The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1. Prepare los diferentes medios de comunicación y componentes adicionales <ol><li> Antes del experimento, que consiste en preparar los medios DMEM u otros medios especificados con la adición de FBS normales, tratado con carbón vegetal FBS, o FBS tratado con carbón vegetal más el componente a ensayar. Tenga en cuenta que varios componentes pueden ser probados en cada experimento. </li><li> Pesar y diluir las hormonas, factores de crecimiento o citoquinas apropiadamente para ser disueltos en el suero tratado con carbón vegetal a la concentración fisiológica. </li></ol> 2. Preparar la matriz de colágeno en el hielo <ol><li> Preparar 2 ml de colágeno I matriz a 2,2 mg / ml mediante la adición de los siguientes componentes filtrados estériles en hielo: 200 l 10x PBS (pH 7,4), 5,4 l de NaOH 1 N, 600 l de doble H 2 O destilada y 1,2 ml de colágeno I (a 3,63 mg / ml). </li><li> Mantenga solución de colágeno I en hielo hasta que esté listo a la placa. </li></ol> 3. Preparar las placas de la migración / invasión de ensayo &lt;/ P&gt; <ol><li> Para cada línea celular a ensayar, utilizar una placa de cámara de 24 pocillos en la que 12-pocillos contienen insertos. Usa los 12 pozos adicionales que no contienen insertos para la adición de los medios de comunicación quimioatrayentes y transferir los insertos para el conjunto experimental arriba. NOTA: una matriz de colágeno en placas con un tereftalato de polietileno (PET) de la membrana y 8 micras de tamaño de poro es óptimo para las líneas celulares utilizan aquí. Sin embargo, una matriz de Matrigel en placas prerrevestidas también puede estar sustituido con el tamaño de poro disminuido de acuerdo con la línea celular siendo investigado. </li><li> Etiquetar claramente la placa, utilizando 3 pozos por condición de ser analizados (migración FBS, migración CS-FBS, de invasión de SFB, y la invasión CS-FBS, así como FBS a la migración para un control, línea celular no invasivo). Ensayo de migración por el movimiento a través de poros en una membrana de PET, y la invasión de ensayo por el movimiento a través de una matriz de colágeno o Matrigel y luego a través de poros en la membrana. Utilice diferentes marcadores de color para cada condición de la celda a unIdentificación en el proceso de recubrimiento. </li></ol> 4. Vierta la Matriz Invasion <ol><li> Cuidadosamente pipeta 75 l de la solución matriz de colágeno en los insertos que se utilizarán para ensayos de invasión. Tenga cuidado para evitar burbujas. Dispersar las burbujas de mediante la aplicación de una punta de pipeta invertida a la superficie. </li><li> Transferir la placa con los insertos recubiertos con colágeno a una incubadora de CO 2 37 ° C y 5% durante 30 min para permitir que el gel se solidifique. </li></ol> 5. Placa de las células en la membrana o matriz Invasion <ol><li> Mientras tanto, trypsinize células y añadir medios con 10% de SFB. Centrifugar las células a 200 xg durante 5 minutos en una centrífuga de mesa y enjuague 3x en medio libre de suero. </li><li> Volver a suspender en medio libre de suero. Contar las células con un hemocitómetro o un contador de diapositivas automatizada. Añadir medio libre de suero a una concentración final de 5 x 10 4 células / ml. </li><li> Cuando la matriz de colágeno se ha solidificado (después de 30 minutos), añadir 700 l de los medios de comunicación, ya sea con2% FBS o definidos con carbón vegetal FBS a cada pocillo. De las 12 inserciones por placa: <ul><li> 3 insertos tienen colágeno y pozos con los medios de comunicación + FBS al 2% </li><li> 3 insertos tienen colágeno y pozos con los medios de comunicación + 2% CS-FBS </li><li> 3 insertos no tienen colágeno y pozos con los medios de + 2% de FBS </li><li> 3 insertos no tienen colágeno y pozos con los medios de comunicación + 2% CS-FBS </li></ul><ol><li> Use platos adicionales dependiendo del número de factores siendo probado y con un control sin colágeno correspondiente a cada condición. </li></ol></li><li> Añadir suspensión celular a los insertos en 5 x 10 4 células / ml, placas 500 células l por inserción en todas las inserciones de migración e invasión. </li><li> Se incuban las células durante 22 horas a 37 ° C. </li></ol> 6. cuantificar el número de Migración y células invasoras <ol><li> Configure tinción de pozos utilizando fijador metanol, eosina y hematoxilina, en pocillos separados. </li><li> Use hisopos de algodón para eliminar las células y la matriz de cada pocillo.Rrepeat con segunda aplicación hisopo para cada pozo. </li><li> Con unas pinzas, sumerja cada inserto 5 veces durante 1 seg en cada una de las 3 soluciones en sucesión. </li><li> Permitir inserciones se sequen O / N. </li><li> O bien i) eliminar filtros con un bisturí, cortar cuidadosamente alrededor de los bordes y montar en un portaobjetos con cubreobjetos y de inmersión en aceite, o ii) permitir que las inserciones se sequen O / N invierte y se utilizan los insertos directamente para microscopía. </li><li> El día siguiente, ver diapositivas o insertos bajo un microscopio con un objetivo de 20X y tomar 5 imágenes de diferentes regiones del filtro. Para mejorar la consistencia, tome 4 campos exteriores y uno central. </li><li> Contar las células para todas las condiciones utilizando el software ImageJ y se aplican a las siguientes fórmulas. </li><li> Determinar la invasión por ciento de la siguiente manera: La media de # de células invasoras a través de colágeno inserto = a La media de # de células que migran a través de inserción de control = b Invasion% = (a / b) * 100 </li><li> Determinar el índice de invasión en 2% de FBS como sigue: % Invasión de las células se sometió a ensayo (en 2% FBS) = c % Invasión de las células no invasivas de control en (2% FBS) = d Invasión Index (FBS) = (c / d) </li><li> Determinar el índice de invasión en 2% CS-FBS como sigue: % Invasión de las células se sometió a ensayo (en 2% CS-FBS) = e % Invasión de las células no invasivas de control en (2% CS-FBS) = f Invasión Index (CS-FBS) = (E / F) </li></ol> 7. Repita Protocolo experimental Comparando con carbón vegetal FBS al FBS despojado-carbón + X n con múltiples factores combinados <ol><li> Repetir el procedimiento varias veces como sea necesario el uso de diferentes componentes para &quot;X&quot; o una combinación de componentes. </li><li> Aplicar los cálculos para determinar la contribución de cada factor &quot;X&quot; para los efectos de la migración y la invasión. </li></ol>

