Work in Dr. Ogunwobi&#8217;s laboratory is supported by a Research Centers in Minority Institutions Program grant from the National Institute on Minority Health and Health Disparities (MD007599) of the National Institutes of Health. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIMHD or the NIH.

Declaración de Ética: Todos los estudios con animales fueron aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) de Hunter College de la City University de Nueva York. 1. Procedimientos Pre-experimento <ol><li> A su llegada a las instalaciones de animales, albergará inicialmente 3 ratones Balb / c con una jaula con una ropa de cama de arriba y astillas de madera filtrada. Asegúrese de que los ratones tienen libre acceso a la alimentación, purina comida para roedores y agua. Ropa de cama y comida deben cambiarse dos veces por semana, mientras que las jaulas deben limpiarse una vez a la semana. </li><li> Inducir la anestesia general en ratones usando un vaporizador de isoflurano junto con oxígeno a una velocidad de flujo constante de 1 L por minuto. El sistema es autosuficiente e incluye un vaporizador de precisión. </li><li> Posición animal en una caja de plástico que tiene el vaporizador precisión y gas / sistema de anestésico conectado a él. Inicialmente, ajuste el vaporizador para 2,5 hasta animal es inconsciente, en cuyo punto más bajo del vaporizador a 2. </li><li> En ese momento, cambie ellínea de válvulas, sellado a la caja y abrirla al cono de la nariz. </li><li> Mueva el animal a la zona de preparación, con el cono de la nariz colocado adecuadamente y conectado (para la anestesia de mantenimiento). </li><li> Preparar 5 × 10 6 células HCC ratón BNL 1ME A.7R.1 para su implantación por ratón. BNL 1ME A.7R.1 debería estar creciendo en un medio (10% FBS inactivado por calor, 1% de penicilina / estreptomicina y 2 mM L-Glutamina) incubar en una placa de cultivo celular a 37 ° C en un incubador humidificado con aire y 5% CO 2. </li><li> Con una jeringa que tiene una aguja G 20, el implante 100 l de PBS que contenía 5 x 10 6 células de carcinoma hepatocelular ratón BNL 1ME A.7R.1 en un lóbulo de los hígados de los ratones BALB / c para producir los tumores primarios. Post-implantación, la casa de los ratones BALB / c en una jaula con los mismos requisitos de las casas como de pre-implantación. </li><li> En el punto final experimental, evidencia clínica de carcinoma hepatocelular es observable como la reducción significativa en la condición corporalanotar (usualmente detectada después de 4 semanas). En este punto, hay una probabilidad de que los tumores primarios han desarrollado en el hígado mientras que los depósitos metastásicos se han formado en los pulmones. A partir de entonces, comenzará el proceso de aislamiento y la propagación de células tumorales circulantes. </li></ol> 2. Recolección de sangre entera para hacer circular Tumor Aislamiento celular NOTA: correctamente desinfectado la zona de la cirugía y asegúrese de que está libre de desorden. Autoclave todos los instrumentos quirúrgicos o sumergirse en un desinfectante de acuerdo con las recomendaciones del fabricante <ol><li> Sacrificar los ratones por eutanasia humanitaria al experimental de punto final (evidencia clínica de desarrollo de tumores). La eutanasia de los ratones debería involucrar asfixia de CO 2. Observar los ratones adecuadamente para confirmar la anestesia se distribuye correctamente. El dióxido de carbono debe ser liberado lentamente en la cámara durante un período de 5 - 10 min, causando paro respiratorio. Siga por desangrado intracardiaca y CervicAl dislocación. 5 </li><li> Use una aguja 25 G unida a una jeringa de 1 ml para la recogida de sangre completa. Heparanize la jeringa con 0,01 ml de heparina. Punción inmediatamente la piel con la aguja inferior al xyphisternum aproximadamente a 45 ° de ángulo. Avanzar la aguja lentamente para perforar el corazón, y luego bajar el ángulo de la aguja. Constantemente mantener la aguja y una jeringa en su lugar, y extraer 500 l - 1.000 l de sangre entera desde el corazón. </li><li> Retire la jeringa y la aguja y la transferencia de la sangre en un tubo libre de pirógenos pre-marcado con 1,5 ml. Centrifugar la sangre entera recogida en 1,5 ml tubo libre de pirógenos a 1.167 xg durante 5 min. NOTA: La centrifugación separa la muestra de sangre en tres capas: la capa inferior o el hematocrito que consiste en células rojas de la sangre, una capa delgada intermedia de la capa leucocitaria, y una capa de plasma superior. </li><li> Retire el plasma con cuidado con la pipeta y recoger en un tubo de 1,5 ml pirógenos separado para furthexperimentos er como la detección de ácidos nucleicos libres de células 7. </li><li> Para el aislamiento de células tumorales circulantes (CTC), recoger la capa leucocitaria en un tubo libre de pirógenos 1,5 ml por separado en la preparación de glóbulos rojos (RBC) de lisis. Esto es necesario para eliminar los glóbulos rojos residuales en la capa de la capa leucocitaria. </li></ol> 3. Receta de glóbulos rojos (RBC) tampón de lisis <ol><li> Hacer una solución 1 L de tampón de lisis RBC. </li><li> Hacer una 500 ml de cloruro de amonio y 500 ml de Tris. A continuación, mezclar para crear una solución de 1 l de tampón de lisis RBC con una concentración final de 155 mM de cloruro de amonio y 10 mM de Tris. </li><li> Amortiguar la solución a un pH de 7,5. Almacene el tampón de lisis RBC entre +2 y +8 ° C, y llevar a RT inmediatamente antes de su uso. </li></ol> 4. RBC lisis de Buffy Coat <ol><li> Añadir 1 ml de tampón de lisis RBC al tubo de 1,5 ml estéril libre de pirógenos que contiene la capa leucocitaria recogida. </li><li> Mezclar el contenido del tubo porinvirtiendo varias veces. Incubar los contenidos mezclados del tubo durante 5 minutos a temperatura ambiente en el banquillo. Centrifugar los contenidos mezclados durante 5 min a 1167 x g. </li><li> Después de la centrifugación, se obtendrá un pellet blanquecino. Descartar el sobrenadante rojizo lentamente y con cuidado en 10% de lejía sin ninguna interrupción al sedimento. Si la pastilla no es blanquecina, volver a hacer los pasos 3.1 a 3.5. El aumento del tiempo de incubación en tampón de lisis RBC a 10 - 15 min también puede ser útil. </li><li> Deseche los residuos biológicos vez descontaminación de desechos biológicos en el 10% de lejía se completa después de 24 horas. </li></ol> 5. propagación en cultivo celular NOTA: Las células tumorales son típicamente crecen rápidamente las células de glóbulos blancos que son abundantes en la mezcla original de células sembradas en el plato. Tras el cambio repetido de medio, y los pasajes posteriores de CTC, todas las células blancas de la sangre se retiran y una población relativamente pura de CTC permanece. <ol><li> Despuésdescartando el sobrenadante en 10% de lejía, lavar el sedimento blanquecino en 1 solución salina tamponada con fosfato ml (PBS) al menos una vez, y resuspender en medio de cultivo completo para la propagación en cultivo celular. </li><li> Una vez que se ha completado lavado, añadir 1 ml de medio de cultivo A.7R.1 completa BNL 1ME (10% FBS inactivado por calor, 1% de penicilina / estreptomicina y 2 mM L-Glutamina) al sedimento y resuspender el precipitado en el medio de cultivo. </li><li> Sembrar la mezcla se volvieron a suspender las células en una placa de cultivo celular. Incubar la placa de cultivo celular a 37 ° C en un incubador humidificado con aire y 5% de CO 2. </li><li> Cambiar el medio de cultivo celular cada dos o tres días. Después de cinco a siete días, los CTC se han adherido a la placa de cultivo celular y comenzó a proliferar. Estas células se mantendrán viable más allá de 25 pasajes y después de congelación y descongelación ciclos repetidos 5. </li></ol> 6. Verificación de carcinoma hepatocelular circulantes línea de células tumorales <ol><li> Realizar apolymerase reacción en cadena (PCR) método basado, para amplificar solamente un segmento de ADN específica del gen β-globina de ratón para confirmar que la novela estableció líneas celulares de CTC son células de ratón como la línea de BNL 1ME A.7R.1 HCC original implantada. Método fue originalmente descrito y validado por Steube et al. 5,8 </li><li> Realizar la inmunotinción para el elemento de respuesta a cAMP marcador específico de hepatocitos proteína de unión 3-like 3 (CREB3L3) mediante el uso de un anticuerpo específico. Esto confirmará que la novela CTC establecidos son de hecho los hepatocitos como el implantado línea original de células BNL 1ME A.7R.1. 5 </li></ol>

