Por esta razón Matrigel se denomina de aquí en adelante como &quot;materiales de la membrana basal.&quot; Estos materiales proporcionan ligandos de la membrana basal esenciales necesarios para integrina adherencia durante la invasión de células de cáncer 10, además de muchas otras proteínas que ejercen una variedad de efectos sobre el comportamiento celular 11. En comparación, el colágeno tipo I, comúnmente preparada a partir de las digestiones ácidas de tendones y otras estructuras colágenas densas 12, es un material de matriz mucho más simple que sirve como un elemento estructural importante de los tejidos tejidos y estroma de sostén conectivos y órganos del cuerpo. Se ha demostrado que las características físicas de colágeno pueden regular una serie de características de la motilidad celular; por ejemplo, la alineación de las fibrillas de colágeno en la interfase tumor estromal permite las células cancerosas para migrar posteriormente a lo largo de las fibrillas al invadir el estroma 13 en. En el ensayo que aquí se presenta, tanto colágeno tipo I y los materiales de la membrana basal se utilizan como herramientas para estudiar el cáncer 3D interacciones célula-estroma. El efecto de la inhibición o estimulación de las vías que controlan la invasión se puede controlar después de que las células se han incrustado en la matriz 3D. Las células pueden ser pretratadas durante el crecimiento en las gotas colgantes o después de la transferencia a la cultura 3D, dependiendo de si se requiere un tratamiento prolongado para modular la invasión. Para los tratamientos más cortos, se recomienda que el fármaco se mezcla con la suspensión esferoide después de la recogida, así como los medios de comunicación que rodeará los cultivos 3D, para facilitar la exposición adecuada al fármaco a las células. A continuación, las células normales o asociados a tumores estromales se pueden mezclar con el material de matriz para evaluar su papel en la modulación de invasión de células tumorales, o para determinar cómo el comportamiento celular influencias de señalización paracrina y autocrina. Esta idea se demostró en un estudio en el que el co-cultivo de la colsobre el cáncer y las células endoteliales en gotas colgantes conducido a una red vascular dentro de los esferoides 14. Finalmente, los componentes de ECM también pueden ser alterados, como la invasión de células de cáncer se ve afectado por diferentes sustratos 15. El método presentado a continuación será por lo tanto proporcionar un marco para evaluar la invasión de células de cáncer bajo una variedad de condiciones. En general se encontró que no todas las líneas celulares crearán esferoides en las gotas colgantes y líneas de células epiteliales de aspecto típicamente formar esferas regulares.

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

a cancer cell spheroid assay to assess invasion in a 3d setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

Usando el ensayo de invasión esferoide (Figura 1), un panel de líneas celulares de cáncer se ensayó para determinar su capacidad para formar esferoides, así como para la cantidad de egreso celular exhibido después de la implantación en una matriz 3D que consiste en materiales membrana basal y colágeno tipo I (Tabla 1). Estos resultados demuestran que no todas las línea celular de cáncer creará esferoides bien formadas, donde las líneas celulares que poseen una morfología ep...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Un Cancer Cell esferoide Ensayo para evaluar la invasión en un entorno 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

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32.3K vistas.  Georgetown University. This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored. 

Video: A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

A tiempo real Técnica de Impedancia Eléctrica base para medir la invasión de monocapa de células endoteliales de las células cancerosas

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Investigación

Medicina

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

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

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

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

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

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Ensayo esferoide para medir TGF-β inducida por invasión

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelado y de imagen de 3 dimensiones invasión celular colectiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

La cuantificación de la invasividad de las células del cáncer de mama El uso de un modelo (3D) Tres Dimensiones

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Un Modificado<em&gt; In vitro</em&gt; Invasión Ensayo para determinar el papel potencial de hormonas, citoquinas y / o factores de crecimiento en la invasión de células del cáncer de Mediación

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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 ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Enfoque Formación: Un análisis basado en células para determinar el potencial oncogénico de un gen

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...