El financiamiento para esta investigación es de Cancer Research del Reino Unido.

1. Etiquetado retroviral de células con proteínas fluorescentes <ol><li> Células de la placa de embalaje retrovirales (por ejemplo, Phoenix) de un total de 0,25 x 10 6 células por pocillo de una 6-y plato fetal al 10% de suero bovino (FBS) / DMEM. </li><li> Transfectar las células con el ADN retroviral 48 horas después, las células con Effectene de acuerdo a las instrucciones del fabricante. </li><li> Enjuague pocillos dos veces con medio de 24 horas después, a continuación, agregar 1,5 ml de 10% de SFB / DMEM por pozo. </li><li> Recoger virus en cultivo de tejidos empaquetados medio 48 horas más tarde con la pipeta y transferir a tubos de 2 ml microcentrífuga. </li><li> Centrifugar a 1600 rpm durante 5 minutos para que sedimenten las células. </li><li> Aspirar el sobrenadante a un tubo limpio y almacenar a -80 ° C. </li><li> Las células que se retroviralmente transducidas se colocan fuera de 1,5 x 10 5 células por pocillo de una placa de 6 pocillos y se colocan en un incubador humidificado a 37 º C durante la noche. </li><li> Al día siguiente, retire el papel de las células y add 1 ml de caldo de virus suplementado con 4 polibreno l (2 mg / mL) a cada pocillo. Vuelva a colocar las placas en la incubadora. </li><li> Después de la incubación durante 5-6 horas, añadir 2 ml de 10% de SFB / DMEM a cada pocillo y las placas se sustituyen en 37 ° C durante la noche inbubator. </li><li> Al día siguiente, reemplazar los medios de comunicación y permiten que las células 24 horas antes de la adición de medios selectivos apropiados. </li><li> Cuando confluentes (que depende de la tasa de crecimiento de las células, y la eficiencia de la transducción retroviral, pero suele tardar entre 4-14 días), trypsinize células y tipo de células con fluorescencia no transducidas de referencia, recopilar y células de la piscina con fluorescencia mayor que los no-células transducidas y las alícuotas se congelan a -80 ° C para su uso futuro. </li></ol> 2. Inversa Matrigel ensayo de invasión <ol><li> Poco a poco descongelar una alícuota de Matrigel completa (es decir, que contienen factores de crecimiento) en el hielo (Figura 1A). </li><li> Una vez descongelado, diluido 1:1 en Matrigel PBS enfriado con hielo (junto con otras adicionalestratamientos en la concentración de 2x en el PBS antes de la dilución; fig. 1B). Para que sea más fácil de manejar Matrigel antes de la polimerización, todos los recipientes de plástico (por ejemplo, consejos, tubos, etc) debe ser fría como el hielo. </li><li> Inserte el mayor número 8 micras de poro 6,5 mm de diámetro sin revestimiento transwells como se requiere en los pocillos de una placa de 24 y cultivo de tejidos, luego cuidadosamente Pipetear 100 L de la Matrigel diluido en los pocillos y se deja incubar durante ~ 30 minutos a 37 ° C para solidificar (fig. 1C). </li><li> Durante este tiempo, preparar uno o más fluorescente marcada con suspensiones celulares de entre 1 y 4 x 10 5 células por ml, en función de la línea celular, de cada estado de pre-tratamiento (e. g. SiRNA, el tratamiento de drogas) en su crecimiento normal medio (Figura 1D). </li><li> Cuando el Matrigel se ha solidificado, invertir la transwells y Pipetear 100 L de la suspensión de células en la parte de abajo miran hacia arriba del filtro (Fig. 1E). </li><li> Con cuidado, cubrir el transwells con la base de un tejido de 24 pocillosplaca de cultivo, toma de contacto con cada gota de suspensión celular (Figura 1 F). </li><li> Se incuba la placa en el estado invertido durante 4 horas para permitir la unión de las células (Figura 1G). </li><li> Después de este tiempo, a su vez las placas con el lado derecho hacia arriba y lavar cada Transwell secuencial por inmersión en medio de 2 x 1 ml de suero libre (Figura 2A). </li><li> Deje el transwells en un tercer pozo que contiene alguna droga o tratamientos requeridos (Figura 2B). </li><li> Suavemente Pipetear 100 L de 10% de SFB / DMEM más quimioatrayente (e. g. FEAG a 25 ng / ml) en el Transwell en la parte superior de la Matrigel solidificado / mezcla de PBS, coloque la tapa e incubar durante 3-5 días a 37 º C con CO2 al 5% (Fig. 2C). </li></ol> 3. Tinción y visualización <ol><li> Las células que expresan proteínas fluorescentes puede saltarse los pasos a continuación antes de la microscopía confocal </li><li> A la imagen no fluorescentes las células invasoras Matrigel, transwells lugar en fresco 24 platos bien y pipeta de 1 ml de 4 MCalceína AM solución mancha en la parte superior de cada plug Matrigel, permitiendo que se derrame por los costados y las manchas de la parte superior e inferior. Calceína AM (éster acetoximetil de calceína) es un tinte de células vivas que las manchas de las células de todo el verde y no requiere la