Los autores no tienen nada que revelar.

1. Ensayo clonogénico <ol><li> Preparar un solo suspensiones de células de líneas celulares de cáncer de mama estrógeno-dependientes células MCF-7 y T47D utilizando los pasos descritos a continuación <ol><li> Aspirar el medio de cultivo (DMEM / 10% inactivado por calor suero fetal de ternera / glutamina y penicilina / estreptomicina) a partir de una placa de cultivo de tejido de 10 cm que es no confluente más de 50% con las células T-47D MCF-7 o. Enjuague con PBS. Incubar con tripsina 0,25% / EDTA 2,21 mM disuelto en DMEM de alta glucosa a 37 ° C durante 1-4 min. </li><li> Compruebe células a intervalos de 1 min bajo un microscopio de contraste de fase para asegurar una distribución de una sola célula. Resuspender las células con una pipeta 2 ml pipeteando arriba y abajo varias veces para interrumpir el contacto célula-célula para lograr una, el estado de célula única casi invariable. </li><li> Continuar incubar células en tripsina a 37 ° C durante un máximo de 4 minutos si observa grupos de células después de sólo 2 minutos de incubación. No utilizar estas células para estudios clonogénicos si permanecen adheridos al correoach otra después de 4 minutos de tripsinización porque el error se introducirá en el rendimiento número de colonias. NOTA: Si se agrupan las células, el número de colonias formadas reflejará el producto de menor número de células que el número se incubaron. Si las células se tratan con tripsina en exceso, su potencial clonogénico puede verse disminuida. </li><li> Preparar una suspensión de células de 1.500 células / ml de medio de cultivo para placas de 24 pocillos, o menos, (+ 500 células / ml, dependiendo del tipo de célula o número de pases), mediante diluciones en serie en un tubo maestro que contienen todo el volumen necesario para todas las variables en el experimento. NOTA: El objetivo es una densidad final de células de 800 células / cm 2 (rango de aproximadamente 500 a 1100 células / cm 2). El objetivo es producir aproximadamente 100 + 50 colonias, que permite relativamente fácil de contar, evita el hacinamiento y permite colonias suficientes para dar lugar a diferencias estadísticas significativas cuando las colonias se aumentan o disminuyen por perturba experimentalciones. </li></ol></li><li> Se incuban las células en Clonigénicas densidad utilizando pasos descritos a continuación <ol><li> Se incuban las células en pocillos por cuadruplicado en 24 placas recubiertas de fibronectina pocillos a una densidad clonogénico de 1500 células / pocillo de un tubo de suspensión de células individuales maestro de 1500 células / ml. Se tritura el medio que contiene células con una pipeta 5 ml mediante la elaboración de 3 ml y dispensación de 1 ml de medio en cada uno de 2 pozos. NOTA: recubiertos de fibronectina placas se deben comprar pre-recubierto de un proveedor comercial. Placas de revestimiento fuera de una calidad controlada, los resultados de proceso automatizado en una superficie desigual inadecuado para este ensayo. </li><li> Mezclar la suspensión pipeteando arriba y abajo con una pipeta de 5 ml, elaborar 3 ml de suspensión celular y llenar 2 pozos con 1 ml cada una. Llenar sólo 2 pozos en un momento dado de una pipeta. Devuelva el volumen restante en la pipeta a la suspensión de células en el tubo principal, volver a suspender las células de nuevo pipeteando arriba y abajo y elaborar otros 3 ml para llenar otro 2 wells con 1 ml cada uno. NOTA: La mezcla continua del tubo principal es necesaria porque las células se sedimentar continuamente. La elaboración de un volumen suficiente para llenar sólo 2 pozos es necesario añadir el número de células similares a cada pocillo porque las células sedimentan en la pipeta también. </li><li> Trabajar rápidamente para distribuir los grandes volúmenes de células porque las células permitiendo a los sientan en suspensión a temperatura ambiente y CO 2 concentración modulará sus clonogénicas potenciales (observaciones no publicadas). </li><li> Optimizar la distribución espacial de las células durante el acto de pipeteado ellos en pocillos para ensayos de colonias. Hacerlo pipeteando lentamente la suspensión que contiene la concentración final de células en el medio de la bien. No exponga la placa para promover el movimiento antes de las células depositan en el fondo. No girar el plato porque mezcla circular centrifugar efectivamente las células al perímetro de las así creando altas densidades de células y colonias confluentes incontables a los 6 días. NOTA: No mezcle menos que sea necesario ya que esto es menos deseable que ninguna mezcla en absoluto después de la introducción del volumen con las células en suspensión. Si es necesario mezclar las