This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

1. Cultivo tridimensional de células de cáncer de mama en Sótano matriz de la membrana (La Técnica de empotramiento) <ol><li> Manejo de la matriz de la membrana basal Matrigel: Descongelar en hielo durante la noche a 4 ° C. Matriz de la membrana basal es líquido a baja temperatura pero solidifica a temperatura ambiente. Mantener la matriz de la membrana basal en hielo (figuras 1A-B). </li><li> Cubra el plato No.1 Confocal con fondo de vidrio con 50 l de la matriz de la membrana basal por medio de la difusión de la matriz usando la punta de un P-200 Pipetman en forma de espiral (Figura 1C). Tenga cuidado cuando la difusión de la matriz de la membrana basal para evitar la formación de burbujas de aire. Del mismo modo, evitar la propagación de la matriz a cerca de los bordes de la placa con fondo de cristal para evitar la formación de menisco. Si matriz de la membrana de manipulación inexperta sótano, a continuación, preenfriado el plato, P-200 Pipetman y pipetas puede ser en hielo (alternativamente dejó durante la noche en una nevera a 4 ° C). Este paso ofertastiempo adicional para ampliar la matriz de la membrana basal antes de la solidificación. Coloque el plato (s) en una incubadora de cultivo celular (a 37 ° C con 5% de CO 2) durante al menos 30 minutos para permitir que la matriz de la membrana basal para solidificar (Figura 1D). </li><li> Mientras que la matriz está experimentando la solidificación, trypsinize un confluente de 100 mm de placa de las células del 70-80%. Una vez que las células han comenzado de elevación fuera de la placa, volver a suspender en 10 ml de medio RPMI 1640 que contiene 10% (v / v) de suero bovino fetal (FBS), para inactivar la tripsina (Figura 1E). A continuación, transferir las células resuspendidas en un tubo cónico de 15 ml (Figura 1F). </li><li> Haga girar las células (presentes en tubo cónico) a 100 xg durante 3 minutos en una centrífuga de cultivo celular específico (Figuras 1G-H). </li><li> Mientras que las células se centrifugan, alícuota de 50 l de la matriz en un tubo de microcentrífuga de 1 ml (nota: cada plato con fondo de cristal se asigna un tubo de microcentrífuga, por lo que, siel experimento requiere de 3 platos, entonces se deduce que 3 tubos de microcentrífuga son necesarias también), y luego colocar el tubo en hielo (Figura 1B). </li><li> Aspirar el medio desde el tubo cónico de la etapa 1.4, dejando el sedimento inalteradas (Figura 1I). Resuspender las células (que se han hecho girar hacia abajo) en 1 ml de FBS suplementado con medio RPMI (Figura 1J). </li><li> Una vez que las células se contaron usando un hemocitómetro (Figura 1K), o un contador de partículas alícuota de células 2,5 x 10 4 células en el tubo de microcentrífuga y por si fuera poco el uso de los medios adecuados para obtener el volumen total de 50 l (Figura 1D). </li><li> Mezclar las células de la etapa 1.7 (25.000 células en 50 l) con el tubo de microcentrífuga que contiene matriz de la etapa 1.5 en una proporción de 1:1; volumen final será de 100 l (Figura 1M). </li><li> Suavemente la placa 100 l de la matriz: mezcla de células de la etapa 1.8 en el solidified matriz recubiertos con membrana basal plato de la etapa 1.2 (Figura 1 N). Esto permite que las células se pueden incrustar en la matriz de la membrana basal. </li><li> Transferir el plato (s) en la incubadora de cultivo celular (a 37 ° C con 5% de CO 2) y permitir que la matriz: mezcla de células se solidifique durante al menos 30 min. </li><li> Una vez que la matriz: mezcla de células se solidifica, añadir 2 ml de medio RPMI suplementados