Este trabajo contó con el generoso apoyo de fondos de la Fundación del Cáncer Infantil (Baltimore, MD), Brandon Fundación Lee Carrington (Silver Spring, MD), DOD (W81XWH-10-1-0137) y el NIH (R01CA108641). Nos gustaría dar las gracias al Dr. Anton Wellstein y miembros de su laboratorio para compartir sus experiencias y ayudarnos a optimizar los métodos presentados.

1. Preparación <ol><li> Todos los pasos deben realizarse en condiciones estériles en una campana de cultivo de tejidos. </li><li> La estación de xCELLigence se coloca en una incubadora de 37 ° C en presencia de 5% de CO 2. </li><li> Usa las células HUVEC de paso bajo, preferiblemente no más que el paso 6. Además, asegúrese de que las células no son más del 75% de confluencia en el tiempo de la cosecha. </li><li> Las células HUVEC se cultivan en medios EGM-2 reconstituidos con kit de EGM-2 bala (Lonza Biosciences) que contiene factores de crecimiento, suplementos y 5% de suero bovino fetal. </li><li> Todos los rastros de tripsina deben ser retirados de las suspensiones de células haciendo girar las células a 200xg, lavando una vez con PBS, y resuspensión de ellos a densidades celulares indicadas. </li><li> Escudo xCELLigence E-placa de 16 con 0,1% de gelatina durante 1 hora a 37 ° C o durante la noche a 4 ° C. Lave la placa con PBS y añadir 100μL EGM-2 reconstituido medios. Realizar una lectura en blanco en el sistema para medir xCELLigence fondoimpedancia en ausencia de células. </li></ol> 2. Generación de HUVEC monocapa <ol><li> Coseche un frasco sub-confluente de células HUVEC mediante tripsinización. Resuspender las células en reconstituidas EGM-2 medio a una densidad final de x 10 5 células / ml 2.5. </li><li> A cada pocillo de un fondo calibrado E-placa que contiene 100μL EGM-2 los medios de comunicación, añadir 100μL de la suspensión de células HUVEC (10 4 células HUVEC 2,5 x). </li><li> Instale inmediatamente E-Plate en el instrumento xCELLigence. </li><li> Software xCELLigence programa para realizar lecturas de impedancia en intervalos de 10 minutos. </li><li> Deje que las células endoteliales crecen para 18-21 horas hasta que formen una monocapa, como se evidencia por un aplanamiento de índice de células (Figura 2). </li></ol> 3. La adición de las células y la normalización de datos invasor. <ol><li> Una vez que una monocapa HUVEC estable se ha formado, la cosecha células tumorales mediante tratamiento con tripsina y se resuspenden a una las casas de finalesdad de 1 x 10 5 células / ml en los medios utilizados para cultivar las células tumorales (tal como RPMI o DMEM que contiene 10% de FBS) </li><li> Lecturas de impedancia pausa en el software xCELLigence y eliminar E-Placa de la estación. </li><li> Retire EGM-2 medio por aspiración. Añadir 100μL de la suspensión de células tumorales que contiene 10 células x 1 4. En general, una proporción de células tumorales 1:2,5: células endoteliales sembradas, que funciona mejor para el ensayo. Sin embargo, la relación puede ser optimizado para las líneas celulares individuales. </li><li> Coloque la placa de E-en el sistema de xCELLigence en la incubadora y continuar lecturas de impedancia a intervalos de 10 minutos. </li><li> La invasión se puede monitorizar en tiempo real, en los próximos 6-12 horas como una caída en el índice de la célula debido a la retracción de las uniones endoteliales y la penetración por invasión de células tumorales. </li><li> Normalizar los resultados al momento de la adición de invasión de las células tumorales. El software permite la normalización xCELLigence a cualquier punto del tiempo y los resultadosse puede ver directamente en la ventana del programa. </li></ol> 4. Los resultados representativos: Un ejemplo de monocapa HUVEC invasión por las células metastásicas y no metastásico se muestra en la Figura 3. K7M2 es una línea celular de osteosarcoma metastásico. Estas células expresan altos niveles de la proteína del citoesqueleto ezrina enlazador, que representa las propiedades invasivas de esta línea celular 13,14. Cuando las células HUVEC se estimularon con células K7M2, hay una caída en la resistencia eléctrica dentro de las 6 horas. Esta caída en la resistencia eléctrica representa la invasión de la monocapa HUVEC por las células tumorales invasoras. La línea de células K12, por otra parte, expresa niveles mucho más bajos de ezrina y es por consiguiente menos metastásico 13. Desafiante la monocapa HUVEC con K12 células da como resultado una menos fuerte caída en la resistencia a medida que las células son incapaces de adherirse o invadir a través de la monocapa HUVEC (Figura 3). <imgsrc = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot; alt = &quot;Figura 1&quot; /&gt; Figura 1. Diagrama esquemático de proceso de flujo de trabajo para la invasión de las células endoteliales por células tumorales. Las células HUVEC se sembraron en E-placas recubiertas de gelatina y se dejaron formar una monocapa confluente. Las células malignas se añaden una vez que se ha formado una monocapa y la invasión se monitoriza en tiempo real, como el índice de la célula cambia debido a la invasión de la monocapa endotelial. <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="La figura 2"/> Figura 2. Formación monocapa HUVEC. Los cambios en el índice de células como las células HUVEC se unen a y se extienden sobre electrodos de oro recubiertas de gelatina. La formación de una monocapa confluente está representado por una estabilización de índice de células después de 18 horas. <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="Figura 3"/> Figura 3. La invasión de las células HUVEC por las células de osteosarcoma K7M2 y K12. Una fuerte disminución de la cell-índice se produce dentro de unas pocas horas de la introducción de las células altamente metastásicas K7M2. Células K12, por otro lado, son menos metastásico y son incapaces de penetrar en la monocapa endotelial tan eficientemente como las células K7M2. Esto está representado por una disminución menos pronunciada en el índice de la célula cuando la monocapa HUVEC se desafió con células K12. De control representa la impedancia de la monocapa HUVEC en ausencia de células cancerosas. Los experimentos se realizaron por triplicado.