células, hacerlo moviendo la placa hacia atrás y adelante en direcciones perpendiculares mientras descansa sobre una superficie plana. </li><li> Se incuban las células a 37 ° C 5% de CO 2 sin cambio de medio durante 6 días. El pequeño número de células en un pozo después de 6 días no impactará significativamente la composición de nutrientes o de citoquinas ni el pH del medio original. </li><li> Diseñar el curso de tiempo del ensayo como sigue: Se incuban las células en la fibronectina recubierto de sustratos en el día 1. Reemplazar medio existente con 1 ml de medio fresco o medio fresco que contiene 10 ng / ml en el día 0. células mancha en día 6 como debajo de 2 FGF- en 1.4). Realizar cualquier perturbaciones experimentales en el día 3, ya continuación en 1.3). </li></ol></li><li> Configurar perturbaciones experimentales de Molecular Signaling o moléculas de adhesión <ol><li> El día 3, añadir 10056; l de una solución que contiene 10 veces más de la concentración deseada final del agente perturbador al medio de 1 ml en los pocillos. No mezclar. Continuar incubar las células a 37 ° C, 5% de CO 2 durante otros 3 días. NOTA: agentes Perturbar puede incluir una variedad de inhibidores y agentes de bloqueo de moléculas de adhesión, receptores u otras proteínas de superficie, inhibidores de las vías de señalización intracelulares, moléculas, factores, cofactores o proteínas estructurales, que pueden jugar un papel en el apoyo al estado latente. </li><li> Colonias de la mancha en el día 6, de la siguiente manera. </li></ol></li><li> Las colonias de la mancha <ol><li> Stain células después de 6 días de cultivo con un 0,1% de cristal violeta recién hecho en 2% de etanol / borato de sodio 10 mM (pH 9,0) solución. Medios Aspirar y añadir solución de cristal violeta uno ml a cada pocillo durante 20 min. </li><li> Lavar platos sumergiéndolos con las aberturas así mirando hacia abajo en un ángulo agudo en un cubo de hielo que desborda con grifo abierto de forma continuaagua en el fregadero. Incline la placa a un ángulo horizontal una vez bajo el agua, así la apertura hacia abajo, y luego inclinarlo de nuevo a un ángulo agudo al retirar en un movimiento que fluye suave. </li><li> Repita la inmersión 2 o 3 veces hasta que el agua en el fondo de los pozos ya no es de color azul. Lavados vigorosos pueden eliminar las células o colonias que son menos adherente debido a la intervención experimental, añadiendo de manera significativa al error en el conteo y los datos. </li><li> Placas secas durante la noche colocándolos boca abajo sobre toallas en la mesa de trabajo junto a sus correspondientes tapas marcadas. </li></ol></li><li> Cuente Colonias <ol><li> Contar el número de colonias que crecen y latentes en cada pocillo después de 6 días de incubación, la tinción y el secado. Recuento de las colonias de manera óptima a 40 aumentos en un microscopio de contraste de fase invertida. Contar las colonias de&gt; 30 células como crecer y colonias de 12 o menos células, con la apariencia morfológica de gran tamaño en comparación con el cultivo de células, amplio expacitoplasma nded con gran citoplasma al núcleo proporciones, mostradas en la Figura 1 7,15. NOTA: Los racimos de 13-29 células normalmente no se cuentan como no son muy frecuentes en los ensayos de inactividad sencillas. Ellos se pueden contar si las perturbaciones desplazan el potencial de crecimiento de cualquiera de las células en crecimiento o latentes. Estos resultados entonces necesitará estar correlacionado con importancia biológica. </li></ol></li></ol> 2. Clonigénicas Incubación de Estudios de inmunofluorescencia <ol><li> Ajustar el número de células a aproximadamente 7,500-8,000 células / pocillo en placas de 6 pocillos, para corresponder a la celda / números de área de superficie análoga a 24 experimentos bien. </li><li> Coloque una ronda,, cubierta de resbalón fibronectina recubierto estéril en cada base pocillo de placas de 6 pocillos para los estudios de imagen antes de la adición de células. Células de pipeta en 3 ml volúmenes en cada pocillo 2 pozos en un momento en concentraciones describen, como se describe para los experimentos clonogénicas más arriba en 1.2). </li><li> Se incuban las célulasdurante 6 días, como anteriormente en 1.2.4 y 1.2.5. Añadir factores perturbadores en días 3 a 10x concentraciones en 300 volúmenes mu l, como se describe en los procedimientos de ensayo colonia en 1.3). </li><li> Células mancha en día 6 con anticuerpos para moléculas de adhesión celular tales como integrinas α4, α5, α6, β1, β3, por ejemplo, de adhesión focal moléculas complejas FAK, paxillin y vinculina, por ejemplo, proteínas implicadas en la motilidad tales como α-tubulina, por ejemplo, los miembros de la vía de señalización