con FBS al plato (Figura 1O) y tomar el plato de nuevo la incubadora donde se almacenará durante el resto del experimento (Figura 1P). </li><li> Cambio de medio cada día durante un período de 5 días (o según requerido para un ensayo; la duración del ensayo para MDA-MB-231 células es de 5 días de duración). </li><li> El uso de un microscopio de luz, toma de contraste de interferencia diferencial (DIC) imágenes de los MDA-MB-231 colonias suspendidas en la matriz de la membrana basal (Figura 3A). Imagen 20 áreas representativas a 10X objetivo una vez al díadurante cinco días para determinar colonia morfogénesis (Figura 3C). </li><li> Analizar las imágenes a ciegas para determinar la formación de colonias de células estrelladas (Figura 3B). Una colonia se considera que es estrelladas si se perciben una o más proyecciones de la esferoide de células. Para determinar el porcentaje de colonias estrelladas invasivos, dividir el número de colonias estrelladas por el número total de colonias de células por imagen adquirida, y que la media de las colonias estrelladas porcentajes de las 20 imágenes para cada día. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> Figura 1. Cultivo tridimensional de células MDA-MB-231 células de cáncer de mama en matriz de membrana basal (la técnica de empotramiento). AB) un esquema del montaje experimental (realizado en la campana de humos). C) El pozo de la placa de fondo de vidrio recubiertocon 50 l de matriz de membrana basal. D) Plato (ES) colocado en una incubadora de cultivo celular (a 37 ° C con 5% de CO 2) para permitir la matriz para solidificar durante al menos 30 min se trataron con tripsina. E) Células. F ) Se resuspendieron las células en un tubo cónico de 15 ml. GH) Las células (presentes en tubo cónico) se centrifugaron a 100 xg durante 3 min en una centrífuga de cultivo de tejidos. I) sedimento celular. J) Las células (que han sido hechos girar hacia abajo) se resuspendieron en 1 ml de medio RPMI suplementado-FBS. K) Las células se contaron usando un hemocitómetro. L) 2,5 x 10 4 células en alícuotas en tubo de microcentrífuga y rematado apagado utilizando medios apropiados con el fin de obtener el volumen total de 50 l. M ) Mezclar las células de la etapa 1.7 (25.000 células en 50 l) con el tubo de microcentrífuga que contiene matriz de la etapa 1.5 en una proporción de 1:1; volumen final será de 100 μ.; L N) placa suavemente 100 l de la matriz: mezcla de células desde el paso 8 en el plato de la matriz con recubrimiento solidificado desde el paso 1.2 O) Una vez que la matriz: mezcla de células se solidifica, añadir 2 ml de medio RPMI suplementados con FBS al. el plato. P) colocar la cápsula en la incubadora, donde se almacenará para el resto del experimento. 2. Examen de las Características Morfogénicas de Culturas 3D con inmunofluorescencia <ol><li> Configurar una bandeja de hielo, fosfato fría solución tamponada (PBS), 20% de acetona: 80% de metanol (solución de fijación), y 3% de albúmina de suero bovino (BSA; solución de bloqueo) (Figura 2A) antes de sacar el plato (ES) de la incubadora. </li><li> Coloque el recipiente (es) en el soporte de la bandeja de hielo y aspirar, lavar 3 veces con 2 ml de PBS frío (Figura 2B). </li><li> Una vez que el último lavado PBS se aspira, añadir 2 ml de 20% de acetona: solución de metanol al 80% en el plato (es) (Figura 2C </strong&gt;) para fijar las células durante 20 min a 4 ° C (ya sea en hielo o en un frigorífico) (Figura 2D). </li><li> Una vez que el 20 min de fijación se termina, llevar los platos de nuevo a temperatura ambiente, aspirar el fijador, y posteriormente lavar 3x con PBS. Una