Name: lung tumor cell recruitment assay
Uploaded: 2019-02-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Todos los procedimientos realizados con ratones fueron aprobados por la Universidad de medicina Animal investigación Comité de Tokio de las mujeres.
1 inyección en la vena cola de pulmón de Lewis (LLC) células
<ol><li>Mantener las células de la LLC en DMEM suplementado con 10% FCS, en humidificado 5% CO2 incubadora a 37 ° C. Células deben contener un sistema de expresión de la proteína fluorescente (p. ej., GFP).</li>
	<li>Eliminar las células de la placa de cu...

<ol>
	<li>Valastyan, S., 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=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Kaplan, R. N., 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=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438 (7069), 820-827 (2005).</li><li>Hiratsuka, S., Watanabe, A., Aburatani, H., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-mediated+upregulation+of+chemoattractants+and+recruitment+of+myeloid+cells+predetermines+lung+metastasis.">Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.</a> Nature Cell Biology. 8 (12), 1369-1375 (2006).</li><li>Hiratsuka, S., 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=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nature Cell Biology. 10 (11), 1349-1355 (2008).</li><li>Tomita, T., Sakurai, Y., Ishibashi, S., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Clara+cell-mediated+homeostatic+inflammation+is+involved+in+lung+metastasis.">Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis.</a> Oncogene. 30 (31), 3429-3439 (2011).</li><li>Deguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+amyloid+A3+binds+MD-2+to+activate+p38+and+NF-&#954;B+pathways+in+a+MyD88-dependent+manner.">Serum amyloid A3 binds MD-2 to activate p38 and NF-&#954;B pathways in a MyD88-dependent manner.</a> Journal of Immunology. 191 (4), 1856-1864 (2013).</li><li>Erler, J. T., 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=Hypoxia-induced+lysyl+oxidase+is+a+critical+mediator+of+bone+marrow+cell+recruitment+to+form+the+premetastatic+niche.">Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.</a> Cancer Cell. 15, 35-44 (2009).</li><li>Huang, Y., 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=Pulmonary+vascular+destabilization+in+the+premetastatic+phase+facilitates+lung+metastasis.">Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.</a> Cancer Research. 69 (19), 7292-7237 (2009).</li><li>Sceneay, J., Smyth, M. J., M&#246;ller, 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+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Reviews. 32 (3-4), 449-464 (2013).</li><li>Castle, J., Shaker, H., Morris, K., Tugwood, J. D., Kirwan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+circulating+tumor+cells+in+breast+cancer:+A+review.">The significance of circulating tumor cells in breast cancer: A review.</a> The Breast. 23, 552-560 (2014).</li><li>Wolf, M. J., 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=Endothelial+CCR2+signaling+induced+by+colon+carcinoma+cells+enables+extravasation+via+the+JAK2-Stat5+and+p38MAPK+pathway.">Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway.</a> Cancer Cell. 22 (1), 91-105 (2012).</li><li>Hiratsuka, S., 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=Primary+tumours+modulate+innate+immune+signalling+to+create+pre-metastatic+vascular+hyperpermeability+foci.">Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci.</a> Nature Communications. 4, 1853 (2013).</li><li>Deguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eritoran+inhibits+S100A8-mediated+TLR4/MD-2+activation+and+tumor+growth+by+changing+the+immune+microenvironment.">Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.</a> Oncogene. 35 (19), 1445-1456 (2016).</li><li>Ieguchi, 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=ADAM12-cleaved+ephrin-A1+contributes+to+lung+metastasis.">ADAM12-cleaved ephrin-A1 contributes to lung metastasis.</a> Oncogene. 33 (17), 2179-2190 (2014).</li></ol>

Pretratamiento de ratones con anticuerpos, medicamentos, una dieta alta en grasas o medio condicionado por el tumor antes de la inyección de células de tumor es posible. El paso más difícil en este análisis es la inyección de la vena de la cola. Inyección incompleta da lugar a datos no concluyentes. La vena de la cola en C57BL/6 es particularmente difícil de identificar, dando por resultado las inyecciones fallidas. Colocando el ratón sobre una almohadilla de calefacción (37 ° C) ayuda a dilatar la vena de la ...