<ol>
	<li>Klein, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer.+The+metastasis+cascade.">Cancer. The metastasis cascade.</a> Science. 321, 1785-1787 (2008).</li><li>Chiang, A. C., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+metastasis.">Molecular basis of metastasis.</a> N Engl J Med. 359, 2814-2823 (2008).</li><li>Orr, F. W., Wang, H. H., Lafrenie, R. M., Scherbarth, S., Nance, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+cancer+cells+and+the+endothelium+in+metastasis.">Interactions between cancer cells and the endothelium in metastasis.</a> J Pathol. 190, 310-329 (2000).</li><li>Bacac, M., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+cancer+cell.">Metastatic cancer cell.</a> Annu Rev Pathol. 3, 221-247 (2008).</li><li>Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+signals+from+the+invasive+front.">New signals from the invasive front.</a> Nature. 441, 444-450 (2006).</li><li>Kramer, R. H., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+tumor+cells+with+vascular+endothelial+cell+monolayers:+a+model+for+metastatic+invasion.">Interactions of tumor cells with vascular endothelial cell monolayers: a model for metastatic invasion.</a> Proc Natl Acad Sci U S A. 76, 5704-5708 (1979).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proc Natl Acad Sci U S A. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromotion+of+mammalian+cells+measured+electrically.">Micromotion of mammalian cells measured electrically.</a> Proc Natl Acad Sci U S A. 88, 7896-7900 (1991).</li><li>Tiruppathi, C., Malik, A. B., Del Vecchio, P. J., Keese, C. R., Giaever, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+method+for+detection+of+endothelial+cell+shape+change+in+real+time:+assessment+of+endothelial+barrier+function.">Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.</a> Proc Natl Acad Sci U S A. 89, 7919-7923 (1992).</li><li>Whipple, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+promotes+tubulin+Detyrosination+and+Microtentacles+that+enhance+endothelial+engagement.">Epithelial-to-mesenchymal transition promotes tubulin Detyrosination and Microtentacles that enhance endothelial engagement.</a> Cancer Res. 70, 8127-8137 (2010).</li><li>Boyd, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-microelectronic+sensing+technique+for+profiling+cytotoxicity+of+chemicals.">A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.</a> Anal Chim Acta. 615, 80-87 (2008).</li><li>Khanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane-cytoskeleton+linker+ezrin+is+necessary+for+osteosarcoma+metastasis.">The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.</a> Nat Med. 10, 182-186 (2004).</li><li>Ren, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin-cytoskeleton+linker+protein+ezrin+is+regulated+during+osteosarcoma+metastasis+by+PKC.">The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.</a> Oncogene. 28, 792-802 (2009).</li></ol>

El costo de la publicación de este manuscrito fue proporcionado amablemente por Roche Applied Science.

