Name: moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol
Uploaded: 2018-03-12T13:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Este trabajo fue apoyado por los institutos nacionales de salud (NIMHD-G12MD007581) a través del RCMI-centro de salud ambiental en la Universidad Estatal de Jackson y el Departamento de defensa, Premio Idea 11-1-0205.

Nota: Este protocolo se describe en McArdle et al. 11. una línea de tiempo se proporciona en la figura 1.
1. preparación de la placa de cultivo
<ol><li>Lave la placa de cultivo de 35 mm que contiene un fondo de cristal de 10 mm con 1 mL de 1 M de ácido clorhídrico (HCl) durante 15 minutos reducir la hidrofobicidad de la parte inferior de cristal.</li>
	<li>Lave la placa de cultivo dos veces con 1 mL de solución salina buffer fosfato (PBS) para deshacerse de la HCl.</li>
	<li>Lave la placa de cultivo dos veces con 1 mL de etanol al 70%.</li>
	<li>Enjuague la placa de cultivo con 1 mL de medio de cultivo específico celular (aquí uso α-MEM suplementado con 10% de calor inactiva FBS y los de aminoácidos no esenciales 1%). Nota: La placa de cultivo tratado que contiene el medio de cultivo se puede almacenar en la incubadora de CO2 con un humidificador a 37 ° C hasta el día siguiente.</li>
	<li>Crecen las células (MSCs y MCF-7 co de la cultura) en el fondo de cristal tratado de la placa de cultivo de 35mm a 100% de confluencia. Co de la cultura células MSC 35.000 y 75.000 de células MCF7 durante 24 h.</li>
</ol>2. preparación del gel de colágeno
<ol><li>Guarde las puntas de micropipeta utilizadas para pipetear el gel de colágeno en el congelador para evitar la polimerización del colágeno al entrar en contacto. Nota: Este es el paso más crítico.</li>
	<li>Determinar un volumen de colágeno de cola de rata I utilizar basado en la concentración de la población de colágeno. Preparar 100 μl de gel de colágeno para este ensayo. Nota: Alta concentración de colágeno de la cola de rata que stock funciona mejor. Determinar el volumen de 10 x medio modificado Eagle de Dulbecco para diluir el colágeno por consiguiente.</li>
	<li>Combinar en el colágeno de cola de rata de hielo I (acción de 3,8 μg/mL), modificado Eagle de 10 x Dulbecco mediana y 1 x celular medio de cultivo suplementado con 2% de suero bovino fetal y la chemoattractant correspondiente a una concentración final de colágeno de 2.4 mg/mL.</li>
	<li>Añadir a la mezcla 4,5% de solución de bicarbonato de sodio para neutralizar el gel de colágeno. Nota: El volumen de solución de bicarbonato de sodio añadido puede variar según el pH exacto de los otros reactivos. Es mejor probar la mezcla con tiras de pH para garantizar la solución neutral, o usar un indicador de pH en el medio de cultivo.</li>
	<li>Pone 80 μl de la mezcla de colágeno sobre las células en el fondo de cristal de la placa de cultivo.</li>
	<li>Permita que el gel de colágeno polimerizar durante 30 minutos en un incubador de CO2 con un humidificador a 37 ° C.</li>
	<li>Cubrir el gel de colágeno con 2 mL de medio de celular con suero reducido (2%) e incubar a las células a 37 ° C en un incubador de2 CO con un humidificador para 24, 48 o 72 h. PRECAUCIÓN: La placa debe manejarse con cuidado para evitar que el gel de colágeno extraer desde el fondo de cristal.</li>