Metástasis del tumor responsable de alta mortalidad en enfermedades relacionadas con el cáncer. Desde el punto de vista de las investigaciones a nivel molecular, la metástasis se puede dividir en varios pasos: iniciación del tumor en el sitio del tumor primario, el crecimiento del tumor primario e invasión en alrededor de los tejidos, intravasación, circulación a través de los vasos sanguíneos , extravasación en órganos distantes y re-crecimiento del tumor. Cada paso implica diferentes conjuntos de moléculas/señales1.
Estudios hasta la fecha han estado preocupados con los eventos que ocurren en órganos distantes antes las células tumorales circulantes en la sangre, que también es conocida como la fase pre-metastásica2,3,4,5, 6. Nuestros estudios con ratones modelo revelaron que la fase pre-metastásica facilita la metástasis del pulmón mediante la creación de larga duración y bajo nivel de las respuestas inflamatorias en los pulmones. La fase pre-metastásica se caracteriza por hiperpermeabilidad de themicrovasculature y reclutamiento de células de la médula ósea derivada (BMDC), upregulation de citoquinas/quimioquinas-como moléculas incluyendo S100A8 y SAA33,4 . Se ha divulgado que esa lisil oxidasa modifica la matriz extracelular en los pulmones para reclutar BMDCs7. Otro informe ha revelado la importancia de Angpt2, MMP3 y MMP10 en la fase pre-metastásica8. En otras palabras, las intrincadas interacciones entre tumores primarios, médula ósea y órganos remotos necesitan ser comprendidos y modulada correctamente (por ejemplo, mediante el uso de fármacos que atacan las proteínas implicadas en estas interacciones) para evitar futuras metástasis9 . Para regular la fase pre-metastásica, debe primero visualizar y evaluar las intervenciones diagnósticas o terapéuticas. Aquí, Divulgamos un ensayo de reclutamiento de células de tumor de pulmón que permite el estudio de la fase pre-metastásico en los pulmones.
Las células del tumor inyectadas directamente en el vaso sanguíneo del ratón son similares a circulación las células del tumor en pacientes con cáncer, aunque puede haber diferencias entre las células cultivadas y células tumorales circulantes. Células tumorales circulantes se experimentan algunos cambios característicos cuando entran en circulación desde el tumor primario. Sin embargo, las células cultivadas pueden formar tumores en ratones inmunodeficientes cuando se inyectan en la vena de la cola. Además, en el sistema de modelo de ratón, fluorescencia marcada de las células del tumor puede utilizarse que permiten el seguimiento de estas células en el cuerpo. El comportamiento de las células tumorales en circulación es fundamental ya que determinan las consecuencias finales en cáncer pacientes10. Sangre no es un buen lugar para la supervivencia de las células de tumor2. Que necesitan para escapar del ataque por el sistema de inmune del huésped y necesita señales anti-apoptosis para evitar la apoptosis debido a la falta de soporte físico. Las células del tumor con mayores habilidades de iniciación de tumor en sitios metastáticos generan más nódulos del tumor. Si las células tumorales muestran tropismo de órgano específico, se deben observar metástasis en los órganos particulares. En este ensayo, los pasos iniciales de la cascada metastásica (crecimiento del tumor primario y la invasión) no están incluidos, y por lo tanto el foco está en la interacción entre la circulación de las células tumorales y las células del estroma (células endoteliales, células epiteliales y células de la sangre). En estudios de inyección de células de tumor convencional, los investigadores tienen que esperar hasta que los nódulos del tumor crecen hasta un tamaño tangible para detectar la metástasis. En este caso, no se puede cuantificar el número exacto de las células tumorales en los vasos sanguíneos. Además, este proceso incluye una fase de rebrote del tumor, indicando que otros factores en las células del tumor pueden afectar el resultado.
Conteo de las células del tumor bajo un estereomicroscopio de fluorescencia de etiquetado es un proceso simple. El estereomicroscopio proporciona un amplio campo de visión que puede analizarse el pulmón entero. Citometría de flujo es también una herramienta útil que al instante le da un número exacto de las células tumorales en el tejido. Conteo de células fluorescentes en el tejido bajo un microscopio confocal, es posible y muestra las imágenes más precisas de las células del tumor metastásico, pero requiere tiempo y puede dar resultados sesgados si sólo se cuenta un área seleccionada. El objetivo final de este ensayo es observar cómo fácilmente se acumulan las células del tumor en los pulmones. Como se informó anteriormente de3,4, si los pulmones están en fase de pre-metastásico debido a la señalización inflamatoria del tumor primario, las células tumorales inyectadas son propensas a acumular en los pulmones, por lo tanto, lo que implica mayor probabilidad de metástasis del pulmón. Otras condiciones (artritis reumatoide, asma, etc.) y su tratamiento con agentes antiinflamatorios (como anti-TNFα) pueden atenuar la metástasis del pulmón. Por lo tanto, concluimos que este análisis es una poderosa herramienta para evaluar la posibilidad de metástasis del pulmón.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>DN-25</td>
			<td>trace amount</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td>40 micrometer mesh</td>
		</tr>
		<tr>
			<td>Fluorescence labeling kit</td>
			<td>Sigma</td>
			<td>MIN26-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>17597K</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
	</tbody>
</table>