fijación. </li><li> Incubar 1 hora a 37 ° C en 5% de CO2 atmósfera húmeda, en la que las células están totalmente manchados punto y listo para ser captado por microscopía confocal. </li><li> Por otra parte, no fluorescente las células invasoras Matrigel se fijaron y se tiñeron como se describe en 3.5 a 3.9. </li><li> Transferencia de cada Transwell a un nuevo máximo de 24 pocillos. Superposición de 1 ml de 4% para-formaldehyde/0.2% Triton X-100. </li><li> Incube a temperatura ambiente durante 0,5 horas. </li><li> Quitar fijador y lavar 3 veces con 1 ml de PBS. </li><li> Quitar ARN citoplásmico con 30 min de tratamiento con RNasa 100 ug / ml RNasa. Lavar dos veces con PBS. </li><li> Para la visualización de añadir 0,01 mg / ml de yoduro de propidio (PI) diluido en PBS y dejar a temperatura ambiente en el dark durante 0,5 horas. Lavar 3 veces con PBS. En esta etapa, PI manchas Transwell se puede mantener a temperatura ambiente en la oscuridad durante al menos 1 mes. </li><li> Métodos precisos de imagen por microscopía confocal son dependientes de los recursos disponibles. Utilizando un microscopio invertido confocal, Transwell lugar con una pequeña cantidad de PBS residual en cubreobjetos grande (asegurarse de que no hay burbujas) sobre los no-inmersión 20 X objetivo y la captura de secciones ópticas de 10-15 m desde el fondo del enchufe matrigel. Individuales rebanadas de ópticas se pueden utilizar para cuantificar el grado de invasión (Figura 3) o en la construcción de objetos en 3 dimensiones usando el software apropiado, como Volocity (Figura 3B). </li></ol> 4. Resultados representante Un ejemplo de un Z-serie de cortes ópticos se muestra en la Figura 3. En este caso, las células fueron teñidas con calceína AM y el número de células invasoras desde el filtro se puede ver que disminuyen con la distancia. Cuantificación de lavasion se puede hacer mediante el análisis de la relación de calceína AM píxeles positivos a negativos píxeles en cada intervalo, o utilizando el método de fijación / tinción se detalla más arriba y contando núcleos PI positiva en cada posición. Una de las ventajas de calceína AM tinción es que las reconstrucciones en 3 dimensiones de la invasión de células se puede montar utilizando software como Volocity, dando una representación visual de la modalidad de la invasión (Figura 3B). Si las células son etiquetados por la expresión de proteínas fluorescentes, entonces las posiciones de cada celda de color pueden ser visualizados en tres dimensiones reconstrucciones, ya sea visto desde un lado (Figura 3C) o haciendo cortes a través de la reconstrucción (Figura 3D). <img alt="Figura 1" src="/files/ftp_upload/3525/3525fig1.jpg" /> Figura 1. Esquema de los pasos involucrados en la creación de la invasión de ensayo inversa. A) Matrigel ECM descongeladas en hielo. B) Matrigel se diluye 1:1 con PBS que contenía los tratamientos de drogas en la concentración de dos veces al final. C)<html> Inserciones Transwell se colocan en placas con pocillos múltiples, y Matrigel pipeta en cada uno. D) Las suspensiones celulares realizados en la concentración deseada. E) Una vez que el Matrigel se ha puesto, la placa se invierte y se retira, las células se colocan en el filtro inferior de las inserciones Transwell. F) En la posición invertida, la placa de paredes múltiples se coloca cuidadosamente sobre inserciones Transwell, haciendo contacto con la suspensión celular. G) Las células pueden adherirse al filtro durante 4 horas. <img alt="Figura 2" src="/files/ftp_upload/3525/3525fig2.jpg" /> Figura 2. Continuación del esquema para el ensayo de invasión inversa. A) Una vez que las células se han adherido, sumerja cada Transwell en medio libre de suero dos veces para eliminar las células sueltas. B) Lugar lavado Transwell los medios de comunicación en un final bien contiene más tratamientos según sea necesario. C) los medios de comunicación que contiene quimioatrayente (por ejemplo, 10% de suero fetal bovino) con los tratamientos que requiere cuidado en capas en Matrigel. ent &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/3525/3525fig3.jpg" /> Figura 3. Imágenes representativas de los resultados del ensayo de invasión inversa. A) las secciones de óptica de las células teñidas con calceína AM invadir Matrigel, tomadas a intervalos de 15 micras por microscopía confocal. B) Reconstrucción de una reconstrucción en 3 dimensiones de la invasión de células de una pila de confocal de la serie Z imágenes, vista desde el lado. Reproducido de la referencia 10. C) La reconstrucción tridimensional de las células de las buenas prácticas agrarias y RFP etiqueta que invaden Matrigel visto desde un lado. D) corte óptico a través de la reconstrucción en 3 dimensiones de las células de las buenas prácticas agrarias y RFP etiquetados. Reproducido de la referencia 10.

