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><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></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...

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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

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JoVE Journal

Medicine

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

14.7K 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

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 ...

Investigación

Medicina

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Generación de tumores pancreáticos ortotópicos y caracterización ex vivo de la citotoxicidad de células T infiltrantes en tumores

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Investigación sobre el cáncer

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

Pruebas de terapias dirigidas en cáncer mediante análisis de alteración estructural del ADN y xenoinjertos derivados del paciente

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Uso combinado de ensayos de metástasis de vena de cola e imágenes en tiempo real en vivo en vivo para cuantificar la colonización metastásica del cáncer de mama y la carga en los pulmones

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Medición de la respuesta de fármacos en tiempo real en rebanadas de tejido tumoral organotípico

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Trasplante ortotópico de tumores de mama como modelos preclínicos para el cáncer de mama

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

Explantes de tumores derivados de pacientes como una plataforma preclínica "viva" para predecir la resistencia a los medicamentos en pacientes

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Perfil de la sensibilidad a las terapias dirigidas en organoides derivados de pacientes con NSCLC con mutación de EGFR

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Detección multiparamétrica de fármacos organoides tumorales mediante imágenes de células vivas de campo amplio para análisis a granel y de un solo organoide

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Evaluación de la efectividad del medicamento para el cáncer Sensibilización<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

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

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