tales como fosfo-Akt, fosfo-ERK, fosfo-p38, fosfo-JNK, por ejemplo, o cualquier otra proteína que es el objetivo de la investigación por su papel en la latencia, utilizando técnicas estándar para directo o indirecto la tinción de inmunofluorescencia. <ol><li> Eliminar diapositivas con fórceps día 6. Fijar en acetona / metanol 1: 1 a -20 ° C durante 20 min y secar al aire. Un fijador alternativo, tal como paraformaldehído, se puede utilizar, si es necesario. Permeabilizar las células con 0,1% Triton X-100, 0,1% de citrato de sodio para 2 min fo la detección de antígenos intracelulares. Lavar las células con PBS. </li><li> Para la tinción immunofluorecence indirecto, las diapositivas de bloque para 1 hr a temperatura ambiente con 5% de BSA o con 10% de suero preinmune de la especie en la que se generó el anticuerpo secundario. </li><li> Incubar durante la noche a 4 ° C con anticuerpos primarios a la célula moléculas de adhesión, moléculas complejas focales, señalización de los miembros de la vía o cualquier otra proteína que es el objetivo de la investigación por su papel en la latencia diluido a diluciones específicas recomendadas por el fabricante en PBS 0,1% de Triton X -100. </li><li> Lavar 3 veces con PBS. Incubar cubreobjetos con anticuerpos-fluoróforo conjugado a temperatura ambiente durante 2 hr. Como un ejemplo, Alexa Fluor 488 burro anti-IgG de ratón de anticuerpos puede ser utilizado para detectar anticuerpos primarios monoclonales murinos. Monte cubreobjetos células hacia abajo sobre portaobjetos de vidrio utilizando un agente antifade con DAPI. Selle los perímetros con esmalte de uñas. </li><li> Por inmunofluorescencia directa, llevar a cabo la BSAbloqueo como anteriormente en 2.4.2), incubar con el anticuerpo primario-fluoróforo conjugado durante la noche a 4 ° C. Lavar 3 veces con PBS. Incubar los portaobjetos con un agente antifade y Dapi y sello con el esmalte de uñas. </li><li> Bandejas deslizantes cubierta con papel de aluminio y se almacena a 4 ° C para formación de imágenes y la fotografía en cualquier momento hasta varias semanas más tarde. Ver y fotografiar las células utilizando cualquier sistema de formación de imágenes microscópicas de fluorescencia equipado con una cámara en 1.000 x magnificación. </li><li> Para la tinción de actina fibrilar, diapositivas de bloque en 1% de albúmina de suero bovino (BSA) durante 30 min y se incuban en BODIPY FL-Phallacidin (verde) o rodamina faloidina (rojo) a temperatura ambiente durante 20 min. Añadir un agente antifade y sellar el anterior. </li></ol></li></ol> 3. Clonigénicas Incubación de Estudios Moleculares <ol><li> Transferencias Western <ol><li> Incubar cáncer de mama ER + células MCF-7 o T47D a densidades clonogénicos de 20.000 células / 60 mm plato y 50.000 células / placa de 100 mm en fibroneclata-revestido placas a 37 ° C en 5% de CO 2. </li><li> Se incuban las células a densidades ligeramente más altos de 75.000 células / placa de 100 mm para los estudios moleculares que requieren cantidades mg de proteína de lisados. Utilice un máximo de diez planchas por punto experimental para recoger suficientes proteínas para los estudios moleculares utilizando transferencias Western o para el aislamiento de ARN para transferencias Northern. </li><li> El número de células de carga de gel con base es un complemento necesario para la comparación de la expresión de proteínas de muy diferentes tamaños células de cultivo y latentes. Recoger las células por tripsinización y contar una parte alícuota en un hemocitómetro en 0,2% de azul de tripano para la preparación de lisados ​​para transferencias de Western en lugar de raspado de las células con una sola pala de borde. </li><li> Centrifugar las células a 10.000 xg durante 2 min, eliminar los medios por aspiración, añadir 200 l de tampón de lisis, sonicar las células en tampón de lisis y determinar la concentración de proteína. </li><li> Calcular la cantidad de proteína por célula dividiendo el rendimiento de proteína por el número de célulasque genera esa cantidad. Cargar el lisado en cada pocillo de geles de poliacrilamida separadas que representa tanto una) cantidades equivalentes de proteína y b) las cantidades de proteínas que representan el número de células equivalentes. Al menos 25 g de proteína debe ser cargado en cada pocillo. NOTA: Esto permitirá comparaciones entre crecimiento y latentes células, que son significativamente diferentes en tamaño y contenido de proteína (Figura 1). </li></ol></li><li> Citometría De Flujo <ol><li> Recoger las células de placas de 100 mm por tripsinización, como en 3.1 y analizar por células activadas por fluorescencia estándar (FACS) utilizando protocolos de tinción de inmunofluorescencia primaria o secundaria, ya sea para antígenos intracelulares o extracelulares, como se indica en 2.4. NOTA: Separación de células a partir de placas de cultivo de tejidos por tripsinización no afecta a la concentración de proteínas de membrana como se determina mediante el etiquetado de anticuerpos. </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+temporal+and+spatial+gene+expression+of+fibroblast+growth+factor+family+members+during+mouse+mammary+gland+development.">Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development.