<ol>
	<li>Weinberg, 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=.">.</a> The Biology of Cancer. , (2013).</li><li>Hanahan, D., Weinberg, 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=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Hanahan, D., Weinberg, 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=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100 (1), 57-70 (2000).</li><li>Valastyan, S., Weinberg, 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=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Dang, Z. C., Lowik, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+serum+factors+by+charcoal+treatment+promotes+adipogenesis+via+a+MAPK-dependent+pathway.">Removal of serum factors by charcoal treatment promotes adipogenesis via a MAPK-dependent pathway.</a> Mol Cell Biochem. 268 (1-2), 159-167 (2005).</li><li>Yohay, D. A., Zhang, J., Thrailkill, K. M., Arthur, J. M., Quarles, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+serum+in+the+developmental+expression+of+alkaline+phosphatase+in+MC3T3-E1+osteoblasts.">Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts.</a> J Cell Physiol. 158 (3), 467-475 (1994).</li><li>Zucker, S. N., Bancroft, T. A., Place, D. E., Des Soye, ., Bagati, B., Berezney, A., R, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dominant+negative+Cx43+mutant+differentially+affects+tumorigenic+and+invasive+properties+in+human+metastatic+melanoma+cells.">A dominant negative Cx43 mutant differentially affects tumorigenic and invasive properties in human metastatic melanoma cells.</a> J Cell Physiol. 228 (4), 853-859 (2013).</li><li>Fox, E. M., Andrade, J., Shupnik, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+actions+of+estrogen+to+promote+proliferation:+integration+of+cytoplasmic+and+nuclear+pathways.">Novel actions of estrogen to promote proliferation: integration of cytoplasmic and nuclear pathways.</a> Steroids. 74 (7), 622-627 (2009).</li><li>Meierjohann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP13+mediates+cell+cycle+progression+in+melanocytes+and+melanoma+cells:+in+vitro+studies+of+migration+and+proliferation.">MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation.</a> Molecular Cancer. 9, 201 (2010).</li><li>Smalley, K. S., Haass, N. K., Brafford, P. A., Lioni, M., Flaherty, K. T., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+signaling+pathways+must+be+targeted+to+overcome+drug+resistance+in+cell+lines+derived+from+melanoma+metastases.">Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases.</a> Mol Cancer Ther. 5 (5), 1136-1144 (2006).</li><li>Both, N. J., Vermey, M., Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+evaluation+of+various+invasion+assays+testing+colon+carcinoma+cell+lines.">A comparative evaluation of various invasion assays testing colon carcinoma cell lines.</a> Br J Cancer. 81 (6), 934-941 (1999).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+cancer+metastasis:+the+'seed+and+soil'+hypothesis+revisited.">The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited.</a> Nat. Rev. Cancer. 3, 453-458 (2003).</li><li>Huang, Y., et al. <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+in+human+breast+epithelial+cells+transformed+by+17beta-estradiol.">Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol.</a> Cancer Res. 67 (23), 11147-11157 (2007).</li><li>Huang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+regulation+of+miR-21+enhances+migration+and+invasion+of+hepatoma.">Thyroid hormone regulation of miR-21 enhances migration and invasion of hepatoma.</a> Cancer Res. 73 (8), 2505-2517 (2013).</li><li>Scheel, C., Weinberg, 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=Cancer+stem+cells+and+epithelial-mesenchymal+transition:+concepts+and+molecular+links.">Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.</a> Semin. Cancer Biol. 22 (5-6), 396-403 (2012).</li><li>Kojima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+TGF-beta+and+stromal+cell-derived+factor-1+(SDF-1)+signaling+drives+the+evolution+of+tumor-promoting+mammary+stromal+myofibroblasts.Proc.">Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts.Proc.</a> Natl Acad Sci U S A. 107 (46), 20009-20014 (2010).</li><li>Maehara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transforming+growth+factor-&#946;1+in+invasion+and+metastasis+in+gastric+carcinoma.">Role of transforming growth factor-&#946;1 in invasion and metastasis in gastric carcinoma.</a> J. Clin. Oncology. 17 (2), 607-614 (1999).</li><li>Lu, Z., Jiang, G., Blume-Jensen, P., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor-induced+tumor+cell+invasion+and+metastasis+initiated+by+dephosphorylation+and+downregulation+of+focal+adhesion+kinase.">Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase.</a> Mol. Cell Biol. 21 (12), 4016-4031 (2001).</li><li>Korkaya, H., Liu, S., Wicha, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+stem+cells,+cytokine+networks,+and+the+tumor+microenvironment.">Breast Cancer stem cells, cytokine networks, and the tumor microenvironment.</a> J. Clin. Invest. 121 (10), 3804-3809 (2011).</li><li>Rosen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interrogation+of+HGF-SF+with+other+cytokines+in+tumor+invasion+and+metastasis.">The interrogation of HGF-SF with other cytokines in tumor invasion and metastasis.</a> EXS. 65, 301-310 (1993).</li><li>Sanz-Moreno, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+and+JAK1+signaling+cooperate+to+control+actomyosin+contractility+in+tumor+cells+and+stroma.">ROCK and JAK1 signaling cooperate to control actomyosin contractility in tumor cells and stroma.</a> Cancer Cell. 20 (2), 229-245 (2011).</li><li>Fleming, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+interactions+between+primary+human+human+fibroblasts+enhance+murine+mammary+gland+humanization+in+vivo.">Paracrine interactions between primary human human fibroblasts enhance murine mammary gland humanization in vivo.</a> Breast Cancer Res. 14 (3), (2012).</li><li>Sikora, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M.J.,+V.,+M.E.,+M.D.,+J.M.">M.J., V., M.E., M.D., J.M.</a> Mechanisms of estrogen-independent breast cancer growth driven by low estrogen concentrations are unique versus complete estrogen deprivation.Breast Cancer Res Treat. 134 (3), 1027-1039 (2012).</li></ol>