lung tumor cell recruitment assay

Para investigar los mecanismos moleculares que rigen la metástasis del tumor, se han propuesto varias pruebas utilizando el ratón como un animal modelo. Aquí, demostramos un simple ensayo para evaluar extravasación de células tumorales o micrometástasis. En este ensayo, las células del tumor fueron inyectadas en la vena de la cola, y después de un corto período, los pulmones fueron disecados y digeridos para contar las células tumorales marcadas acumulada. Este análisis omite el paso inicial de la invasión del tumor primario en el vaso sanguíneo y facilita el estudio de los acontecimientos en el lejano órgano donde se produce la metástasis del tumor. El número de células que inyecta en los vasos sanguíneos puede ser optimizado para observar un número limitado de metástasis. Se ha reportado que las células estromales en el órgano distante contribuyen a la metástasis. Así, este análisis podrían ser una herramienta útil para explorar potenciales medicamentos o dispositivos para la prevención de metástasis del tumor.

Los pulmones presentan muchas células GFP-LLC 2 h después de la inyección (figura 1A). Cabe señalar que los puntos fluorescentes también detectados en el filtro rojo deben ser excluidos de la cuenta del número de celular. La gran mayoría de las células LLC desaparece de los pulmones 24 h después de la inyección (figura 1).
Para confirmar el número de la GFP-L...

Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Lung Tumor Cell Recruitment Assay

