The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

NOTA: la obtención de tejidos del paciente fue autorizada mediante junta de revisión institucional (IRB): aprobado muestras biológicas y Protocolos Clínicos (números 09-121 y 11-041 de Protocolo, respectivamente) en el Centro de Cáncer Memorial Sloan Kettering. Adquisiciones 1. Tissue <ol><li> Adquisición de paciente del tumor primario / metástasis NOTA: Hasta la fecha, este protocolo se ha realizado sobre tipos pancreáticos, gástricos y de tumor de mama extirpado quirúrgicamente, así como, las metástasis de linfoma. <ol><li> Dirigir el equipo quirúrgico para entregar la muestra por el mensajero o el sistema de tubo neumático al departamento de Patología en bien cerrado y estéril bolsa espécimen de plástico a prueba de fugas en contenedores a prueba de derrames estéril. Dirigir el equipo quirúrgico para el transporte de la muestra en estado fresco sin formalina o fijador. </li><li> Dirigir el patólogo o patólogo asistente para cosechar las muestras frescas utilizando una técnica estéril, que incuso ludes de guantes y los instrumentos estériles en una campana de flujo laminar. </li><li> Registre el tiempo de recolección de la muestra adquiridos, que se mantiene rigurosamente en 30 minutos de la finalización de la intervención quirúrgica. Teniendo en cuenta los efectos de la isquemia fría, no tome muestras de las piezas resecadas con un tiempo de isquemia fría&gt; 30 min 18. </li><li> Dirigir el personal del Departamento de Patología de quitar una muestra de tumor primario de aproximadamente 0,5 cm 3 a 1,0 cm 3 en una campana de flujo laminar para mantener un ambiente estéril. Cuando sea posible, elija el tejido tumoral de la periferia de la lesión índice para evitar posibles franca necrosis central (muerte celular). NOTA: El tejido necrótico puede ser groseramente reconocible por cualquiera de los siguientes criterios: pérdida de color o palidez del tejido; pérdida de fuerza en la que el tejido necrótico es blando y friable; una demarcación clara entre el tejido necrótico y viable. </li><li> Dirigir el pathologist o asistente patólogo para proporcionar el tejido periférico que está en exceso (descarte quirúrgico) después del muestreo inmediata del tumor para la evaluación de diagnóstico. Colocar la muestra en un tubo de centrífuga cónico de 15 ml estéril que contiene aproximadamente 5 ml de medio esencial mínimo (MEM) que contenía 1% de antibióticos (penicilina y estreptomicina). </li><li> Si está disponible (por ejemplo, los ganglios linfáticos de la cola axilar de la muestra de mastectomía), procurar un espécimen extremadamente positivos los ganglios linfáticos asociados del mismo tamaño (0,5 cm 3-1,0 cm 3) y se compara con la respuesta al fármaco de tumor primario. Al igual que la muestra de tumor primario, colocar la muestra de ganglios linfáticos asociados en un tubo de centrífuga cónico de 15 ml estéril que contiene aproximadamente 5 ml de medio esencial mínimo (MEM) que contenía 1% de antibióticos (penicilina y estreptomicina). </li><li> Si el tamaño de los permisos de la pieza quirúrgica (por ejemplo, muestras de mastectomía), eliminar (distante del tumor primario) una muestra de tejido normal (es decir,/ parénquima normal denso fibroso de mama), y meterlo en un tubo de centrífuga cónico de 15 ml estéril que contiene aproximadamente 5 ml de medio esencial mínimo (MEM) que contenía 1% de antibióticos sólo con fines de transferencia. Después de la transferencia de las