The authors have nothing to disclose.

Metástasis tumoral es un proceso de varios pasos. Las células deben romper a través de la membrana basal, intravasate en cualquiera del sistema linfático o de la sangre microvasos, en los que se transporten a sitios distantes. Las células tumorales entonces extravasan y colonizan en un macrometástasis 12. La progresión a través de la epitelial a mesenquimal (EMT), invasión tumoral y la metástasis se ha mejorado por las hormonas esteroides 13,14, factores de crecimiento 15-18, 19-21</...

Un nuevo ensayo de invasión se ha desarrollado con el objetivo de identificar la participación de hormonas específicas, citoquinas y / o factores de crecimiento como quimioatrayentes en la invasión de células de cáncer. La capacidad de las células tumorales para invadir a través de la matriz extracelular (ECM) es una característica del fenotipo metastásico 1-3. Cámaras de invasión se han utilizado ampliamente como herramientas in vitro para estudiar la invasión de células de cáncer y la migración y pueden proporcionar conocimiento en cuanto a los mecanismos de invasión tumoral in vivo y la metástasis en. Las cámaras consisten en insertos de cultivo de células cilíndricas anidadas dentro de los pocillos de placas de cultivo celular. Los fondos de los insertos son de policarbonato o poliestireno membranas semi-permeables de tamaños de poro definidos. En el ensayo de invasión estándar, las células se sembraron sobre la membrana inserto con medio libre de suero y se colocaron en pocillos de cultivo celular que se llenan con suero o sin suero como quimioatrayentes para set un grupo de quimioatractivas. Esto lleva a la fuerza de las células para moverse a través de la membrana semi-permeable (migración) o, alternativamente, a través de una capa de matriz extracelular (invasión), que luego se pueden teñir usando tintes convencionales para detectar y cuantificar el número de células. El número de células se normaliza entonces de acuerdo con la medida de la migración y la invasión en una línea celular no invasivo. Este método permite a un investigador para evaluar el potencial invasivo de diferentes tipos celulares bajo una variedad de condiciones, incluyendo manipulaciones genéticas. Hormonas, citoquinas y factores de crecimiento son componentes críticos de suero y cada vez más se están demostrado estar asociados con la conducción del fenotipo invasivo 4. Sin embargo, la naturaleza, la función y la especificidad de estas moléculas séricas quimioatractivas en la mediación de la invasión sigue siendo difícil de alcanzar. El reto sigue siendo para determinar qué factores específicos en suero o suero como quimioatrayentes son responsables de la conduccióninvasión de células cancerosas, así como qué moléculas pueden inhibir el proceso de invasión. En este reporte se describe una metodología para evaluar la posible participación de las hormonas, citoquinas y / o factores de crecimiento presentes en el suero, como moléculas que impulsan la quimioatracción y la invasión de las células cancerosas. En este protocolo modificado propuesto, el decapado de carbón se usa para eliminar las hormonas, citoquinas y factores de crecimiento de 2% de suero bovino fetal (FBS) para generar FBS 2% tratado con carbón vegetal (CS-FBS). Tanto FBS al 2% y 2% CS-FBS se utilizan como agentes para establecer la fuerza de quimioatrayentes para conducir la invasión de células de cáncer. Uso de 2% CS-FBS como un agente quimioatrayentes en comparación a la normalidad 2% de FBS proporciona varios beneficios, incluyendo en primer lugar, y lo más prominente, la capacidad de estudiar el fenotipo de la invasión del cáncer en el reducido / ausencia relativa de hormonas, citoquinas y factores de crecimiento. También permite al investigador evaluar si la eliminación