</a> Molecular Endocrinology. 8 (2), 218-229 (1994).</li><li>Plath, A., 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=Expression+and+localization+of+members+of+the+fibroblast+growth+factor+family+in+the+bovine+mammary+gland.">Expression and localization of members of the fibroblast growth factor family in the bovine mammary gland.</a> J Dairy Science. 81 (10), 2604-2613 (1998).</li><li>Lavandero, S., Chappuzeau, A., Sapag-Hagar, M., Oka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+evidence+of+basic+fibroblast+growth+factor+action+in+mouse+mammary+gland+development.">In vivo and in vitro evidence of basic fibroblast growth factor action in mouse mammary gland development.</a> FEBS letters. 439 (3), 351-356 (1998).</li><li>Sinowatz, F., Schams, D., Plath, A., Kolle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+localization+of+growth+factors+during+mammary+gland+development.">Expression and localization of growth factors during mammary gland development.</a> Adv in Exp Med &amp; Biol. 480, 19-27 (2000).</li><li>Korah, R., Sysounthone, V., Scheff, E., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+FGF-2+promotes+differentiation+in+T47-D+breast+cancer+cells.">Intracellular FGF-2 promotes differentiation in T47-D breast cancer cells.</a> Biochem Biophys Res Comm. 277 (1), 255-260 (2000).</li><li>Korah, R., Das, K., Lindy, M. E., Hameed, M., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinate+loss+of+FGF-2+and+laminin+5+expression+during+neoplastic+progression+of+mammary+duct+epithelium.">Co-ordinate loss of FGF-2 and laminin 5 expression during neoplastic progression of mammary duct epithelium.</a> Human Pathology. 38 (1), 154-160 (2007).</li><li>Wieder, R., 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=Overexpression+of+basic+fibroblast+growth+factor+in+MCF-7+human+breast+cancer+cells:+lack+of+correlation+between+inhibition+of+cell+growth+and+MAP+kinase+activation.">Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation.</a> J. Cellular Physiology. 177, 411-425 (1998).</li><li>Brunner, G., Gabrilove, J., Rifkin, D. B., Wilson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+release+of+basic+fibroblast+growth+factor+from+human+bone+marrow+cultures+as+a+biologically+active+complex+with+a+phosphatidylinositol-anchored+heparan+sulfate+proteoglycan.">Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.</a> J Cell Biol. 114 (6), 1275-1283 (1991).</li><li>Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., Gabrilove, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+stimulates+myelopoiesis+in+long-term+human+bone+marrow+cultures.">Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures.</a> Blood. 77 (5), 954-960 (1991).</li><li>Gupta, P., McCarthy, J. B., Verfaillie, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblast+heparan+sulfate+is+required+for+cytokine-mediated+ex+vivo+maintenance+of+human+long-term+culture-initiating+cells.">Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.</a> Blood. 87 (8), 3229-3236 (1996).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Farnesyl+transferase+inhibitor+treatment+of+breast+cancer+cells+leads+to+altered+RhoA+and+RhoC+GTPase+activity+and+induces+a+dormant+phenotype.">Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype.</a> Int J Cancer. 129 (1), 61-69 (2011).</li><li>Korah, R., Sysounthone, V., Golowa, Y., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+confers+a+more+differentiated+phenotype+in+MDA-MB-231+human+breast+cancer+cells.">Basic fibroblast growth factor confers a more differentiated phenotype in MDA-MB-231 human breast cancer cells.</a> Cancer Res. 60 (3), 733-740 (2000).</li><li>Dontu, G., 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=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes &amp; Dev. 17 (10), 1253-1270 (2003).</li><li>Tivari, S., Lu, H., Dasgupta, T., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+dormant+estrogen-dependent+breast+cancer+micrometastases+by+bone+marrow+stromal+injury+in+an+in+vitro+model+of+dormancy.">Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy.</a> Proceedings of the 105th Annual Meeting of the American Association for Cancer Research. , (2014).</li></ol>