instalaciones de laboratorio, transferir la muestra &quot;normal&quot; para una congelación criovial &#39;snap&#39; y guárdelo en un -80 ° C congelador para futuros análisis moleculares. </li><li> Coloque todas las muestras en hielo húmedo y transportar inmediatamente al espacio de laboratorio para ex vivo seccionar los tejidos frescos. No la sección de la muestra normal, sino más bien almacenar en un congelador -80 ° C para futuros análisis moleculares. Transferir una porción del tumor primario y la metástasis asociada a una congelación criovial &#39;snap&#39; y guárdelo en un -80 ° C congelador para futuros análisis moleculares. </li></ol></li><li> Adquisición de tejido a partir de estudios clínicos (por ejemplo, las biopsias con aguja gruesa opcional (CNB)) NOTA: Nosotros obtenemos los tejidos tumorales de pacientesregistrados en protocolos clínicos que dan su consentimiento para someterse a un CNB de una metástasis de tumores accesibles antes del inicio de la terapia. CNB, hasta la fecha, se han realizado en pacientes con cáncer de mama, linfoma de la zona marginal, linfoma de células del manto, paraganglioma metastásico, el cuello uterino (células escamosas) y cáncer de ovario. <ol><li> Haga que el personal clínico de radiología intervencionista o departamento pertinente realizar una biopsia con aguja gruesa de pre-tratamiento y colocar la muestra en un 15 ml tubo de centrífuga cónico estéril que contiene aproximadamente 5 ml de medio esencial mínimo (MEM) que contenía 1% de antibióticos (penicilina y estreptomicina). </li><li> Coloque la muestra de biopsia en el hielo húmedo y transportar inmediatamente al espacio de laboratorio para ex vivo seccionar los tejidos frescos. </li></ol></li></ol> 2 Preparación de Materiales para Ajustes de sección y Vibratome <ol><li> Preparación de los materiales para seccionar <ol><li> Hacer una solución de agarosa al 4% mediante la adición de 4 g de AGAse elevó a 100 ml de PBS. Autoclave (ajustes de ciclo: líquidos, 121 ° C, 30 min, 15 libras por pulgada cuadrada) la solución antes del primer uso. Para usos posteriores, coloque la agarosa al 4% en el microondas para licuar (fuego alto durante intervalos de 30 segundos) antes de cada uso y enfriar adecuadamente a ~ 25 ° C (es decir, caliente al tacto) consciente de que agarosa solidifica a temperatura ambiente. </li><li> Utilice la agarosa al 4% como media del tejido incrustar no permanente. Cuando la agarosa al 4% se ha enfriado lo suficiente y todavía está en un estado líquido, con una pinza estéril colocar la muestra de tejido en un molde de inclusión y se vierte en la agarosa al 4%. </li><li> Coloque la muestra de tejido estrechos (~ 2 mm de distancia), pero no directamente en la pared de la superficie de corte del molde de inclusión. La agarosa 4% empezará a solidificar rápidamente, por lo tanto, posicionar apropiadamente la muestra de tejido rápidamente. Durante el corte, mantenga la agarosa al 4% en un 55 ° C baño de agua para mantener en solución. </li><li> Para una placa de 24 pocillos para el análisis de la sección, añadir 1 ml of medio completo (es decir, MEM + 10% de suero fetal bovino + 1% de antibióticos) a cada pocillo. Mantenga la placa de 24 pocillos a temperatura ambiente (~ 25 ° C) durante toda la duración de la sección. </li></ol></li><li> Preparación de Configuración Vibratome <ol><li> Usando el microtomo vibrante, ajustar la velocidad a la que la cuchilla se acerca la muestra [0 (0,00 mm / s) a 10 (2,5 mm / seg)] y la frecuencia