</ol>3. ejemplo de elaboración de colágeno gel usando un stock de colágeno de cola de rata de 3,8 μg/ml
<ol><li>Con hielo, combinar 114.5 μl de colágeno de cola de rata, 16.6 μl de 10 x DMEM y 33,1 μl del medio de cultivo que contiene la chemoattractant.</li>
	<li>Neutralizar la mezcla de colágeno con 14.0 μL de bicarbonato de sodio.</li>
	<li>Continuar como en el paso 2.5.</li>
</ol>4. la proyección de imagen de las células
<ol><li>Retire con cuidado los medios de comunicación de la fuente.</li>
	<li>Enjuague suavemente el gel con 1 mL de PBS.</li>
	<li>Fijar las células con 1 mL de paraformaldehído al 4% durante 15 minutos.</li>
	<li>Lavan las células dos veces con 1 mL de PBS.</li>
	<li>Se tiñen las células con solución DAPI (100 ng/mL) durante 20 min a temperatura ambiente.</li>
	<li>Imagen de las células utilizando un microscopio confocal en diferentes lugares y tomando imágenes de z-stack de 400 μm de cada ubicación en 16 pasos de μm. El microscopio utilizado tiene un disco central que permite la proyección de imagen confocal usando una luz blanca, fuente de excitación del arco de la exploración. Utilizar un objetivo de X 20 utilizado con apertura numérica de 0,75.</li>
	<li>Imágenes de reconstituir.<ol>
<li>Abrir imágenes de un gel determinado (alrededor de 25 imágenes) y crear una pila de imagen pulsando en imagen → pila → imágenes para apilar. La primera imagen corresponde a la parte inferior del gel (z = 0 μm), la segunda imagen corresponde al primer paso en la dirección z (z = 16 μm), la tercera imagen corresponde al segundo paso en la dirección z (z = 32 μm). Evaluar cada núcleo en cada profundidad de imagen y designar la profundidad en que el núcleo estaba en el foco más agudo como la profundidad de invasión para ese celular particular.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li><li>Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Wolf, K., 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho/ROCK+signalling+and+extracellular+proteolysis.">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li><li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro.">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198 (2008).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li><li>Poujade, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li><li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-induced+cancer+cell+fusion:+a+mechanism+of+breast+cancer+metastasis.">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li><li>McArdle, T. J., Ogle, B. M., Noubissi, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types.">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li><li>Noubissi, F. K., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Cell+Fusion:+Mechanisms+Slowly+Unravel.">Cancer Cell Fusion: Mechanisms Slowly Unravel.</a> Int J Mol Sci. 17, (2016).</li><li>Meyvantsson, I., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+models+in+microfluidic+systems.">Cell culture models in microfluidic systems.</a> Annu Rev Anal Chem. 1, 423-449 (2008).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods In Enzymol. 406, 625-643 (2006).</li><li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+memory+and+dosing+influence+stem+cell+fate.">Mechanical memory and dosing influence stem cell fate.</a> Nat Materials. 13, 645-652 (2014).</li></ol>

