Este trabalho foi generosamente apoiado por fundos da Fundação do Câncer Infantil (Baltimore, MD), Brandon Lee Carrington Foundation (Silver Spring, MD), DOD (W81XWH-10-1-0137) e NIH (R01CA108641). Gostaríamos de agradecer ao Dr. Anton Wellstein e membros de seu laboratório para compartilhar sua experiência e nos ajudando a otimizar métodos apresentados.

1. Preparação <ol><li> Todos os passos devem ser realizados em condições estéreis, numa câmara de cultura de tecidos. </li><li> A estação xCELLigence é colocada numa incubadora de 37 ° C na presença de 5% de CO 2. </li><li> Use células HUVEC passagem baixas, de preferência, não mais do que a passagem 6. Além disso, certifique-se de que as células não são mais do que 75% confluente no momento da colheita. </li><li> As células HUVEC são cultivadas em meios EGM-2 reconstituídos com kit EGM-2 bala (Lonza Biosciences) contendo factores de crescimento, suplementos e 5% de soro fetal bovino. </li><li> Todos os vestígios de tripsina devem ser removidos a partir de suspensões de células por centrifugação a 200xg células, lavagem uma vez com PBS, e ressuspensão de densidades celulares indicadas. </li><li> Revestimento xCELLigence E-chapa 16 com 0,1% de gelatina durante 1 hora a 37 ° C ou durante a noite a 4 ° C. Lavar a placa com PBS e adicionar 100 uL reconstituído mídia EGM-2. Realizar uma leitura em branco no sistema xCELLigence para medir fundoimpedância na ausência de células. </li></ol> 2. Gerando monocamada HUVEC <ol><li> Colher um frasco de sub-confluente de células HUVEC por tripsinização. Ressuspender as células em reconstituídos EGM-2 Meios para uma densidade final de 2,5 x 10 5 células / ml. </li><li> A cada poço de uma base calibrado E-placa contendo 100 ul de meio EGM-2, adicionar 100 uL da suspensão de células HUVEC (2,5 x 4 10 células HUVEC). </li><li> Instalar imediatamente E-Plate no instrumento xCELLigence. </li><li> Software xCELLigence programa para realizar leituras de impedância em intervalos de 10 minutos. </li><li> Permitir que as células endoteliais para crescer 18-21 horas até se formar uma monocamada, como evidenciado por um achatamento do índice de células (Figura 2). </li></ol> 3. Além das células e normalização de dados invasor. <ol><li> Uma vez que uma monocamada de HUVEC estável formado, a colheita de células tumorais por tripsinização e ressuspender a um tocas finaisdade de 1 x 10 5 células / ml em meios utilizados para crescer células tumorais (tais como meios RPMI ou DMEM contendo 10% de FBS) </li><li> Leituras de impedância pausa do software xCELLigence e remover E-Plate da estação. </li><li> Remover EGM-2 media por aspiração. Adicionar 100 uL de suspensão de células de tumor contendo 1 x 4 10 células. Geralmente, uma proporção de células de tumor de 1:2,5: células endoteliais semeadas, funciona melhor para o ensaio. No entanto, a proporção pode ser optimizado para as linhas de células individuais. </li><li> Colocar a placa de E no sistema xCELLigence na incubadora e continuar leituras de impedância em intervalos de 10 minutos. </li><li> Invasão pode ser monitorado em tempo real ao longo dos próximos 6-12 horas como uma queda no índice de células devido à retração das junções endoteliais e penetração de invadir as células tumorais. </li><li> Normalizar os resultados para o momento da adição de invadir as células tumorais. O software xCELLigence permite a normalização de qualquer ponto de tempo e os resultadospode ser visto directamente na janela do software. </li></ol> 4. Resultados representativos: Um exemplo de monocamada HUVEC invasão por células metastáticas e não metastáticas é mostrado na Figura 3. K7M2 é uma linha de células de osteossarcoma metastático. Estas células expressam níveis elevados da proteína do citoesqueleto ezrina ligante, que representa as propriedades invasivas de esta linha celular 13,14. Quando as células HUVEC são desafiados com K7M2 células, há uma queda na resistência eléctrica dentro de 6 horas. Esta queda na resistência eléctrica representa a invasão de monocamada HUVEC pelas células do tumor invasoras. A linha de células K12, por outro lado, expressa níveis muito mais baixos de ezrina e é, consequentemente, menos metastático 13. Um desafio a monocamada de HUVEC com K12 células resulta em uma diminuição menos acentuada da resistência à medida que as células são incapazes de aderir ou invadir através da monocamada de HUVEC (Figura 3). <imgsrc = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot; alt = &quot;Figura 1&quot; /&gt; Figura 1. Diagrama esquemático do processo de fluxo de trabalho para a invasão de células endoteliais por células tumorais. As células HUVEC foram semeadas em placas de E-revestidos de gelatina para permitir a formação de uma monocamada confluente. As células malignas são adicionados uma vez por monocamada formou e invasão é monitorado em tempo real como o índice da célula muda devido à invasão da monocamada endotelial. <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="Figura 2"/> Figura 2. Formação de monocamada HUVEC. Mudanças no índice de células como células HUVEC juntar-se e espalhar sobre eletrodos de ouro gelatina revestidos. A formação de uma monocamada confluente é representado por uma estabilização do índice de células após 18 horas. <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="Figura 3"/> Figura 3. Invasão de células HUVEC por K7M2 e K12 células de osteossarcoma. A queda acentuada no cell-índice ocorre dentro de poucas horas após a introdução das células altamente metastáticas K7M2. K12 células, por outro lado, são menos metastático e são incapazes de penetrar na monocamada endotelial tão eficientemente quanto K7M2 células. Isto é representado por uma diminuição no índice de menos acentuada quando a monocamada de células HUVEC é desafiado com células K12. Controlo representa uma impedância de monocamada HUVEC, na ausência de células de cancro. Os experimentos foram realizados em triplicata.