de la cuchilla de [0 (0 Hz) a 10 (100 Hz) ]. Ajuste la configuración de acuerdo a la textura del tejido (es decir, la densidad / firmeza). </li><li> Para un tumor primario de mama de carcinoma ductal invasivo, ajustar la configuración a 6 y 3, la velocidad y frecuencia, respectivamente. Para el tejido tumoral menos denso / firme, reducir la velocidad y aumentar la frecuencia en consecuencia. </li><li> Ajuste el espesor de la sección dentro de los límites del instrumento (1-999 micras). Para estos análisis, establecer el espesor de corte a 200 micras. </li><li> Montar el espécimen de agarosa embebido usando una capa delgada de adhesivo líquido sobre la platina del that se sumerge en un depósito de papel que está encerrado en hielo húmedo. </li></ol></li></ol> 3. Tissue seccionamiento, Tratamiento y Análisis <ol><li> Retire las secciones de precisión en rodajas inmediatamente después de seccionar utilizando una pinza estéril y colocar en una placa de múltiples pocillos con 1 ml de medio completo. Cuando se adquieren secciones suficientes para la respuesta y análisis correlativos, colocar la placa de múltiples pocillos para la respuesta al tratamiento en un incubador de cultivo de tejido fijado en condiciones de cultivo de tejidos estándar. </li><li> Tome varias secciones de control que SUJETALIBROS las secciones tratadas en un esfuerzo para confirmar la respuesta dependiente de la dosis en comparación con los artefactos cultivadas. Realizar tejido seccionado rápidamente (~ 30 - 40 min), bien dentro de los efectos perjudiciales del tiempo de isquemia fría (~ 2 - 4 horas) 18. Realizar el tratamiento de las secciones sólo después de un período de aclimatación de 1 hora. </li><li> Tras el periodo de aclimatación de 1 hora, añadir dosis crecientes (terapéuticamente relevante; <em.&gt; Por ejemplo, 0 - 10 mM) concentraciones de drogas a las secciones y la casa en condiciones estándar de cultivo de tejidos para el período de tratamiento. Para este estudio, utilizar 24 y / o 48 períodos de evaluación hr. Sin embargo, el control (vehículo solo) secciones de tejido seguirán siendo viables, según las pruebas realizadas en este estudio, por períodos de hasta e incluyendo los períodos de evaluación de 96 hr. </li><li> Después del tratamiento, retirar todas las secciones en condiciones estériles y formalina-fix mediante la colocación de la sección de tejido en un tubo que contenía 10% de formalina tamponada y lugar a TA O / N o en un tubo que contiene 4% de paraformaldehído (16% de paraformaldehído diluir de un vial con PBS para una concentración final de 4%) y el lugar a TA durante 2 h. <ol><li> Fijar secciones de tejido más grandes (por ejemplo., ~ 6 mm x 4 mm) 200 m de espesor en 10% de formalina tamponada O / N a TA. Fijar secciones más pequeñas (por ejemplo., 1 mm x 1 mm) 200 m de espesor en paraformaldehído al 4% durante 2 horas a RT. NOTA: Para la penetración eficiente de fijador el volumen de fixative debe 20x el volumen de tejido. Dado que este protocolo resulta en tamaños muy pequeños de tejido (por ejemplo, 6 mm 3 tejido = 6 l de volumen), para la garantía de uso adecuado de fijación 1 ml de fijador para todos los tamaños de tejido. </li><li> Tras la fijación tienen las muestras de tejido en parafina embebidos por el personal del Departamento de Patología. Sección espécimen bloques de parafina sobre portaobjetos cargados a ser hematoxilina y eosina tiñen y evaluada por inmunohistoquímica (por ejemplo, la apoptosis). </li></ol></li></ol>