<ol>
	<li>Ashworth, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+of+cancer+in+which+cells+similar+to+those+in+the+tumors+were+seen+in+the+blood+after+death.">A case of cancer in which cells similar to those in the tumors were seen in the blood after death.</a> Australian Medical Journal. 1869, 146-147 (14).</li><li>van de Stolpe, A., den Toonder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Tumor+Cells:+What+Is+in+It+for+the+Patient?+A+Vision+towards+the+Future.">Circulating Tumor Cells: What Is in It for the Patient? A Vision towards the Future.</a> Cancers. 6, 1195-1207 (2014).</li><li>Sheng, W., 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=Capture,+release+and+culture+of+circulating+tumor+cells+from+pancreatic+cancer+patients+using+an+enhanced+mixing+chip.">Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip.</a> Lab on a chip. 14, 89-98 (2014).</li><li>George, T. J., Ogunwobi, O. O., Sheng, W., Fan, Z. H., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#34;Tissue+is+the+issue&#34;:+circulating+tumor+cells+in+pancreatic+cancer.">&#34;Tissue is the issue&#34;: circulating tumor cells in pancreatic cancer.</a> Journal of gastrointestinal cancer. , (2014).</li><li>Ogunwobi, O. O., Puszyk, W., Dong, H. J., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+upregulation+of+HGF+and+c-Met+drives+metastasis+in+hepatocellular+carcinoma.">Epigenetic upregulation of HGF and c-Met drives metastasis in hepatocellular carcinoma.</a> PloS one. 8, e63765 (2013).</li><li>Cheng, B., 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=Transparent,+biocompatible+nanostructured+surfaces+for+cancer+cell+capture+and+culture.">Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture.</a> International journal of nanomedicine. 9, 2569-2580 (2014).</li><li>Yu, M., Stott, S., Toner, M., Maheswaran, S., Haber, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of cell biology. 192, 373-382 (2011).</li><li>Steube, K. G., Koelz, A. L., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+verification+of+rodent+cell+lines+by+polymerase+chain+reaction.">Identification and verification of rodent cell lines by polymerase chain reaction.</a> Cytotechnology. 56, 49-56 (2008).</li><li>Pantel, K., Alix-Panabieres, C., Riethdorf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+micrometastases.">Cancer micrometastases.</a> Nature reviews. 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The authors declare that they have no competing financial interests.

