Damos las gracias a Ken Iwata (OSI Pharmaceuticals, Nueva York, EE.UU.) para los reactivos y Fred Miller (Barbara Ann Intitute Cáncer Karmanos, en Detroit, EE.UU.) para las líneas celulares.

1. Preparación de la solución Methocel (500 ml) <ol><li> Autoclave de 6 g de metilcelulosa en una botella de 500 ml con agitador magnético. </li><li> Disolver la metilcelulosa en 250 ml de DMEM/F12 sin suero precalentado (60 ° C) durante 20 min. </li><li> Añadir 250 ml de DMEM/F12 (RT), que contiene 10% de suero de caballo. Mezcla durante la noche (4 ° C). </li><li> Clara la solución mediante centrifugación (5000 g, 2 h, 4 ° C). </li><li> Tome la solución clara de alta viscosidad a un nuevo tubo (aproximadamente el 90-95% de la solución madre). </li><li> Almacena a 4 º C hasta su uso. </li></ol> 2. Cultura esferoide <ol><li> Prepare una solución al 20% metocel (10 ml metocel y 40 ml de medio de crecimiento). </li><li> Lavar las células dos veces con PBS, añadir tripsina al 0,1% y las células se incuban a 37 ° C </li><li> Resuspender las células en medio de crecimiento y el recuento de las células. </li><li> Preparar una suspensión de 10 000 células por ml en el 20% metocel. </li><li> Añadir la suspensión celular a la s 96 de fondo redondo yplacas USPENSIÓN (100 l por pocillo). </li><li> Al día siguiente, consulte la formación esferoide en las placas de suspensión. Esferoides estará listo para su uso después de 1-3 días. </li></ol> 3. Preparación de colágeno <ol><li> Prepare en el hielo una solución de colágeno de 8 ml, 1 ml de PBS 10x, 1 ml de NaOH 0,1 N y 3 gotas de HCl 0,1 N. Compruebe si el pH es de 7.4 por manchar algo de la solución en las tiras indicadoras del pH. Ajustar el pH si fuera de rango. </li><li> Añadir 50 l de solución de colágeno neutralizado en los pocillos de una placa de 96 pocillos </li><li> Se incuba a 37 ° C hasta que el colágeno se solidifica (90 min). </li><li> Prepare metocel-ligando colágeno soluciones en el hielo: para cada pipeta ligando una parte de la solución de colágeno. Agregar 20 ng por ml de TGF-β colágeno. Después de la dilución con metocel y difusión sobre las diferentes capas, la concentración final de TGF-β será de 5 ng / ml Mezclar por agitación. Añadir una parte de metocel solución. Mezclar mediante agitación. </li></ol> 4. Esferoide incrustarding <ol><li> Coloque una punta de 200 l que tiene aproximadamente 5 mm de la punta cortada, en una pipeta de 200 microlitros. Chupar un esferoide, con la pipeta y cuidadosamente dispensar el medio en un plato vacío. Ver si el esferoide se mantiene dentro de la punta. El esferoide es visible como un punto blanco. Sin soltar el émbolo, absorben 100 l colágeno metocel mezcla y vierta el esferoide en la mezcla de colágeno en el colágeno recubiertas de 96. Haga esto durante 12 esferoides para cada condición. </li><li> Deje que el colágeno se solidifican al menos 30 minutos a 37 ° C </li><li> Eliminar las burbujas de aire con una punta de 200 microlitros. </li><li> Añadir 50 l de medio con el 1,6% de suero y los inhibidores disueltos en DMSO. Para SB-431542 añadir 4 l de solución madre (10 mm) a 1 ml de medio. Después de la difusión, la concentración final de inhibidor de la será de 10 micras. TGF-β y los inhibidores mantendrá el curso del experimento. Tome fotos de los esferoides en el microscopio con aumento de 40x. </li><li> Después de 48h de la estimulación con TGF-β y / o inhibidores, tomar fotos de los esferoides en el microscopio con aumento de 40x. </li><li> Medir el área de los esferoides invasores después de 2 días y restar la zona de salida. La medición de la superficie de un esferoide: Abrir la imagen del esferoide en Adobe Photoshop Extended (figura 2A) y seleccionar la herramienta de selección rápida (Figura 2B). El puntero del ratón se transforma en un círculo con un &#39;plus&#39; signo (+) (figura 2c). Mientras arrastra el cursor sobre el esferoide, Photoshop se reconocen las fronteras del esferoide. Si un área fuera del esferoide se selecciona, esta región puede ser excluido de la selección pulsando la tecla Alt o mediante el uso de la herramienta de selección negativa que se indica mediante un signo - en la barra de herramientas (). El cursor se convierte en un círculo con un &quot;menos&quot; signo (-) y arrastrando el cursor sobre el área seleccionada erróneamente, esta zona se retira de la selección (figura 2D). Si las regiones es necesario, pueden seleccionar múltiples pulsando el Shbotón ift. Para trabajar con mayor precisión, el tamaño del puntero del ratón se puede ajustar cambiando el diámetro del cepillo en la barra de herramientas (Figura 2E). Cuando el esferoide se selecciona todo el área de la selección se calcula a través de análisis, medida en la barra de menú, o pulsando Ctrl + Mayús + M (figura 2F). La ventana de grabación de medición aparecerá mostrando el nombre del archivo y el área de la selección en píxeles, así como otros datos (figura 2G). Si se miden varias selecciones, la primera línea muestra la suma de todas las áreas. Las medidas de las selecciones individuales se presentan en las líneas de abajo. Los datos de todas las mediciones se pueden exportar a un archivo de texto y se abre en Excel. </li></ol> 5. Los resultados representativos: Un ejemplo de la prueba esferoide con MCF10A1 (M1), las células epiteliales de mama normales, H-RAS transformó MCF10AneoT (M2) y las células derivadas de MCF10CA1a M2 (M4) se da en la figura 3. Células M1 mostró sólo la invasión débil sinestimulación, pero invadido significativamente mejor a la estimulación por TGF-β. En contraste, el RAS-transforma M1-M2 derivados invadido ya de manera eficiente sin la adición de estímulos, lo que aumentó aún más a 4.5 veces el TGF-β tratamiento. Células M4 mostraron la mayor invasión, con y sin TGF-β además. A continuación, examinó si se podría interferir con el TGF-β inducida por la invasión de este modelo esferoide el uso de inhibidores químicos. Para ello se utiliza el SB-431542, un análogo de la ATP y el inhibidor selectivo de la actividad quinasa de TβRI, así como la activina tipo IB receptor (ActRIB) y activin receptor de tipo quinasa (ALK) 7 12. Como era de esperar, SB-431542 potentemente inhibida TGF-β inducida por invasión de M1, M2 y M4 células (Fig. 4), lo que indica que el TGF-β invasión inducida es TβRI-quinasa dependiente. Además, la invasión basal de los esferoides también fue fuertemente inhibida, lo que sugiere que la invasión basal es también deindependiente de TGF-β. <img alt="Figura 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> Figura 1. Diagrama de flujo del ensayo esferoide 3D. En primer lugar, las células se colocan en condiciones de no adherentes en forma de U placas suspensiones. Células aggregrate en esferoides multcellular y puede ser embebido en una matriz de colágeno. Dentro de esta matriz de colágeno, que son capaces de invadir. <img alt="Figura 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> Figura 2. Cuantificación de la zona de los esferoides con Adobe Photoshop Extended. (A) La imagen del esferoide se abre en Adobe Photoshop Extendend (B) Selección de la herramienta de selección rápida (C) Al arrastrar el cursor sobre el esferoide para seleccionar el área de la esferoidal (D) La eliminación de un área wongly incluyó el uso de la negativo herramienta de selección rápida (E) Ajuste de el tamaño del pincel de la herramienta de selección rápida para seleccionar con mayor precisión la zona (F) La selección de lamedición de comando que desea grabar (G) Ejemplo de un registro de medición. <img alt="Figura 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> Figura 3. TGF-β inducida por invasión de esferoides de forma espontánea inmortalizada MCF10A1 (M1) (A), RAS-transforma MCF10AneoT (M2 (B) y sus derivados MCF10CA1a metastásico (M4) (C). M1, M2 y M4 esferoides incrustado en colágeno fueron tratados con 5ng/ml de TGF-β durante 48 horas como se indica. AC imágenes representativas tomadas en el día de la incorporación y dos días después. invasión D relativa se cuantificó como la diferencia de área en el día 2 menos el día 0. Los resultados se expresan como media ± desviación estándar Adaptado de 13. <img alt="Figura 4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="Figura 4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> Figura 4. Invasión esferoide basal y TGF-β inducida se inhibe la by SB-431542. Esferoides M1, M2 y M4 fueron incorporados en el colágeno y tratados con TGF-β 5ng/ml y / o 10 M SB-431542, como se indica. Imágenes representativas tomadas después de 2 días (A). Invasión relativa se cuantificó como la diferencia de área en el día 2 menos el día 0 (B). Los resultados se expresan como media ± DE Adaptado de 13.