Apoyado por el Departamento de Becas de Defensa DAMD17-01-C-0343 y DAMD17-03-1-0524, la Comisión Estatal de New Jersey para la Investigación del Cáncer 02-1140-CCR-E0 y Ruth Estrin Goldberg Memorial para la Investigación del Cáncer (RW)

Nuestro modelo se compone de varios elementos clave de la latencia en la médula ósea. Se compone de células sensibles a estrógenos, que son el tipo propensos a permanecer en estado latente en la médula ósea durante períodos prolongados 10, se compone de fibronectina, un elemento estructural clave de la médula ósea, FGF-2, un factor de crecimiento abundantemente sintetizado por el estroma de la médula ósea y fuertemente depositado en la matriz extracelular de la médula ósea 31,32 y la i...

Células de cáncer de mama metástasis en la médula ósea antes de la enfermedad es detectable 1, tan pronto como los tumores se desarrollan pequeños vasos sanguíneos 2,3. El proceso metastásico es rápida pero ineficiente. Las células entran en los vasos sanguíneos nuevos rápidamente, en millones por día 4 pero pocos sobreviven el viaje a órganos distantes 5. Sin embargo, algunos micrometástasis sobrevivir en el hueso y se pueden encontrar como células individuales o grupos de células pequeñas en aspirados de médula ósea de los pacientes recién diagnosticados 1. Estas células resisten a la quimioterapia adyuvante, que es administrado con el propósito de eliminarlas 6. Esta resistencia está dotado, sustancialmente, por la supervivencia de señalización iniciada por la interacción con el microambiente de la médula ósea 7,8. Las micrometástasis se pueden encontrar en aproximadamente un tercio de las mujeres con cáncer de mama localizado y representan un indicador independiente de la supervivencia cuando se analizaron por análisis univariado 9. Algunos micrometastases son el crecimiento iniciado, pero los patrones de recurrencia dependen del tipo de célula. Los pacientes con cáncer de mama triple negativo tienden a repetirse entre 1 a 4 años, lo que sugiere un mal control sobre el estado de latencia. Otros tipos de células, incluyendo las células ER / PR +, pueden permanecer latentes durante un máximo de 20 años, con un ritmo constante y continua de recurrencia 10. Mientras que las diferencias en la latencia firmas de expresión génica entre ER + y ER- líneas celulares de mama y tumores reflejan diferentes potenciales de latencia 11, las interacciones con estroma de la médula ósea probablemente representan una contribución significativa a la latencia. El estudio de la latencia in vivo es excepcionalmente difícil debido a micrometástasis son raros y son superados en número por las células hematopoyéticas en más de un 10 6 -fold. Por lo tanto, los modelos relevantes se deben generar que proporcionan datos in vitro que puede sugerir mecanismos y generar hipótesis comprobables en vivo. Un número de modelos de latencia, incluyendo mathemodelos matemá- 12,13, en modelos in vitro, in vivo 7,8,14,15 modelos de xenoinjerto 16, combinaciones de in vitro y modelos de xenoinjerto 17,18 y tumoral y la metástasis modelos espontáneos 19, han producido una cierta penetración en la latencia de células de cáncer 20 . Cada uno de estos modelos tienen sus propias limitaciones y son en sí mismos principalmente útil para generar hipótesis sobre la señalización molecular y las interacciones que rigen la latencia a ensayar en modelos más biológicamente relevantes. Con el objetivo general de la definición de los mecanismos moleculares de la inactividad, las interacciones con el microambiente que da lugar a la detención del ciclo, rediferenciación y resistencia terapéutica y mecanismos que dan lugar a la recurrencia en las células ER +, hemos desarrollado un modelo in vitro que proporciona selecciona los elementos pertinentes de la estroma microambiente 7. Este modelo, si bien es relativamente escasa en su componentes, es suficientemente robusto para permitir a los investigadores a derivar mecanismos moleculares específicos que afectan a las funciones significativas de latencia. Estos experimentos generan hipótesis que se pueden probar directamente in vivo. El modelo se basa en una serie de elementos clave que hemos demostrado ser relevante en la latencia. Estos incluyen el uso de células de cáncer de mama dependientes de estrógenos, cultivo de células a una densidad clonogénico donde su interacción es principalmente con el sustrato y los componentes solubles del medio, un sustrato de fibronectina y la presencia de factor de crecimiento básico de fibroblastos (FGF-2) en el medio. Hemos caracterizado los mecanismos que