<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>

O custo para a publicação desse manuscrito foi gentilmente cedido pela Roche Applied Science.

O ensaio de invasão HUVEC em tempo real é um método simples, mas poderosa para a invasão de monitoramento. Ele fornece resultados em tempo real, o que é uma vantagem significativa em relação a outras técnicas tradicionais, que se baseiam na análise de ponto final. O ensaio pode ser modificado para testar drogas, anticorpos, ligandos e proteínas como inibidores ou estimuladores da metástase. O efeito de diferentes agentes na endotelial / célula cancerosa conversa cruzada pode ser avaliada, bem. Quando a introdução de agentes exógenos, tais como factores de crescimento e os medicamentos nos meios de cultura, é importante para garantir que eles não afectam a integridade da monocamada de HUVEC. Perturbação da monocamada HUVEC pode ser testada através da introdução do agente exógeno na ausência de células invasoras, e garantir que não existe qualquer alteração no índice de células. Assim, os controles apropriados devem ser incluídos como parte do projeto experimental. Linhas celulares diferentes demonstrar diferentes curvas de índice de células, que formam um confmonocamada luent. A forma da curva, bem como o valor máximo do índice da célula atingida, é do tipo de célula específico. Células HUVEC mostram uma característica transitória achatamento do índice de célula de 4-6 horas após a semeadura, seguido por outro de estabilização em um índice maior de células após 16-18 horas. Por isso, é importante para permitir que as células formam uma monocamada confluente durante pelo menos 18 horas antes da adição de células tumorais. Uma vez que o índice de células está dependente do ambiente iónico no eléctrodo / células de interfase, por vários factores tais como a morfologia celular e qualidade de adesões célula-célula influenciar o índice de células, para além da área do poço inferior coberta. Assim, a diminuição no índice de célula, que ocorre após a invasão de células de tumor, é uma função de diversos factores, que incluem a retracção das junções endoteliais e invasão de células de tumor subsequente. O instrumento xCELLigence é fabricado em três diferentes configurações: analisador de células em tempo real(RTCA) SP, RTCA MP e RTCA DP. O instrumento RTCA SP detém uma de 96 poços E-plate Considerando que o instrumento RTCA MP pode conter até seis de 96 poços E-chapas simultaneamente. Isto permite o rastreio de alto rendimento de drogas e compostos. Os experimentos deste artigo são realizadas utilizando o instrumento DP RTCA, que pode realizar três de 16 poços E-placas. Cada estação de realização E-plate pode ser operado de forma independente, o que permite que múltiplos usuários para executar experiências em simultâneo. O instrumento RTCA DP pode também ser usado para estudar a migração usando CIM-chapas. Estes são pratos de 16 poços com câmaras superior e inferior separadas por um tereftalato de polietileno (PET) de membrana com um tamanho de poro médio do 8um. Os sensores electrónicos estão localizados no lado de baixo da membrana porosa da câmara superior. A câmara inferior serve como um reservatório para o quimioatraente para células na câmara superior. No entanto, uma das principais vantagens do ensaio de invasão HUVEC nas placas CIM é que a invasão de células tumorais na ex-inão é restringida pelo tamanho do poro, como a invasão de células endoteliais depende de conversa cruzada entre as células tumorais e endoteliais. O ensaio de invasão de HUVEC também pode ser realizada em ECIS Z instrumento, fabricado por Biofísica Aplicada (Troy, NY). O Z instrumento ECIS utiliza uma tecnologia semelhante ao instrumento xCELLigence, com diferenças nas configurações da matriz e eletrodo. Temos realizado com sucesso testes de invasão HUVEC usando as matrizes ECIS oito poços. É importante notar que a área de superfície bem inferior é maior nas matrizes ECIS, o que requer um maior número de células endoteliais e de tumor seja banhado: 2,5 x 10 5 a 1 x 10 5 células, 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