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Hemos establecido un modelo esferoide en el que MCF10A1 células y sus derivados malignos invaden en una matriz de colágeno de manera TGF-β-dependiente. En primer lugar, esferoides se forman en 96 pozos de placas de suspensión de fondo redondo, con la presencia de metilcelulosa. Por supuesto, también en forma de U poli-HEMA placas recubiertas se pueden utilizar para este propósito. La metilcelulosa es absolutamente necesario en este paso, ya que las células se mueren en la ausencia de metilcelulosa. TGF-β se aña...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Metil celulosa </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avanzada Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> tiras indicadoras del pH </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96 y la suspensión placas </td><td> Greiner Bio-One </td><td> 650 185 </td><td></td></tr><tr><td> 96-TC y la adhesión placas </td><td> Greiner Bio-One </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-β ha opuesto papel en la progresión del cáncer de mama al actuar como un supresor tumoral en la fase inicial, pero estimulando la invasión y metástasis en 1,2 más adelante. Por otra parte, el TGF-β es frecuentemente sobreexpresado en el cáncer de mama y su expresión se correlaciona con mal pronóstico y la metástasis 3,4. Los mecanismos por los que el TGF-β induce la invasión no se comprenden bien. TGF-β induce la respuesta celular a través del tipo II de TGF-β (TβRII) y el tipo I (TβRI) receptores. TGF-β sobre la inducida por la formación de complejos heteroméricos, TβRII fosforila la TβRI. El TβRI activado inicia su vía de señalización intracelular canónico por la fosforilación de Smads receptor (R-Smads), es decir, Smad2 y Smad3. Estas activa R-Smads formar complejos con heteroméricos Smad4, que se acumulan en el núcleo y regulan la transcripción de genes diana 5. Además de los anteriores described vía Smad, los resultados de la activación del receptor en la activación de varias otras organizaciones no Smad vías de señalización, por ejemplo la proteína quinasa activadas por mitógeno (MAPK) vías 6. Para estudiar el papel del TGF-β en las diferentes etapas del cáncer de mama, hemos hecho uso del sistema de células MCF10A. Este sistema se compone de manera espontánea inmortalizado células de la mama MCF10A1 (M1) del epitelio 7, el H-RAS transformó M1 derivados MCF10AneoT (M2), que produce lesiones premalignas en ratones 8, y el MCF10CA1a M2 derivados (M4), que se estableció a partir xenoinjertos M2 y formas de alta carcinomas de grado con la posibilidad de metástasis en el pulmón 9. Esta serie MCF10A ofrece la posibilidad de estudiar las respuestas de las células con diferentes grados de malignidad que no están sesgados por una base genética diferente. Para el análisis de TGF-β inducida por la invasión, se generó homotípicos MCF10A células esferoide embe culturasñadido en una matriz de colágeno 3D in vitro (Fig. 1). Estos modelos se parecen mucho a los tumores humanos in vivo mediante el establecimiento de un gradiente de oxígeno y nutrientes, lo que resulta en células activas y las operaciones en el exterior y las células en reposo o incluso necrosis en el interior del esferoide 10. Esferoide ensayos basados ​​también han demostrado una mejor resistencia a los medicamentos que recapitular monocapas 11. Este sistema MCF10 modelo 3D nos permitió investigar el impacto de TGF-β señalización en las propiedades de las células de mama invasivo en las diferentes etapas de malignidad.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-ÃŽÂ²-induced Invasion at JoVE.com