gobiernan el sistema in vitro, incluyendo la inducción de la detención del ciclo celular por FGF-2 21, mediada a través de TGF 22, la supervivencia de señalización a través de PI3 quinasa ERK 7,8 y 8 y la diferenciación morfogénica a un fenotipo epitelial, que dependía de RhoA inactivación, la integrina45; 5β1 upregulation y la ligadura de la fibronectina del estroma para la supervivencia 7,15 (Figura 1). Los efectos del ciclo celular in vitro de FGF-2 en células MCF-7 células comienzan a concentraciones al menos un log por debajo de 10 ng / ml 21,23. La razón se basa en el control temporal de FGF-2 expresión que rige la morfogénesis ductal mamario, la expansión cíclica y la recesión en un número de sistemas de mamíferos 24-27. Hemos demostrado que el FGF-2 induce la diferenciación, incluyendo la morfogénesis ductal en la cultura 3D 28, y que el FGF-2 de expresión se pierden generalmente con la transformación maligna de los tumores humanos 29. La expresión de FGR1 permaneció intacta en los carcinomas de mama encuestados 29 y MCF-7 células continúan para expresar todos los 4 receptores de FGF 30. En el contexto de la latencia, FGF-2 se exporta por y fuertemente deposita sobre estroma de médula ósea 31,32 donde funciona en la preservación de las células madre hematopoyéticas 33. Nos demonstrated que FGF-2 induce un estado latente en cáncer de mama ER + células cultivadas sobre sustratos de fibronectina, también abundante en la médula ósea, donde se induce la diferenciación morfogénica 7. En el modelo, las células de cáncer de mama son el crecimiento inhibido, inactivar Rho A través de la RhoGAP GRAF, redifferentiate a un fenotipo epitelial y re-expresa lost integrinas α5β1 con la progresión maligna. Ellos se unen a través de fibronectina α5β1 integrina y activan la supervivencia de señalización que hacerlos resistentes a la terapia citotóxica 7,8,15 (Figura 1). La inhibición de la Rho GTPasas de clase se ha demostrado previamente para inducir un fenotipo inactivo 34. Aquí vamos a esbozar los procedimientos específicos que permitan a los investigadores a establecer el modelo y estudiar los mecanismos moleculares y celulares específicas que regulan la latencia de las células de cáncer de mama ER +. En los experimentos presentados aquí para ilustrar el uso del modelo, Nos centramos en la vía PI3K (Figura 1B) con un inhibidor de Akt y un inhibidor de PI3K y todos los miembros de la familia Rho (Figura 1B) con un inhibidor pan-Rho y un (ROCK) inhibidor de Rho quinasa.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MCF-7 cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>T47D cells&#160;</td>
			<td>ATCC</td>
			<td>HTB-123</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td>
			<td>Corning</td>
			<td>354411</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 60 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354403</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 100 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354451</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td>
			<td>Corning</td>
			<td>354088</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well tissue culture plate</td>
			<td>CellTreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td>
			<td>Corning Life Sciences</td>
			<td>50-013-PB</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated, Fetal Bovine Serum</td>
			<td>Serum Source International</td>
			<td>FB02-500HI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin/2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, 100x, Liquid</td>
			<td>Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF&#160;basic</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td>
			<td>CalBiochem</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr>
			<td>LY294002&#160;</td>
			<td>CalBiochem</td>
			<td>19-142</td>
			<td>Chenical PI3K inhibitor</td>
		</tr>
		<tr>
			<td>C3 transferase&#160;</td>
			<td>Cytoskeleton</td>
			<td>CTO3</td>
			<td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>129830-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY FL-Phallacidin (green)&#160;</td>
			<td>Molecular Probes</td>
			<td>B607</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>BODIPY FL-Rhodamine phalloidin (red)&#160;</td>
			<td>Molecular Probes</td>
			<td>R415</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&#160;</td>
			<td>Molecular Probes</td>
			<td>R37114</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI&#160;</td>
			<td>Molecular Probes</td>
			<td>P-36931</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