Los autores han declarado que ningún interés competencia existen.

Aquí, describimos un ensayo de invasión vertical invertido simple que es conveniente para una variedad de tipos de células incluyendo las células que son raros o dependiente en su microambiente. En este ensayo de invasión 3D, las células permanecen en su microentorno original y están cubiertas por el gel de colágeno que contiene un chemoattractant. Las células entonces migraran e invaden en el colágeno. En este ensayo, las células pueden ser manchadas y fácilmente monitoreadas en el gel. La simplicidad y repr...

La motilidad celular es un proceso fisiológico y de desarrollo normal de las células. Sin embargo, cambios en el patrón y la capacidad de las células para migrar e invadir están asociados a condiciones patológicas como enfermedades inflamatorias y cáncer metástasis1,2,3. La metástasis es posiblemente el aspecto más mal entendido en cáncer. Para metástasis, las células cancerosas deben degradar la matriz extracelular (ECM), invaden el tejido local y intravasate. Una vez en circulación, las células cancerosas se difundir el sitio distante extravasate y formar tumores secundarios. Por lo tanto, la capacidad de las células para degradar el ECM, a migrar y a invadir se relaciona con su potencial metastásico. Han desarrollado diferentes enfoques para analizar y cuantificar las capacidades de migración y la invasión de las células. Los enfoques más utilizados incluyen la cicatrización ensayo, microscopía Time-lapse y el análisis de la cámara de Boyden. La herida curativo ensayo4 y tiempo lapso microscopia son ensayos simples y convenientes que darían visión preliminar de migración de la célula. Sin embargo, ambos son ensayos 2D que reflejan el entorno 3D en el que las células existen en vivo. Los estudios han demostrado que las células responden de manera diferente a un entorno 3D comparado con un 2D5,6. Por otra parte, ensayos curativos de heridas son difíciles de reproducir y producir resultados cuantitativos7,8,9. El análisis de la zona de exclusión de la célula, que es una modificación 3D de ensayo de las heridas, es un ensayo más fisiológico relevante pero no es adecuada para células bajas en número como células híbridas resultantes de celular fusión eventos10,11 .
El análisis de la cámara de Boyden es un ensayo de 3 dimensiones que provee microambientes pertinentes estudios celulares. Sin embargo, necesita extraerse su microentorno original y ser depositado en la cámara alta del sistema de análisis de Boyden para migrar e invadir hacia abajo hacia el medio que contiene chemoattractant. Este enfoque 3D no es apropiado en el análisis de la capacidad de migración y la invasión de células vulnerables a su microambiente o limitada en número de11,10,12. Un enfoque más reciente para analizar la invasión y migración de la célula es la microfluídica cámara gradiente análisis13. Aunque conveniente y fiable en los estudios de migración y la invasión, este análisis son más costoso y pueden ser técnicamente difícil para un laboratorio típico. Describimos aquí un ensayo de invasión vertical invertido simple que es compatible con muchos tipos de células incluyendo las células sensibles a su microambiente o restringidas en número. Este ensayo fue adaptado del protocolo propuesto por Hooper et al. 14. en este ensayo, las células permanecen en su microentorno original y su migración y la invasión se controla mientras se mueven hacia arriba en el gel de colágeno que contiene un chemoattractant11. Este ensayo es reproducible y ha sido optimizado y validado en la placa de cultivo de 35 mm. Podría adaptarse a las condiciones de ensayo diferentes incluyendo tamaños de célula diferente cultura, diferentes matrices o fuerza de adherencia y diferentes atrayentes. Este análisis podrían servir como una plataforma en vitro para la detección inicial en el proceso de descubrimiento de fármacos.

<table><tbody><tr><td>MatTek P35G-1.5-10C</td><td>MatTek Corporation, Ashland, MA</td><td>P35G-1.5-10-C</td><td>mattek glass bottom dishes</td></tr><tr><td>Rat tail collagen I</td><td>Corning, Corning, NY, USA</td><td>354236</td><td>Collagen gel</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>H1758</td><td>HCl</td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>10010023</td><td>PBS</td></tr><tr><td>10X Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>D2429</td><td>10X DMEM</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Confocal Fluorescent microscope</td><td>Olympus America, Center Valley, PA, USA</td><td>Olympus IX81</td><td>Confocal&amp;nbsp; microscope</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td><td>SH30070.03</td><td>FBS</td></tr><tr><td>Ti:Sapphire multi-photon laser scanning microscope</td><td>Prarie Technologies, Sioux Falls, SD, USA</td><td>40x NA 0.8 objective</td><td>MPLSM</td></tr><tr><td>Imaris software</td><td>Bitplane Inc. Zurich, CH</td><td>Imaris 7.5.2</td><td>Imaris software</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>D9542</td><td>DAPI</td></tr><tr><td>Paraformaldehyde</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>50980487</td><td>PFA</td></tr><tr><td>&amp;alpha;-minimum essential medium</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>M0894</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC; Manassas, VA, USA</td><td>HBT-22</td><td></td></tr><tr><td>MSC</td><td>Trivedi and Hematti, 2007</td><td></td><td></td></tr><tr><td>20X lens UPLAPO</td><td>Olympus America, Center Valley, PA, USA</td><td>36-064</td><td>Olympus UPLSAPO 20X Objective</td></tr></tbody></table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