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No conflicts of interest disclosed.

Biólogos del cáncer se enfrentan a retos significativos al intentar desarrollar estrategias terapéuticas eficaces. Prueba de fármacos en desarrollo en las líneas celulares de cáncer establecidas no puede reflejar con precisión en la respuesta in vivo e in vivo en modelos PDX son mano de obra intensiva y muy caro. Teniendo en cuenta lo anterior, la aplicación de técnicas ex vivo de los tumores primarios del paciente 14, 15 se posiciona al lado del análisis molecular de lín...

El desarrollo de la terapéutica del cáncer eficaces ha demostrado ser extremadamente difícil. Líneas celulares de cáncer y explantes tumorales -, así como xenoinjertos se han utilizado en la investigación del cáncer durante más de medio siglo 1,2,3. Hasta la fecha, el análisis molecular de sensibilidad a los fármacos y la resistencia en las dos líneas celulares de cáncer establecidos y xenoinjertos derivados del paciente (PDX) es indispensable. Sin embargo, las pruebas de los compuestos en líneas celulares de cáncer establecidas a menudo no es predictivo de la eficacia in vivo, y los correspondientes estudios in vivo en animales, especialmente en los modelos de PDX, es muy caro y consume mucho tiempo. Las limitaciones de estos sistemas de modelo, a saber, la incapacidad para informar sobre la influencia del microambiente nativo en la progresión tumoral y la respuesta a estrategias terapéuticas, ha llevado al campo de la investigación para desarrollar métodos adicionales para complementar estos análisis. De reciente, se está prestando mayor atención a ex vivo análisis de tum pacienteo explantes 4, 5, debido a la mayor comprensión de que la respuesta terapéutica del cáncer no es exclusivo de la composición molecular inherente de las células cancerosas, sino que está fuertemente influenciado por el microambiente de las células tumorales 6, 7 una función que no puede ser recapitulado por métodos de cultivo tradicionales y / o PDX. análisis ex vivo en el contexto anterior (es decir., influencia de la célula tumoral adyacente microambiente circundante) implica la evaluación de secciones de tumor / metástasis primarias viables, en lugar de análisis ex vivo de los aislamientos celulares 8, 9. Se presenta aquí en una técnica ex vivo (es decir., Secciones de precisión en rodajas frescas de tejido) de los tumores primarios de pacientes y metástasis asociados (es decir, los ganglios linfáticos) que informa fielmente de la respuesta (IC50), fuera de objetivo efectos y permite molecular análisis de los mecanismos de resistencia y de retroalimentación. Además, un análisis correlativo de therapeUTIC sensibilidad / resistencia en función de biomarcadores y genes perfil de expresión se puede realizar en un esfuerzo por identificar a los pacientes más propensos a responder al fármaco experimental de interés (es decir., respuesta de alta drogas coincide paciente con especial perfil biológico). Aplicación de la técnica ex vivo y la evaluación de una manera multi-parámetro es movimiento hacia la selección de los pacientes y la mejora general de los resultados clínicos. Ex vivo análisis de la respuesta al tratamiento podría convertirse en una herramienta estándar en el desarrollo preclínico y clínico de la terapéutica del cáncer y está concebido como un paso hacia un enfoque de medicina personalizada en las estrategias de desarrollo terapéutico.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:96px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome Leica VT1000s</td>
			<td style="width:96px;">Leica</td>
			<td style="width:119px;">14047235613</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraPure agarose</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">16500500</td>
			<td style="width:167px;">Prepare 4% and 6% before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injector blade</td>
			<td style="width:96px;">Ted Pella</td>
			<td style="width:119px;">121-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MEM with Penicillin&#160; + Streptomycin</td>
			<td style="width:96px;">Media Core Facilities (MSKCC)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The media is prepared at Memorial Sloan Kettering Cancer Center&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scalpel no. 10</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">31-200-32</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable forceps</td>
			<td style="width:96px;">Cole-Parmer</td>
			<td style="width:119px;">84011182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Embedding mold</td>
			<td style="width:96px;">Electron Microscopy Science</td>
			<td style="width:119px;">70181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS (heat inactivated)</td>
			<td style="width:96px;">Gemini</td>
			<td style="width:119px;">100106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well plates</td>
			<td style="width:96px;">Corning</td>
			<td style="width:119px;">3524</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formalin (10%)</td>
			<td style="width:96px;">Sigma Diagnostics</td>
			<td style="width:119px;">SDHT501128&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">16% Formaldehyde solution</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Embedding microsettes</td>
			<td style="width:96px;">Simport</td>
			<td style="width:119px;">M503-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol (70%)</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">A405P-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Waterbath</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">15-462-2SQ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microwave</td>
			<td style="width:96px;">General Electric</td>
			<td style="width:119px;">ModelJES2051DNBB</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adhesive (Ethyl Cyanoacrylate)</td>
			<td style="width:96px;">Sigma-Aldrich</td>
			<td style="width:119px;">E1505-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mm dishes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">353003</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15ml tubes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">352096</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Para este estudio, se utilizó la técnica ex vivo en un análisis correlativo de terapéutica de sensibilidad / resistencia de un inhibidor de la proteína de choque térmico 90 (Hsp90i). En una evaluación preclínica de este Hsp90i, el tumor primario del cáncer de mama, una ER + carcinoma ductal invasivo (IDC), y asociado metástasis en los ganglios linfáticos se analizaron ex vivo para la respuesta al tratamiento &#160; (Figura 1). Múltiples 200 m de serie de se...