El ensayo de invasión de células HUVEC en tiempo real es un método simple pero potente, para la supervisión invasión. Se proporciona resultados en tiempo real, lo cual es una ventaja significativa sobre otras técnicas tradicionales que se basan en el análisis de punto final. El ensayo puede ser modificado para fármacos de ensayo, anticuerpos, ligandos y proteínas como inhibidores o estimuladores de la metástasis. El efecto de diferentes agentes en células endoteliales / cáncer de diafonía puede evaluarse también. Cuando la introducción de agentes exógenos, tales como factores de crecimiento y drogas en los medios de cultivo, es importante para asegurarse de que no afectan a la integridad de la monocapa HUVEC. La interrupción de la monocapa HUVEC se puede probar mediante la introducción del agente exógeno en ausencia de células invasoras, y asegurar que no hay ningún cambio en el índice de la célula. Por lo tanto, los controles apropiados deben ser incluidos como parte del diseño experimental. Líneas celulares diferentes demuestran diferentes curvas de células de índice, ya que forman un confmonocapa luent. La forma de la curva, así como la célula-índice máximo alcanzado, es de tipo celular específico. Células HUVEC muestran una característica transitoria aplanamiento de índice de células 4-6 horas después de la siembra, seguido por otro de estabilización en un índice mayor de células después de 16-18 horas. Por lo tanto, es importante dejar que las células forman una monocapa confluente durante al menos 18 horas antes de la adición de las células tumorales. Dado que el índice de células es dependiente sobre el medio ambiente iónico en la interfase electrodo / célula, varios factores tales como la morfología celular y la calidad de las adherencias célula-célula influyen en el índice de células, además de la zona del fondo del pozo cubierto. Por lo tanto, la disminución en el índice de células, que se produce tras la invasión de células tumorales, es una función de varios factores, que incluyen la retracción de las uniones endoteliales y la posterior invasión de células tumorales. El instrumento xCELLigence se fabrica en tres configuraciones diferentes: analizador de células en tiempo real(RTCA) SP, RTCA MP y DP RTCA. El instrumento RTCA SP tiene un 96-así placa de E-mientras que el instrumento RTCA MP con capacidad para seis de 96 pocillos E-platos simultáneamente. Esto permite la selección de alto rendimiento de fármacos y compuestos. Los experimentos descritos en este artículo se realizaron con el instrumento DP RTCA, con capacidad para tres de 16 y E-platos. Cada estación de soporte E-placa puede funcionar de forma independiente, lo que permite que varios usuarios ejecutar experimentos de forma simultánea. El instrumento DP RTCA también se puede utilizar para estudiar la migración utilizando placas de CIM. Estas son placas de 16 pocillos con cámaras superior e inferior separadas por un tereftalato de polietileno (PET) de membrana con un tamaño medio de poro de 8um. Los sensores electrónicos se encuentran en la parte inferior de la membrana porosa de la cámara superior. La cámara inferior sirve como un depósito para quimioatrayente para las células en la cámara superior. Sin embargo, una ventaja importante de la invasión de ensayo HUVEC sobre las placas de CIM es que la invasión de células tumorales en la antigua is no restringido por el tamaño de poro, como la invasión de células endoteliales depende de la diafonía entre las células tumorales y endoteliales. El ensayo de invasión de células HUVEC también se puede realizar en ECIS Z instrumento, fabricado por Applied Biofísica (Troy, NY). El Z instrumento ECIS utiliza una tecnología similar a la del instrumento xCELLigence, con las diferencias en las configuraciones de matriz y el electrodo. Hemos llevado a cabo con éxito los ensayos de invasión HUVEC utilizando los arrays ECIS 8 pocillos. Es importante tener en cuenta que el área de superficie de pocillos de fondo es mayor en las matrices de ECIS, lo que requiere un mayor número de células endoteliales y tumorales a ser chapado: 2,5 x 10 5 células y 1 x 10 5 respectivamente.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr> 
 <tr>
 	<td>xCELLigence RTCA DP station</td>
 <td>Roche	</td>
 <td>05469759001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>RTCA control unit</td>
 <td>Roche</td>
 <td>05454417001</td>
 <td>Notebook with preinstalled RTCA software</td>
 </tr>
 <tr>
 	<td>E-Plate 16</td>
 <td>Roche</td>
 <td>05469830001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HUVEC cells</td>
 <td>Lonza</td>
 <td>CC-2517</td>
 <td></td>
 </tr>
 <tr>
 	<td>EGM-2 media</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>EGM-2 bullet kit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>0.1% Gelatin in 
sterile H2O
</td>
 <td>Millipore</td>
 <td>MCPROTO045</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells

La diseminación metastásica de las células malignas requiere la degradación de la membrana basal, la unión de células tumorales al endotelio vascular, la retracción de las uniones endoteliales y, finalmente, la invasión y la migración de las células tumorales a través de la capa endotelial para entrar en el torrente sanguíneo como un medio de transporte a sitios distantes en el huésped 1-3. Una vez en el sistema circulatorio, las células cancerosas se adhieren a las paredes capilares y extravasación al tejido circundante para formar tumores metastásicos 4,5. Los diversos componentes de interacción célula-célula endotelial del tumor pueden replicarse in vitro por impugnar una monocapa de células endoteliales de la vena umbilical humana (HUVEC) con células de cáncer. Los estudios realizados con electrones y microscopía de contraste de fase sugieren que la secuencia en vitro de eventos bastante representan el vivo proceso metastásico 6 in. A continuación, describimos una técnica basada en impedancia eléctrica que controla y cuantifica en tiempo real de los INVASiones de las células endoteliales por células tumorales malignas. Giaever y Keese descritos por primera vez una técnica para medir las fluctuaciones en la impedancia cuando una población de células de crecer en la superficie de los electrodos 7,8. El instrumento xCELLigence, fabricado por Roche, utiliza una técnica similar para medir cambios en la impedancia eléctrica como las células se adhieren y propagan en una placa de cultivo cubierto con una matriz de microelectrodos de oro que cubre aproximadamente el 80% de la superficie en el fondo de un pozo. Como las células se adhieren y propagan sobre la superficie del electrodo, que conduce a un aumento de la impedancia eléctrica 9-12. La impedancia se muestra como un parámetro denominado índice de células-adimensional, que es directamente proporcional a la superficie total de cultivo de tejidos bien que está cubierto por las células. Por lo tanto, el índice de célula puede ser usado para monitorear la adhesión celular, la difusión, la morfología y la densidad de las células. La invasión de ensayo descrito en este artículo se basa en los cambiosen la impedancia eléctrica en la interfase electrodo / célula, como una población de células malignas invadir a través de una monocapa de HUVEC (Figura 1). La interrupción de las uniones endoteliales, retracción de la monocapa endotelial y la sustitución por las células tumorales conduce a grandes cambios en la impedancia. Estos cambios se correlacionan directamente con la capacidad invasiva de las células tumorales, es decir, la invasión de células altamente agresivos conducen a grandes cambios en la impedancia celular y viceversa. Esta técnica proporciona una doble ventaja sobre los métodos existentes de la medición de la invasión, tales como cámara de Boyden y ensayos de matrigel: 1) la interacción célula-célula endotelial del tumor imita más estrechamente el proceso in vivo, y 2) se obtienen los datos en tiempo real, tiempo y es más fácilmente cuantificable, en comparación con el análisis de punto final para otros métodos.

Watch this Scientific Journal Video about A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells at JoVE.com

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

En este artículo se describe una<em&gt; In vitro</em&gt; Técnica para las células cancerosas invasoras de vigilancia a través de una monocapa de células endoteliales. Los datos se adquiere en tiempo real como una función de los cambios en la impedancia en la superficie de los electrodos en la parte inferior así.

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 ...

a-real-time-electrical-impedance-based-technique-to-measure-invasion

Research

JoVE Journal

Medicine

27.2K vistas.  Georgetown University. En este artículo se describe una In vitro Técnica para las células cancerosas invasoras de vigilancia a través de una monocapa de células endoteliales. Los datos se adquiere en tiempo real como una función de los cambios en la impedancia en la superficie de los electrodos en la parte inferior así.

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

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

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

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

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

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

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 ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Una plataforma de detección de luciferasa basado multiplexado para interrogar cáncer asociado transducción de señales en las células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

Líquido cefalorraquídeo MicroRNA perfiles utilizando cuantitativa en tiempo real PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

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 ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tumor Ensayo tridimensional (3D) esferoide Invasion

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Aislamiento de células endoteliales linfático humano por Multi-parámetro de clasificación de células activadas por fluorescencia

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...