Disseminação metastática de células malignas requer degradação da membrana basal, a ligação de células de tumor ao endotélio vascular, a retracção das junções endoteliais e, finalmente, a invasão e a migração de células tumorais através da camada endotelial para entrar na corrente sanguínea, como meio de transporte para locais distantes do hospedeiro 1-3. Uma vez que no sistema circulatório, as células cancerígenas aderem à parede capilar e extravasar para o tecido circundante de modo a formar tumores metastáticos 4,5. Os vários componentes de interacção célula-célula endotelial tumoral pode ser replicado in vitro por desafio de uma monocamada de células endoteliais humanas da veia umbilical (HUVEC) com as células cancerosas. Estudos realizados com elétrons e microscopia de contraste de fase sugerem que a seqüência em vitro de eventos representam adequadamente o processo metastático in vivo 6. Aqui, descrevemos uma técnica baseada em impedância elétrica que monitora e quantifica em tempo real, as invasião de células endoteliais de células tumorais malignas. Giaever Keese e descrita pela primeira vez uma técnica para medir as flutuações na impedância quando uma população de células crescem sobre a superfície dos eléctrodos 7,8. O instrumento xCELLigence, fabricado pela Roche, utiliza uma técnica semelhante para medir as variações na impedância eléctrica como células de prender e espalhado num prato de cultura coberta com uma matriz de microeléctrodos de ouro que cobre cerca de 80% da superfície da parte inferior do poço. Como as células anexar e espalhada sobre a superfície do eléctrodo, ele conduz a um aumento na impedância eléctrica 9-12. A impedância é apresentada como um parâmetro sem dimensões denominado índice de células, que é directamente proporcional à área total do poço de cultura de tecidos, que é coberto por células. Assim, o índice de célula pode ser usado para monitorar a adesão celular, espalhamento, a morfologia e a densidade de células. O ensaio de invasão descrito neste artigo é baseado em mudançasna impedância eléctrica no eléctrodo / células de interfase, como uma população de células malignas invadem através de uma monocamada de HUVEC (Figura 1). A ruptura das junções endoteliais, retracção da monocamada de células endoteliais e sua substituição por células tumorais levar a grandes alterações na impedância. Estas alterações directamente correlacionadas com a capacidade invasiva de células tumorais, isto é, a invasão por células altamente agressivos levar a grandes alterações na impedância de células e vice-versa. Esta técnica proporciona uma dupla vantagem em relação aos métodos existentes de medição, tais como invasão câmara de Boyden e ensaios matrigel: 1) a interacção célula-célula endotelial tumoral imita mais de perto o processo in vivo, e 2) os dados são obtidos em tempo real, tempo e é mais facilmente quantificáveis, em oposição a análise de ponto final para outros 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

Este artigo descreve uma<em&gt; In vitro</em&gt; Técnica para células cancerosas invasoras de monitorização por meio de uma monocamada de células endoteliais. Os dados são adquiridos em tempo real, como uma função de alterações na impedância sobre a superfície de eléctrodos no fundo bem.

A técnica de impedância elétrica em tempo real baseado medir Invasão de monocamada de células endoteliais por células cancerosas

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

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Research

JoVE Journal

Medicine

27.2K visualizações.  Georgetown University. Este artigo descreve uma In vitro Técnica para células cancerosas invasoras de monitorização por meio de uma monocamada de células endoteliais. Os dados são adquiridos em tempo real, como uma função de alterações na impedância sobre a superfície de eléctrodos no fundo bem.

Vídeo: 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.

Avaliação de Captação de nanopartículas em tumores em tempo real usando imagens intravital

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

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-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

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.

Ensaio esferóide para TGF-β Medida induzida 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 ...

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.

Modelagem e imagem 3-Dimensional Invasion cela coletiva

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.

A plataforma de triagem baseada luciferase diversificadas para interrogar associados ao câncer transdução de sinal em 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 cefalorraquidiano MicroRNA perfil usando Quantitative PCR em Tempo Real

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.

A modificação<em&gt; In vitro</em&gt; Invasion Ensaio para determinar o possível papel dos hormônios, citocinas e / ou fatores de crescimento na Cancer Mediating Invasion celular

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.

Avaliação do cancro do ovário esferóide Apego e invasão de células mesoteliais em Tempo 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.

Tridimensional (3D) Tumor Esferóide 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 ...

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.

Isolamento de linfático humano endotelial por multi-parâmetro activadas por fluorescência celular

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