Watch this Scientific Journal Video about Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics at JoVE.com

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Establecido líneas celulares de cáncer y xenoinjertos han sido el pilar de la investigación del cáncer durante las últimas décadas. Sin embargo, la evidencia reciente sugiere que la respuesta terapéutica está fuertemente influenciado por el microambiente de las células tumorales. Por lo tanto, hemos desarrollado un análisis ex vivo de las muestras de tumor primario con fines de desarrollo de fármacos.

Ex Vivo respuesta al tratamiento de los tumores primarios y / o Asociados Metástasis de Preclínica y Desarrollo Clínico de Terapéutica

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

ex-vivo-treatment-response-primary-tumors-andor-associated-metastases

Research

JoVE Journal

Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.6K vistas.  Memorial Sloan Kettering Cancer Center.  Establecido líneas celulares de cáncer y xenoinjertos han sido el pilar de la investigación del cáncer durante las últimas décadas. Sin embargo, la evidencia reciente sugiere que la respuesta terapéutica está fuertemente influenciado por el microambiente de las células tumorales. Por lo tanto, hemos desarrollado un análisis ex vivo de las muestras de tumor primario con fines de desarrollo de fármacos. 

Video: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

La ablación térmica para el tratamiento de los tumores abdominales

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

Investigación

Medicina

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Monitoreo metástasis tumorales y lesiones osteolíticas con bioluminiscencia y Micro Imágenes de TC

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Una novela de alta resolución<em&gt; En vivo</em&gt; Técnica de escaneo para el Estudio de la respuesta dinámica de estructuras intracraneales al crecimiento del tumor y Terapéutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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 Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

A preclínicos modelo murino de metástasis hepática

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Aislado hepática de perfusión como un tratamiento para las metástasis hepáticas de melanoma uveal

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Generación de microtumores Uso Humano 3D Biogel sistema de cultivo y las células de glioblastoma derivados de los pacientes para Kinomic de perfiles y pruebas de respuesta a los fármacos

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Evaluación de la Alternancia Espontánea, el Reconocimiento de Objetos Novelos y la Clasificación de Miembros en Modelos de Ratón Transgénico de la Neuropatología Amiloide-β y Tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Treatment of Liver Metastases Using an Internal Target Volume Method for Stereotactic Body Radiotherapy

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For inoperable patients, local ablation therapies, such as stereotactic body radiotherapy (SBRT), can provide effective local tumor control with minimal toxicity. Because of its high precision and accuracy, SBRT delivers a higher radiation dose per fraction, is more effective, and targets smaller irradiation volumes than does conventional radiotherapy. In addition, steep dose gradients from target lesions to surrounding normal tissues are achieved using SBRT; thus, SBRT provides more effective tumor control and exhibits fewer side effects than conventional radiotherapy. The use of SBRT is prevalent for treating intracranial lesions (known as stereotactic radiosurgery); however, it is now also used for treating spinal and adrenal metastases. Because of advancements in image-guided assistance and respiratory motion management, several studies have investigated the use of SBRT for treating lung or liver tumors, which move as a patient breathes. The results of these studies have suggested that SBRT favorably controls tumors in the case of moving lesions.
Four-dimensional computed tomography (4D-CT) with an abdominal compressor (AC) is clinically convenient for effective respiratory motion management. Because this method is noninvasive and allows free breathing, its use reduces complications. Furthermore, patients consider this method convenient. Moreover, it is considered more efficient than other methods of respiratory motion management by physicians and therapists. The use of 4D-CT with an AC for treating pulmonary lesions has also been widely investigated, and the technique is gaining acceptance for treating hepatic lesions. However, the protocols for using 4D-CT with an AC for treating hepatic lesions are different from those used for treating pulmonary lesions. In this article, we describe a new protocol for SBRT with 4D-CT and an AC for treating liver metastases.

Stereotactic body radiotherapy (SBRT) requires rigorous accuracy and precision for delivering high radiation doses per fraction to small treatment volumes to improve tumor control and simultaneously reduce toxicity. Herein, we present a noninvasive and clinically convenient respiratory motion management protocol for SBRT for liver metastases.

Tratamiento de las metástasis hepáticas mediante un método de volumen objetivo interno para radioterapia estereotáctica del cuerpo

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Contracciones de cardiomiocitos humanos derivados de iPSC sincicios medición con un colorante fluorescente sensible al Ca en placas de 384 pocillos temperatura controlada

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...