In this study, how to isolate and propagate CTCs from the whole blood of a syngeneic mouse with HCC were described. The objective of this work is to enhance the ongoing studies of the mechanisms of cancer metastasis. 
A factor that contributes to the poor prognosis of many cancers is the lack of timely detection and consequent widespread dissemination of the malignancy3. CTCs originate from primary cancers and spread via the blood stream to distant organs. As such, CTCs are importan...

The cancer research community has known of the existence of circulating tumor cells (CTCs) since first being observed by Thomas Ashworth in 18691. Since then, CTCs have been shown to be important in tumor metastasis and disease progression2-5. Today, solid tumors are a major cause of morbidity and mortality worldwide. CTCs are rare cells that originate from primary tumors and travel through the blood stream to different organs of which only a small fraction ultimately develop into metastasis2-5. Notably, there is positive correlation between tumor size and CTC number3,4. 
An understanding of CTC biology can contribute to the search for targeted therapy. Furthermore, CTCs may have diagnostic applications. To achieve these potential clinical applications, one needs to overcome some current challenges to studying CTCs. One challenge is related to the fact that CTCs may be present as single cells or as clusters and they may even be able to change their phenotype in response to the blood microenvironment2. Moreover, detection can be very challenging, in part, due to the low count of CTCs (a few to hundreds per milliliter) among one billionhematologic cells per milliliterin the blood6. Nevertheless, in recent years, research into the potential clinical applications of CTCs from solid organ cancers has intensified.
Despite these efforts, the challenges of studying and understanding the role of CTCs persist due to the rarity of CTCs and the inadequacy of the technological tools currently available. Despite these challenges, the tremendous potential for clinical applications continues to be an incentive to pursue research into the role of CTCs in cancer metastasis.
We were recently successful in isolating and propagating in cell culture CTCs from an orthotopic syngeneic mouse model of hepatocellular carcinoma (HCC) metastasis5. The purpose of the current paper is to describe in detail all aspects of the successful methodology. The significance of this methodology lies in the fact that this approach may be modified in order to successfully isolate and propagate in culture human CTCs, thus enhancing the possibility of in vitro studies of CTC biology. 
There are multiple potential clinical applications for the use of CTCs. CTCs may be useful for prognosis, response monitoring, screening, dynamic monitoring of tumor molecular alterations, and personalized therapy4. Therefore, a better understanding of the biology of CTCs has high potential for clinical impact.