colectiva de las hormonas, factores de crecimiento, y Cycitoci- provoca un aumento o disminución en el índice invasiva. El ensayo es entonces diseñado para determinar si la adición de un componente individual, designado como &quot;X&quot; puede inhibir, aumento, descenso o restaurar el índice invasiva a su valor original (véanse las Figuras 2 y 3). Aunque esta metodología es específico para el logro de los niveles fisiológicos del componente que se retira a partir del suero durante el decapado de carbón, también debe tenerse en cuenta que decapado de carbón también puede afectar a vías de transducción de señal. Por ejemplo, se ha informado de que decapado de carbón puede disminuir la actividad de la fosfatasa alcalina de las células osteoprogenitoras e inducir la adipogénesis mediante la reducción de los activadores de MAPK 5. De carbón medios pelados están disponibles comercialmente y se basan en el protocolo descrito que combina carbón de leña con dextrano y se incubaron con suero bovino fetal O / N 6. Este ensayo se desarrolló en RESPONSE a un resultado reportado por Zucker et al. 7 Los autores demostraron que un fenotipo mutante que afecta a la comunicación brecha de la salida demostró un aumento en el índice de invasión al migrar hacia los medios normales con 2% de FBS. Este aumento fue eliminado con la sustitución de suero con carbón vegetal. De este resultado, los autores concluyeron que esta migración afectados mutante hacia una molécula lipófila (más específicamente, una hormona, factor de crecimiento o citoquinas) 7. Es bien sabido que las hormonas, factores de crecimiento y citoquinas median en las vías de señalización implicadas en la promoción de tumores y la invasión 4. Por lo tanto, es importante para los investigadores científicos para determinar qué factor (s) están impulsando la invasión tumoral de sus células de cáncer en particular o mutantes bajo estudio. El ensayo está diseñado para abordar el papel de componentes individuales en su concentración fisiológica como se determina de acuerdo con la diferencia entre la concentración del componente y# 8220; X &quot;en FBS normal y el CS-FBS. Mediante el desarrollo de este ensayo, los investigadores pueden obtener mayor conocimiento en la vía invasiva específica que regule su sistema. Hormonas, factores de crecimiento y citocinas a menudo han sido clasificados como promotores tumorales 8. Algunos de estos factores, tales como EGF se utilizan como fuentes directas para quimioatrayentes en cámaras de invasión 9. Por lo tanto, parece probable que estos representan los principales componentes del suero que la invasión tumoral directa. Este protocolo propone una simple, pero significativa, modificación el ensayo in vitro convencional en la invasión que permite a un investigador para evaluar la participación de las hormonas, factores de crecimiento y citoquinas en la mediación del potencial invasivo de las células cancerosas. Sin embargo, el ensayo está diseñado para proporcionar al investigador con una respuesta de si el procedimiento será eficaz para su estudio en una primera etapa en el análisis, a fin de no utilizar un tiempo precioso y resources si consideran innecesarios. Este método utiliza CS-FBS y se basa en la discreción investigadores en cuanto a que las moléculas de perseguir como candidatos de plomo que potencialmente median invasión de células cancerosas. Los resultados de estos análisis deben demostrar ser útil en la identificación de los componentes del suero que sirven como quimioatrayentes o inhibidores de la línea celular en particular o mutante siendo estudiados. Además, este enfoque puede ayudar al investigador a identificar vías de señalización clave, ya sea que promueven o inhiben la invasión de células de cáncer; dirigiendo así el futuro diseño de fármacos.