<ol>
	<li>Olson, M. 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=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</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. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>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=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

Los autores no tienen nada que revelar.

Los ensayos de invasión Matrigel tradicionalmente han sido establecidos con células colocadas sobre una capa de proteína de matriz extracelular con quimiotáctica inducida por la movilidad hacia ya través de un filtro en la parte inferior. La invasión se calificó como una función de cuántas células se podía contar en la parte inferior del filtro. Aunque en la práctica hay poca diferencia con el ensayo &quot;inversa&quot; invasión se ha descrito anteriormente, hay mucha más información sobre el proceso de i...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> DMEM (Dulbecco Modificado Medio Eagle) </td><td> GIBCO </td><td> 21969 </td></tr><tr><td> El suero fetal bovino </td><td> PAA </td><td> A15-101 </td></tr><tr><td> Penicilina-estreptomicina </td><td> GIBCO </td><td> 15140 </td></tr><tr><td> 200 mM L-glutamina (100 veces) </td><td> GIBCO </td><td> 25050-032 </td></tr><tr><td> Puromicina </td><td> Sigma-Aldrich </td><td> P8833 </td></tr><tr><td> 0,05% de tripsina EDTA </td><td> GIBCO </td><td> 25300 </td></tr><tr><td> Polybrene </td><td> Sigma-Aldrich </td><td> AL-118 </td></tr><tr><td> Lipofectamine 2000 reactivo </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> Transwells 6,5 mm, 8,0 micras de tamaño de poro </td><td> Corning </td><td> 3422 </td></tr><tr><td> Completar Matrigel </td><td> BD Biosciences </td><td> 354234 </td></tr> &lt;tr&gt; <td> Calceína AM </td><td> Invitrogen </td><td> C1430 </td></tr><tr><td> RNasa </td><td> Qiagen </td><td> 19101 </td></tr><tr><td> Yoduro de propidio </td><td> Sigma-Aldrich </td><td> P4864 </td></tr><tr><td> Confocal microcope </td><td> Leica </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