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		<tr>
			<td>Heparin Sodium Salt 1G</td>
			<td>VWR</td>
			<td>89508-852</td>
			<td></td>
		</tr>
		<tr>
			<td>BTX Tube Micro 1.5mL Clear NS</td>
			<td>VWR</td>
			<td>89511-254</td>
			<td>1.5mL&#160; pyrogen-free eppendorf Tubes</td>
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		<tr>
			<td>Needle Sterile Disp BD 25GX1IN</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
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		<tr>
			<td>Slp Tip SRNG 1ml 200 each per pack</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>1mL syringe tip</td>
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		<tr>
			<td>Syringe 1ml leur lok Pk 100</td>
			<td>VWR</td>
			<td>BD309628</td>
			<td>1 mL syringe</td>
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			<td>VWR Forceps Tissue 6</td>
			<td>VWR</td>
			<td>82027-446</td>
			<td>Forceps</td>
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			<td>Cyromold intrm 15X15X5MM PK 100</td>
			<td>VWR</td>
			<td>25608-924</td>
			<td>Cyromold</td>
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			<td>Cryo-oct compund 4oz</td>
			<td>VWR</td>
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			<td>VWR</td>
			<td>48311-703</td>
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			<td>VWR</td>
			<td>48-382-128</td>
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			<td>VWR Slide Box True North Fm Pu</td>
			<td>VWR</td>
			<td>89140-278</td>
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			<td>Super HT PAP Pen</td>
			<td>VWR</td>
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			<td>Water RNASe and DNAse free 2L</td>
			<td>VWR</td>
			<td>101454-204</td>
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			<td>Buffer Tris Ultra Pure Grade 500G</td>
			<td>VWR</td>
			<td>97061-796</td>
			<td>Tris Buffer</td>
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			<td>Ammonium Chloride ACS Grade 2&#160; 5KG</td>
			<td>VWR</td>
			<td>97062-048</td>
			<td>Ammonium Chloride Buffer</td>
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			<td>Falcon Tissue Culture Dish 60 x 15mm Style polystyrene</td>
			<td>VWR</td>
			<td align="right">353002</td>
			<td>Tissue Culture Dish</td>
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			<td>Clorox&#174; Germicidal Bleach, Regular</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Clorox Bleach</td>
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			<td>PBS, 1X (Phosphate-Buffered Saline) without calcium &#38; magnesium (500mL)</td>
			<td>Thermo Fischer Scientific</td>
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			<td>DMEM, 1X&#160; with 4.5 g/L glucose &#38; L-glutamine without sodium pyruvate</td>
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			<td>Fetal Bovine Seru8m</td>
			<td>Thermo Fischer Scientific</td>
			<td>35-010-CV</td>
			<td>FBS</td>
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			<td>Penicillin Streptomycin Solution, 100X</td>
			<td>Thermo Fischer Scientific</td>
			<td>30-002-Cl</td>
			<td>Penicillin Streptomycin</td>
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		<tr>
			<td>Sorvall Biofuge pico</td>
			<td>Thermo Fischer Scientific</td>
			<td align="right">75002411</td>
			<td>13000rpm Centrifuge</td>
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isolation and propagation of circulating tumor cells from a mouse cancer model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

La metodología descrita aquí ha demostrado que las CTC se pueden aislar. Los ratones fueron sacrificados humanitariamente al experimental de punto final. El proceso implicado carbono asfixia con dióxido, desangrado intra-cardiaca, y la dislocación cervical. Un diagrama esquemático de los pasos clave del procedimiento se ilustran en la Figura 2. Muestras de sangre total extraída por desangrado intra-cardiaca se procesaron utilizando el protocolo descrito anteriormente. Después de ...

Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

El aislamiento y la propagación de células tumorales circulantes de un Cáncer de ratón

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

isolation-propagation-circulating-tumor-cells-from-mouse-cancer

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11.9K vistas.  Hunter College of The City University of New York. Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Video: Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

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

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

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

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

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

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

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

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Combinación de radioterapia en un modelo ortotópico de tumor cerebral del ratón

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

La detección de Modificadores melanoma utilizando un modelo de pez cebra Tumor autóctona

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Imágenes en directo de las respuestas de drogas en el microambiente tumoral en modelos de ratón de cáncer de mama

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

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

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

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

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

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

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptación de semiautomatizado de células tumorales circulantes (CTC) Ensayos para aplicaciones clínicas y de investigación preclínica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Un modelo de tumor de Estrés quirúrgico para explorar los mecanismos de inmunosupresión postoperatoria y Evaluar Novel perioperatorias Inmunoterapias Ratón

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

El aislamiento de las células madre del cáncer de muestras de cáncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...