vez que el lavado final de PBS se aspiró, añadir 2 ml de 3% de BSA a la placa (s) para bloquear por lo menos durante 30 min a temperatura ambiente (Figura 2E). </li><li> Durante el período de bloqueo preparar diluciones de primaria (laminina V 1:100) y anticuerpos secundarios (con una dilución adecuada) disueltos en 3% de albúmina de suero bovino (BSA) tampón de bloqueo. Por favor, tenga en cuenta que la solución de 400-500 l &#39;BSA + anticuerpo primario &quot;debe añadirse directamente en la matriz: mezcla de células. </li><li> Una vez que el bloqueo de 30 minutos de haber caducado, añada los anticuerpos primarios y se incuba durante al menos 1 hora a temperatura ambiente (Figura 2F). Por favor, tenga en cuenta que no hay lavados de PBS se deben realizar después de la 30 min blockinperiodo g. </li><li> Sobre la terminación de la etapa 2.6, eliminar la solución &#39;de BSA + anticuerpo primario&#39;, y lavar 3x con PBS. </li><li> Añadir el anticuerpo secundario disuelto en 3% de BSA (a una dilución apropiada) directamente sobre la matriz: mezcla de células, cubrir los platos y se incuba durante 1 hora a temperatura ambiente (Figura 2G). </li><li> Retire la solución &quot;BSA + anticuerpo secundario &#39;, y lavar 3 veces con PBS. </li><li> Añadir 2 ml de Hoechst 33258 (1:10.000) disueltos en PBS para la tinción de los núcleos, y se incuba durante 5 minutos bajo una lámina de aluminio (o cubra con un envase opaco para limitar fotoblanqueo) (Figura 2H). </li><li> Una vez que el 5 min de incubación con Hoechst 33258t han expirado, lavar el plato (es) de 5x con PBS. </li><li> Añadir medio de montaje directamente sobre la matriz: mezcla de células en el plato confocal y cubrir con cubreobjetos de vidrio con cuidado para evitar la inducción de cualquier interrupción de la integridad de las colonias en el Matri membrana basalx: mezcla de células (Figura 2I). </li><li> Dejar que la cápsula (es) que se seque durante la noche (o 24 horas) a temperatura ambiente (Figura 2I). Una vez seco, el plato (s) se puede almacenar a -20 ° C, pero se recomienda que deben ser reflejados lo más rápidamente posible. </li><li> Adquirir imágenes utilizando un microscopio de fluorescencia con longitudes de onda láser adecuados de anticuerpos utilizados (Figuras 3D-E). </li></ol><img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> Figura 2. Examen de las culturas 3D mediante inmunofluorescencia. . A) Un esquema del montaje experimental B) del plato (es) colocado en la bandeja de hielo y de los medios de aspiración; posteriormente las células se lavaron tres veces con 2 ml de PBS frío C) 2 ml de 20% de acetona:. solución de metanol 80% añadido en la cápsula (s) D) Fijar las células para.20 min a 4 ° C (ya sea en hielo, o en un refrigerador). E) 2 ml de 3% de BSA añadido a la placa (s) para bloquear por lo menos durante 30 min a temperatura ambiente. F) Una vez que el 30 min de bloqueo han expirado, añadir los anticuerpos primarios y se incuba durante al menos 1 hora a temperatura ambiente G) Los anticuerpos secundarios disueltos en 3% de BSA (a una dilución apropiada) se añadieron directamente sobre la matriz de membrana basal:. mezcla de células; . posteriormente platos cubrieron y se incubaron durante 1 hora a temperatura ambiente H) 2 ml de Hoechst (1:10.000; disuelto en PBS) añadido a la platos y cubiertos (es) y las células se incubaron durante 5 min bajo papel de aluminio i) Medio de montaje añade directamente. sobre la matriz: mezcla de células en el plato confocal y el plato se cubre con cubreobjetos de vidrio. Plato (ES) dejó secar durante la noche (o 24 horas) a temperatura ambiente.