<html> Se realizaron experimentos para recapitular el ensayo. El curso de tiempo del experimento se muestra en la Figura 2A. Las células se incubaron a densidad clonogénico en el día -1, se añade FGF-2 en medio fresco en días 0 y las células se cultivan hasta el día 6 cuando se tiñen y se cuentan las colonias. Cualquier perturbaciones en el sistema se administran en el día 3 en 100 volúmenes mu l a 10x concentraciones finales deseadas. La Figura 2B muestra la apariencia típica...

Watch this Scientific Journal Video about An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation at JoVE.co

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Un<em&gt; In Vitro</em&gt; Latencia Modelo de cáncer de mama estrógeno-sensibles en la médula ósea: una herramienta de Estudios mecanismo molecular y Hipótesis Generación

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

8.2K vistas.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

Video: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

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 ...

Investigación

Medicina

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

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

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

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

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

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodal de imágenes de la angiogénesis en un modelo de rata desnuda de las metástasis óseas del cáncer de pecho utilizando imágenes de resonancia magnética, tomografía computarizada y la ecografía volumétrica

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

La detección de Modificadores melanoma utilizando un modelo de pez cebra Tumor autóctona

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Usando cuantitativa en tiempo real PCR para determinar quimerismo de células donantes en un modelo competitivo Trasplante de médula ósea murina

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Femoral Aspiración de médula ósea en ratones vivos

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Una<em&gt; Ex vivo</em&gt; Modelo para estudiar la acción hormonal en el pecho humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

En vivo y ex vivo para estudiar Cáncer de ovario metastásico colonización de Estructuras Vía lugar en Peritoneal adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilización del ensayo Soft Agar formación de colonias para identificar inhibidores de tumorigenicidad en células de cáncer de mama

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...