Aunque se han desarrollado ensayos de invasión 3D, el desafío sigue siendo estudiar las células sin afectar la integridad de su microambiente. 3D tradicionales ensayos tales como el compartimiento de Boyden requieren que las células son desplazadas de la ubicación original de la cultura y se trasladó a un nuevo entorno. No sólo hace esto interrumpir los procesos celulares que son intrínsecos al microentorno, pero a menudo resulta en una pérdida de células. Estos problemas son especialmente difíciles cuando se trata de las células que son raros, o extremadamente sensibles a su microambiente. Aquí, describimos el desarrollo de un ensayo de invasión 3D que evita ambos problemas. En este ensayo, las células se platean dentro de un pequeño bien y una matriz del ECM que contiene un chemoattractant se coloca encima de las células. Esto no requiere desplazamiento de la célula y permite a las células invadir hacia arriba en la matriz. En este ensayo, invasión celular y morfología celular puede ser evaluada en el gel de colágeno. Usando este análisis, se caracteriza la capacidad invasiva de las células raras y sensibles; las células híbridas resultantes de la fusión entre las células de cáncer de mama MCF7 y mesenquimales multipotentes/tallo/estroma células (MSCs).

Hemos utilizado una placa de cultivo de 35 mm con un vidrio inferior y rata de cola colágeno I a desarrollar y optimizar una invasión en vitro 3D análisis de protocolo. Usando este análisis, hemos demostrado eso mama células cancerosas MCF7 y MSC células podrían invadir en el gel de colágeno a una distancia media de 0.04 μm ± 1.8 y 41,3 ± 76.0 respectivamente después de 48 h cuando IGF1 (18,6 ng/mL) fue utilizado como chemoattractant (figura 2</st...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Este protocolo describe un ensayo de invasión vertical invertido que podría ser utilizado para cuantificar la capacidad de migración y la invasión de las células en un entorno tridimensional conservando el microambiente celular. Este ensayo es adecuado para las células raras o sensibles.

Mover hacia arriba: Un Simple y Flexible In Vitro invasión tridimensional protocolo

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

moving-upwards-simple-flexible-vitro-three-dimensional-invasion-assay

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

6.0K vistas.  University of Minnesota - Twin Cities. Este protocolo describe un ensayo de invasión vertical invertido que podría ser utilizado para cuantificar la capacidad de migración y la invasión de las células en un entorno tridimensional conservando el microambiente celular. Este ensayo es adecuado para las células raras o sensibles.

Video: Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Ensayo esferoide para medir TGF-β inducida por invasión

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Investigación

Medicina

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelado y de imagen de 3 dimensiones invasión celular colectiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Matriz basada en tumores completamente humana en ensayo de invasión de esferoide tridimensional

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Investigación sobre el cáncer

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Un In vitro Tumor FluoroBlok invasión de ensayo

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Biología

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

En los ensayos de Vitro para evaluar la migración, invasión y proliferación de las líneas de células inmortalizadas trofoblasto humano de primer trimestre

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Biología del Desarrollo

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

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

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

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Un Modificado<em&gt; In vitro</em&gt; Invasión Ensayo para determinar el papel potencial de hormonas, citoquinas y / o factores de crecimiento en la invasión de células del cáncer de Mediación

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Modelos 3D en Biología del Cáncer: Dinámica de Invasión en Heterosferoides

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Autor destacado: Descifrando la diafonía entre el cáncer y las células inmunitarias durante la progresión del cáncer de cabeza y cuello

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Estrategia alternativa para analizar la invasión celular in vitro de cultivos 3D

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

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 ...

Mover hacia arriba: Un Simple y Flexible In Vitro invasión tridimensional protocolo

Resumen

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Estrategia alternativa para analizar la invasión celular in vitro de cultivos 3D

Estrategia alternativa para analizar la invasión celular in vitro de cultivos 3D

Tumor Ensayo tridimensional (3D) esferoide Invasion