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:367px;">Item</td>
			<td style="width:195px;">Vendor</td>
			<td style="width:136px;">Catalog #</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td>BioCoat Control Inserts with 8.0&#181;m PET Membrane 
			(12 in each of two 24-Well Plates)</td>
			<td>Corning</td>
			<td>354578</td>
			<td>polyethylene teraphthalate (PET) membranes&#160;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagen I, rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Source = rat tail tendon, purity = 90%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fixative; Diff-Quick</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9844047</td>
			<td>Methanol-based fixative, Hmatoxylin/Eosin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>12676</td>
			<td>Designated as CS-FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30070</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30068</td>
			<td>Designated as CS-FBS</td>
		</tr>
	</tbody>
</table>

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

Suero de la sangre sirve como un quimioatrayente hacia el cual las células cancerosas migran e invaden, facilitando su intravasación en microvasos. Sin embargo, las moléculas reales hacia el que las células migran siendo difícil de alcanzar. Este ensayo de invasión modificado ha sido desarrollado para identificar objetivos que impulsan la migración celular y la invasión. Esta técnica compara el índice de invasión en tres condiciones para determinar si una hormona específica, factor de crecimiento o citoquina juega un papel en la mediación el potencial invasivo de una célula de cáncer. Estas condiciones incluyen i) de suero bovino fetal normal (FBS), ii) con carbón vegetal FBS (CS-FBS), que elimina hormonas, factores de crecimiento y citoquinas y iii) CS-FBS + molécula (denotado &quot;X&quot;). Un cambio significativo en la invasión de células con CS-FBS en comparación con FBS, indica la implicación de las hormonas, citoquinas o factores de crecimiento en la mediación del cambio. Las moléculas individuales se pueden agregar de nuevo a CS-FBS para ensayar su capacidad de volververso o rescatar el fenotipo invasión. Además, dos o más factores pueden combinarse para evaluar el efectos aditivos o sinérgicos de los múltiples moléculas en la conducción o inhibir la invasión. En general, este método permite al investigador determinar si las hormonas, citoquinas, y / o factores de crecimiento juegan un papel en la invasión de células sirviendo como quimioatrayentes o inhibidores de la invasión para un tipo particular de célula de cáncer o un mutante específico. Mediante la identificación de factores quimiotácticos y los inhibidores específicos, este ensayo de invasión modificado puede ayudar a dilucidar las vías de señalización que dirigen la invasión de células de cáncer.

El índice de invasión se calcula para cada condición de acuerdo a la normalización a una línea celular no invasivo. Para nuestros experimentos, utilizamos la línea celular de melanoma 1205Lu y variantes establecido líneas celulares estables como nuestras líneas invasivos, así como la variante no invasiva premaligna, WM793 de la que se derivaron las células 1205Lu 10 que sirve como un control lógico. También utilizamos colágeno I como la matriz invasión, ya que es el componente principal de la de...

Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

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 ...

a-modified-vitro-invasion-assay-to-determine-potential-role-hormones

Research

JoVE Journal

Medicine

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

10.6K vistas.  Roswell Park Cancer Institute. 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.

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

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

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Un In Vitro Sistema para el Estudio de latencia del tumor y el salto hacia el crecimiento metastásico

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

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 ...

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 ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Establecimiento de un modelo de xenoinjerto Humano mieloma múltiple en el pollo para estudiar el crecimiento del tumor, la invasión y la angiogénesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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 ...