Ensayo de reclutamiento de células de pulmón Tumor

Este manuscrito describe un método para cuantificar la acumulación de células de tumor en los pulmones en un modelo animal de metástasis del tumor.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Este método puede ayudar a responder preguntas clave en el campo de la biología tumoral sobre los mecanismos moleculares de la metástasis. La principal ventaja de esta técnica es que el reclutamiento de células tumorales dentro de un órgano remoto puede ser la cantidad. Comience por usar un reactivo de disociación celular no enzimática para eliminar las células de carcinoma pulmonar Lewis que expresan proteínas fluorescentes, o LLC, de su recipiente de cultivo de acuerdo con los protocolos estándar.Transfiera la solución celular resultante a un tubo cónico de 15 mililitros antes de la centrifugación, y centrifugar los pellets dos veces en cinco mililitros de PBS por lavado. Después del segundo lavado, filtre las células a través de un colador de células de poro de 40 micrómetros y diluya la suspensión a una de cada 10 a las seis células por concentración de mililitro. Luego cargue las células en una jeringa de un mililitro equipada con una aguja del calibre 29 e inyecte 100 microlitros de células en la vena de la cola de cada ratón macho de ocho a 10 semanas de edad, C57-Negro-6.En el punto final experimental adecuado, utilice tijeras para extraer la piel de la superficie ventral de cada ratón, y corte las costillas y el diafragma para exponer la cavidad torácica. Utilice una aguja de infusión alada de 25 calibres y un tubo para lavar 15 mililitros de PBS a través del ventrículo derecho, y retire el corazón y el timo. Agarrar la tráquea con fórceps, tirar hacia arriba, diseccionar los tejidos conectivos alrededor de la tráquea, para cosechar los pulmones.Luego lave los pulmones en PBS y desprenda un lóbulo para la visualización de las células fluorescentes in situ bajo un microscopio de fluorescencia. Para digerir el tejido pulmonar, primero corta el lóbulo caudal con tijeras y coloca las piezas pulmonares en una jeringa de 10 mililitros. Cierre la jeringa con el émbolo y aspire cinco mililitros de solución de digestión en la jeringa, moviendo el émbolo dentro y fuera del eje de la jeringa hasta que el tejido pulmonar se disefunda a lo largo de la solución.Dispensar la suspensión pulmonar en un tubo de 50 mililitros, y digerir el tejido en una coctelera recíproca durante 15 minutos a 37 grados Celsius y 150 rpm. Al final de la incubación, triturar el tejido restante hasta que las células pulmonares estén completamente dispersas, y devolver el tubo al agitador durante 30 minutos adicionales. Luego filtra las células a través de un colador de poro de 40 micrómetros, y recoge las células por centrifugación.Para analizar las células pulmonares aisladas por citometría de flujo, resuspendir el pellet en dos mililitros de tólus de células sanguíneas rojas tampón durante un minuto a temperatura ambiente, y recoger las células por centrifugación. A continuación, centrifugar el pellet dos veces en cinco mililitros de PBS fresco complementado con 1%albúmina sérica bovina por lavado. Después de la segunda centrifugación, resuspender el pellet en un mililitro de PBS fresco más BSA, y filtrar las células a través de un nuevo colador de poro de 40 micrómetros para la cuantificación de las células tumorales por citometría de flujo.Los pulmones presentan muchas células GFP-LLC dos horas después de la inyección, con la gran mayoría de las células desapareciendo de los pulmones dentro de las 24 horas. Tenga en cuenta que los puntos fluorescentes que se detectan dentro del filtro rojo deben excluirse del recuento de celdas. Para confirmar el número de GFP-LLC en los pulmones, uno de los lóbulos se puede utilizar para el análisis citométrico de flujo como se demuestra.Al intentar este procedimiento, es importante recordar que un procedimiento de sección congelada es un método más preciso para observar la ubicación exacta de las células tumorales.

Research

JoVE Journal

Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

Condicional Knockdown de genes en líneas celulares de cáncer para estudiar el reclutamiento de monocitos/macrófagos que el microambiente del Tumor

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Investigación

Investigación sobre el cáncer

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

El establecimiento de un ensayo de colonización pulmonar para la visualización de células tumorales en tejidos pulmonares de la circulación

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Modelo de pulmón acelular y celular para el estudio de metástasis de Tumor

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Análisis de imágenes de células vivas para estudio Glioblastoma cerebral Tumor células madre migración y la invasión

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Digital de la reacción en cadena de polimerasa para la variación genética en un paciente de la poliposis adenomatosa familiar esporádico con el formato de Chip en tubo

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Aislamiento de células madre mesenquimales del tejido de la pulpa y cocultivo con células de cáncer para estudiar sus interacciones

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Tiempo real detección de Vitro Tumor de la célula apoptosis inducida por CD8+ T las células para el estudio de las funciones inmunológicas de supresores de Tumor infiltrante de células mieloides

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Caracterización semiautomática PD-L1 y enumeración de células tumorales circulantes de pacientes con cáncer de pulmón celular no pequeño por inmunofluorescencia

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Ensayo In Vitro para estudiar la interacción tumor-macrofago

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Detección de la progresión del tumor pulmonar en ratones por imágenes por ultrasonido

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...