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

Un ensayo para medir cuantitativamente el factor transformador del crecimiento (TGF)-β-inducida por invasión en geles de colágeno en 3 dimensiones se describe. Este ensayo se aprovecha de la serie MCF10A de líneas celulares, que representan diferentes etapas de desarrollo de cáncer de mama. Este método puede ser aprobada para su uso con otras líneas celulares y podrían ser utilizados para investigar otras posibles activadores o inhibidores de la invasión.

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 ...

spheroid-assay-to-measure-tgf-induced-invasion

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21.8K vistas.  Leiden University Medical Centre. Un ensayo para medir cuantitativamente el factor transformador del crecimiento (TGF)-β-inducida por invasión en geles de colágeno en 3 dimensiones se describe. Este ensayo se aprovecha de la serie MCF10A de líneas celulares, que representan diferentes etapas de desarrollo de cáncer de mama. Este método puede ser aprobada para su uso con otras líneas celulares y podrían ser utilizados para investigar otras posibles activadores o inhibidores de la invasión.

Video: Spheroid Assay to Measure TGF-β-induced Invasion

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

A tiempo real Técnica de Impedancia Eléctrica base para medir la invasión de monocapa de células endoteliales de las células cancerosas

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

Investigación

Medicina

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 ...

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

Método para medir el tono del músculo axial y proximal

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Precisión en Medicina Dental, Una nueva forma de medir veracidad y precisión

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

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 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Evaluación de Cáncer de Ovario esferoide El apego y la invasión de células mesoteliales en Tiempo real

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetría intrauterina para medir la presión del ratón contráctil<em&gt; En Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

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 ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

Ensayo Invasión polluelo corazón para probar la invasividad de las células del cáncer y de la actividad de los compuestos potencialmente anti-invasivos

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Un Cancer Cell esferoide Ensayo para evaluar la invasión en un entorno 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Ensayo esferoide para medir TGF-β inducida por invasión

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