Una característica definitoria de la malignidad del cáncer es una invasión y metástasis. En algunos tipos de cáncer (e. g. Glioma 2), invasión local en el tejido sano circundante es la causa de la enfermedad y la muerte. Para otros tipos de cáncer (e. g. De mama, pulmón, etc.), Es el proceso de metástasis, en el que las células tumorales pasan de una masa tumoral primaria, colonizar los sitios distales y en última instancia, contribuir a la insuficiencia de órganos, que finalmente conduce a la morbilidad y la mortalidad a los 3. Se ha estimado que la invasión y las metástasis son responsables del 90% de las muertes por cáncer 4. Como resultado, ha habido un gran interés en la identificación de los procesos moleculares y mediadores críticos de proteínas de invasión y metástasis a los efectos de mejorar el diagnóstico y tratamiento 5. Un reto para los científicos es desarrollar cáncer de ensayos de invasión que se asemejan bastante a la situación in vivo enpara permitir el modelado preciso enfermedad 6. Dos dimensiones ensayos motilidad celular son sólo informativos acerca de un aspecto de la invasión y no tienen en cuenta la remodelación de proteínas de matriz extracelular (ECM), que es también un elemento crítico. Recientemente, la investigación ha mejorado nuestra comprensión de la invasión de células tumorales, y reveló que las células individuales pueden moverse por los modos de forma alargada o redondeada 7. Además, ha habido un mayor reconocimiento de la contribución de la invasión colectiva, en la que las células invaden en filamentos, hojas y racimos, sobre todo en tumores altamente diferenciadas que mantienen las características del epitelio, a la propagación del cáncer 8. Se presenta un método refinado 9 para examinar los aportes de proteínas candidatas a la invasión de colectivos 10. En particular, por las piscinas de ingeniería independiente de las células para expresar diferentes proteínas fluorescentes, es posible analizar molecularmente las actividades y proteinurians requerido en las células principales en comparación con los requeridos en las celdas siguientes. El uso de ARNi proporciona la herramienta molecular para desmontar experimentalmente los procesos involucrados en la invasión de células individuales, así como en diferentes posiciones de la invasión colectiva. En este procedimiento, las mezclas de las células con fluorescencia etiquetados se colocan en la parte inferior de un inserto de Transwell Matrigel previamente llena de proteínas ECM, luego se deja invadir &quot;hacia arriba&quot; a través del filtro y en el Matrigel. La reconstrucción de las pilas de la imagen de la serie Z, que se obtiene por la imagen confocal, en representaciones tridimensionales permite la visualización de forma colectiva hilos invadir y análisis de la representación de las células con fluorescencia marcado en la dirección frente a las siguientes posiciones.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

Los modelos de la invasión de células tumorales en tres dimensiones de la matriz extracelular para reflejar mejor la<em&gt; En vivo</em&gt; Situación de los ensayos de la motilidad de dos dimensiones. Utilizando los ensayos de invasión de la matriz combinada con la imagen confocal de las células con fluorescencia etiquetado, información detallada sobre los modos de invasión y las diferentes contribuciones de los líderes frente a las siguientes células se pueden obtener.

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 ...

modeling-and-imaging-3-dimensional-collective-cell-invasion

Research

JoVE Journal

Medicine

16.6K vistas.  University of Strathclyde. Los modelos de la invasión de células tumorales en tres dimensiones de la matriz extracelular para reflejar mejor la En vivo Situación de los ensayos de la motilidad de dos dimensiones. Utilizando los ensayos de invasión de la matriz combinada con la imagen confocal de las células con fluorescencia etiquetado, información detallada sobre los modos de invasión y las diferentes contribuciones de los líderes frente a las siguientes células se pueden o...

Video: Modeling and Imaging 3-Dimensional Collective Cell Invasion

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

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

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

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

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

Investigación

Medicina

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

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

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

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

Ensayo esferoide para medir TGF-β inducida por invasión

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

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

3-Dimensional resina de moldeo e Imagen de la vena portal del ratón o Sistema vías biliares intrahepáticas

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Imagen Molecular a Target trasplantó células progenitoras musculares

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Generación y cuantificación de 3 dimensiones de las lesiones arteriales en ratones mediante tomografía óptica de proyección

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Molecular Profiling del microambiente tumoral invasiva en un modelo de 3 dimensiones de las células del cáncer colorrectal y<em&gt; Ex vivo</em&gt; Los fibroblastos

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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 ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modelado espontánea metastásico Carcinoma de células renales (CCR metastásico) en ratones después de la nefrectomía

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

El aislamiento y la inmortalización de líneas celulares derivados de los pacientes a partir de una biopsia muscular para Modelado de Enfermedades

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

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 ...