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No tenemos nada que revelar.

El desarrollo de técnicas de cultivo de células en 3D ha permitido a los investigadores estudiar la transformación de células epiteliales de mama, lo que nos permite visualizar los dramáticos cambios morfológicos. Además de analizar la invasión de células, los esferoides epiteliales mamarias individuales o multicelulares pueden usarse para evaluar los cambios en la adhesión celular, la proliferación, el tamaño, y la polaridad basal-apical. En contraste con las metodologías reportadas previamente donde las c...

La migración y la invasión de células individuales o colectivos son dos características del cáncer, y necesarios para la diseminación metastásica de células de cáncer de 1-4. La capacidad de las células de los cánceres de iniciar la metástasis depende de su capacidad para migrar e invadir en el tejido vecino usando invadopodia para degradar la membrana basal de las células. Invadopodia son protuberancias degradación de la matriz de actina-rica dinámicos que permiten la degradación de la matriz extracelular a través de la liberación de proteasas que degradan la matriz 5. La invasión de células del cáncer implica la degradación de la matriz, seguido de la migración de las células cancerosas y esto va acompañado por una reorganización del medio ambiente matriz tridimensional (3D) 2. Por lo tanto, para penetrar a través de la matriz, una célula debe transformar su forma y interactuar con la matriz extracelular (ECM) 2. El mantenimiento de la integridad del tejido mamario depende fuertemente controlled arquitectura del tejido desde las uniones célula-ECM y célula-célula de adhesión influyen en la expresión génica y la interrupción de la polaridad epitelial puede conducir a la aparición de cáncer de 6-10. Sin embargo, la mayoría vitro de migración y la invasión ensayos in como transwell ensayos de cámara de ensayos o herida-scratch son de dos dimensiones (2D) y por lo tanto éstos descuidan las intrincadas interacciones entre las células y su entorno adyacente 3,6,8,11-14. Diversidades morfológicas y funcionales considerables tales como las variaciones en la morfología celular, la diferenciación celular, las adherencias célula-matriz y patrones de expresión génica han sido detectadas por el cultivo de células en los cultivos 3D que faltan comúnmente en 2D ensayos 2,6,8,11. Por lo tanto, los usos de los ensayos en 3D están significativamente beneficioso en la recapitulación de una forma más fisiológica en condiciones in vivo, lo que lleva a una mejor transposición de los resultados innovadores en la investigación básica a la clínica 6-10. Sin embargo, hay que señalar, A pesar de las muchas ventajas que se obtienen con el uso de cultivos 3D, este modelo no puede capturar todas las complejidades de la microambiente del tumor in vivo en que incluye diversos tipos de células. Sin embargo, es posible incorporar las células del estroma en los modelos 3D (por ejemplo, fibroblastos, leucocitos, y macrófagos) para estudiar el efecto de las interacciones tumor-estroma sobre la adhesión de células de cáncer y la invasión 15-17. Células epiteliales de mama en cultivo crecen con mayor eficacia cuando las proteínas ECM, como la laminina y el colágeno están presentes. Con esta conocida, una mezcla de la matriz disponible comercialmente se ha derivado de Engelbreth-Holm-Swarm (EHS) del tumor murino y se conoce como matriz de la membrana de Matrigel sótano 2,8. Un número de técnicas se han establecido para hacer crecer células epiteliales como colonias 3D en la matriz de la membrana basal 2,8. El modelo de matriz de membrana basal 3D es eficaz para el establecimiento de células de mama tanto maligno y no malignocrecimiento, parecido a lo que está ocurriendo en el entorno in vivo 18,19. Células MCF10A son células epiteliales mamarias no malignas. Cuando se cultiva en la matriz de la membrana basal, estas células exhiben en los rasgos in vivo de las células normales del seno y se someten a control de la proliferación celular, la polarización celular y la apoptosis de establecer el espacio lumen 8,12,20. Además, la aparición de núcleos celulares de las células MCF10A que forman acinos en cultivos 3D se asemejan más a las de las células epiteliales mamarias en el tejido que las cultivadas en monocapa 21. Los estudios realizados por Bissell y sus colegas fueron los primeros en revelar que las células malignas de mama pueden diferenciarse de células de mama no malignas cuando se cultiva en un entorno de laminina-rica, ya que las células malignas muestran un fenotipo altamente desorganizado, aumento de la proliferación, la disminución de la célula-a- la adhesión celular, aumento de la expresión de marcadores mesenquimales y un aumento en el número de estructura invasiva formaron 3,6,22. Anomalías del entorno celular pueden influir en la formación de tumores 20. El método de cultivo 3D se puede utilizar para estudiar con eficacia la comunicación que se produce entre las células tumorales y su ambiente circundante y determinar cómo influye en la expresión de proteínas tales 14,20,21,23 comunicación. En este artículo se presenta una metodología detallada para crecer MDA-MB-231 células de cáncer de mama en las culturas 3D para analizar la invasividad, y para estudiar la pérdida de la morfología epitelial utilizando un laminina marcadores epiteliales, un componente de la membrana basal de células 18,19,24, 25. Los procedimientos detallados proporcionan la capacidad de cuantificar con precisión y de manera reproducible estrelladas (invasiva) formación de la estructura por cualquier célula de cáncer invasivo y no es limitante para las líneas celulares de cáncer de mama común (tales como MDA-MB-231, Hs578T, MCF-7, o T47D ). Por lo tanto, este ensayo puede servir como una plataforma para la evaluación de cómo la expresión de proteínas en las células o el tratamiento con pro-o anti-compuestos invasivos regular degradación de la matriz extracelular, por las células individuales o múltiples.

<table border="0" cellpadding="0" cellspacing="0" width="1062">
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			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

Ahora es bien conocido que el microambiente celular y tisular son reguladores críticos que influyen en la iniciación del tumor y la progresión. Por otra parte, la matriz extracelular (ECM) se ha demostrado que es un regulador crítico de comportamiento de las células en cultivo y la homeostasis in vivo. El enfoque actual de cultivo de células en dos dimensiones (2D), las superficies de plástico resulta en la alteración y la pérdida de interacciones complejas entre las células y su microambiente. Mediante el uso de tres dimensiones (3D) de ensayos de cultivo, se establecieron las condiciones para la interacción de células-microambiente se asemeja a la microentorno in vivo. Este artículo proporciona una metodología detallada para hacer crecer células de cáncer de mama en una matriz de proteína de membrana basal 3D, que ejemplifica el potencial de la cultura 3D en la evaluación de la invasión de células en el medio ambiente circundante. Además, se discutirá cómo estos ensayos 3D tienen el potencial para examinar la pérdida de señalización molecdulos que regulan la morfología del epitelio mediante inmunotinción procedimientos. Estos estudios ayudan a identificar importantes detalles mecanicistas de los procesos que regulan la invasión, necesarios para la propagación del cáncer de mama.

Un ejemplo de las células MDA-MB-231 invasores en la matriz 3D se ilustra en la Figura 3C. Las células están incrustadas en la matriz (Día 1), y se empiezan a formar (estrelladas) estructuras invasoras por Día 3, y completamente invaden la matriz por el día 5 (Figura 3C). El número de colonias formadas estrelladas se cuentan, y se expresó como un porcentaje del número total de colonias por placa (invasivos y no invasivos). Además, ya que las mediciones se realizan al día dura...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

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

Este artículo proporciona metodologías detalladas para el uso de ensayos (3D) en tres dimensiones para cuantificar la invasión de células de cáncer de mama. En concreto, se discuten los procedimientos necesarios para configurar este tipo de ensayos, la cuantificación y el análisis de datos, así como los métodos para examinar la pérdida de integridad de la membrana que se produce cuando las células invaden.

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

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

quantification-breast-cancer-cell-invasiveness-using-three

Research

JoVE Journal

Medicine

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

15.7K vistas.  University of Western Ontario.  Este artículo proporciona metodologías detalladas para el uso de ensayos (3D) en tres dimensiones para cuantificar la invasión de células de cáncer de mama. En concreto, se discuten los procedimientos necesarios para configurar este tipo de ensayos, la cuantificación y el análisis de datos, así como los métodos para examinar la pérdida de integridad de la membrana que se produce cuando las células invaden. 

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Imágenes in vivo de tumores bioluminiscencia Dinámica hipoxia de la metástasis cerebral cáncer de mama en un modelo de ratón

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Investigación

Medicina

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 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotípicos tridimensional<em&gt; In Vitro</em&gt; Modelado de estromal-epitelial interacciones durante la iniciación y progresión del cáncer de ovario

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Un tejido tridimensional Cultura modelo para estudiar primaria médula ósea humana y sus malignidades

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Inicio de la metástasis de carcinoma de mama mediante la Focalización del epitelio ductal con adenovirus Cre: Un modelo de ratón transgénico de Novela del Cáncer de Mama

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Perfiles de microARN regulados por estrógeno en las células del cáncer de mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Tres Culturas dimensionales: una herramienta para estudiar normal Acinar Arquitectura vs transformación maligna de las células de la mama

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensional Modelo Co-cultura para la interacción tumor estromal

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

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 ...