Esta investigación está financiada por el programa Intramural del NIH.

I. Preparación de la célula <ol><li> Las células se crecieron hasta confluencia en DMEM con FBS al 10% sobre un 80% confluente 225 cm 3 matraz por cada 5 ratones para ser implantados. </li><li> Aspirar medios fuera de las células; lavar con 10 ml de PBS sin calcio o magnesio. Aspirar PBS fuera de las células. </li><li> Trypsinize células con 3 ml de tripsina, esperar 10-15 min, y neutralizar la tripsina con 15 ml de medio. </li><li> Pipetee todas las células y los medios en un cónico de 50 ml. Dos matraces grandes encajar en un cónico de 50 ml. </li><li> Girar las células a 1600 rpm durante 7 min a 4 ° C. Totalmente aspirar sobrenadante. Resuspender las células en 10 ml de PBS frío, y enjuague células pipeteando suavemente hacia arriba y hacia abajo. Hacer que el sedimento celular se mezcla uniformemente con el PBS. La suspensión debe ser turbia. </li><li> En este momento un recuento de células se realiza mediante un contador Coulter. Las células se centrifugan de nuevo a 1600 rpm durante 7 min a 4 ° C. </li><li> Después de la centrifugación 2 ª de las células, comopirata todo PBS y resuspender en 1 ml de PBS. </li><li> Las células se hacen girar en la 1 ml de PBS a 3000 rpm durante 10 min a 4 ° C. El PBS es aspirado, y PBS se añade para ajustar la concentración final de 1x10 6 por 5 l para U87 células, otras líneas de células pueden requerir un número diferente y la concentración de células para el crecimiento del tumor óptima. </li><li> Después de que el volumen final y la concentración se obtiene las células se mantuvieron en hielo en un tubo eppendorf. </li></ol> II. Xenoinjerto <ol class="ualpha"><li> Preparación del ratón <ol><li> Los ratones son anestesiados utilizando una mezcla de la inyección de ketamina / xilazina / solución salina. La mezcla es de 120 mg / kg de ketamina, 16 mg / kg de Rompun y solución salina. Los ratones se pesaron individualmente y dado 0,1 ml por 10 g en base a su peso. El anestésico se administra con una aguja 26G unida a una jeringuilla de IP. Tomará aproximadamente 15 minutos para los ratones para llegar a una fase de sueño sin respuesta al dolor. Usted puede comprobar la respuesta al dolor de laratones por pellizcar a su pata. </li><li> Después de que el ratón está totalmente anestesiado, pomada para los ojos se administra a los ojos, para evitar que se sequen. </li><li> El ratón se coloca sobre la cama de la estereotáxica con los dientes delanteros superiores en la muesca de la barra de la boca. El lecho se coloca entonces en el estereotáxico. </li><li> El protector de la nariz se coloca sobre la nariz del ratón para evitar el movimiento de la cabeza, y las barras del oído se coloque suavemente en los canales auditivos de los ratones. Tenga cuidado de no hacer bien la colocación de la barra de la nariz o los oídos barras demasiado apretados, ya que el cráneo se puede fracturar y / o evitar que el ratón de la respiración. </li><li> Asegúrese de que la cabeza del ratón es el nivel de uso de las marcas de graduación en el estereotáxica es de gran ayuda durante este proceso. </li><li> Para preparar el yodo ratón, el alcohol y providine se aplica abundantemente en la cabeza del ratón alterna para un total de 3 aplicación de aplicaciones de alcohol y 3 de yodo. Una aplicación final de una almohadilla de alcohol con 70% se aplica ala cabeza. </li></ol></li><li> El procedimiento quirúrgico <ol><li> Todos los instrumentos en el procedimiento debe ser esterilizado en autoclave o bien remojo instrumentos en etanol al 70% antes de comenzar el procedimiento. </li><li> Se hace una incisión en la parte superior del cráneo, detrás del ojo derecho en diagonal atrás a la parte posterior izquierda del cráneo. La incisión debe ser de aproximadamente 10 mm a 15 mm de largo. </li><li> El uso de un Q-tip, lentamente separar la incisión para exponer el cráneo y limpie la membrana que hay entre el cráneo y la piel. Use un Q-tip para borrar toda la sangre y ver a un sangrado excesivo. </li><li> Exponer la parte superior del cráneo para visualizar el bregma. Utilizando el punto de jeringa Hamilton, colóquelo sobre el bregma. La posición correcta del agujero a perforar en el cráneo es 2 a la derecha mm y 1 mm anterior del bregma (figura 2). Un cirujano rotulador, que se compra estéril, se utiliza para marcar la ubicación del orificio a taladrar. Por favor, tenga en cuenta que el agujero se encuentra cerca de la unalínea de sutura terior. </li><li> A continuación, perfore un pequeño agujero en el cráneo en la posición marcada con la punta de una aguja 22G en un florero de alfiler o alimentado a través de una aguja 18G. </li><li> Cargue el 10 l de vidrio Hamilton jeringa con 5 ml de las células. La jeringa de Hamilton es de 10 ml con una aguja 30G 33G contundente de fijo se le atribuye. Asegúrese de mezclar las células antes de cargarlos, ya sea con la pipeta o usando una jeringa de 1 ml con una aguja 25G adjunto. Después de la jeringa Hamilton se carga, volver a meterla en el brazo para alinearse con el agujero en el cráneo. </li><li> Baje el extremo de la aguja toque a la superficie del cráneo, una vez que la aguja esté a nivel con el cráneo; bajar la aguja hasta que esté en la profundidad de 3 mm por debajo de la superficie del cráneo. </li><li> Después de la aguja es bajada, deberá permanecer allí durante 2 minutos para evitar cualquier trauma importante de la aguja que afecta al cerebro. Implantación de las células es a una velocidad de 1 l por minuto para un total de 6-8 minutos, tener cuidado de cualquier backflow. Después de la última de las células se han implantado, deje la aguja en su lugar durante otros 2 minutos, para permitir que todas las células para resolver pulg </li><li> Retire la aguja en el cerebro, y limpiar el orificio con un Q-tip. Proceder a limpiar la aguja y una jeringa con acetona al 50%, 100% de EtOH, y PBS, 3x en cada solución y en ese orden. </li><li> La piel sobre el cráneo está listo para ser suturado en este punto. Uso de 5-0 PBS cerrará la incisión con cerca de 3 puntos. </li><li> Deje que el ratón para despertar en una almohadilla de calor, y volver a aplicar pomada para los ojos si es necesario. El ratón puede ser devuelto a la jaula después de que muestra el movimiento por sí mismo (es decir, de elevación hasta la cabeza o mover los hombros). </li></ol></li></ol> III. El tratamiento del implante IC por la radiación y Drogas <ol class="ualpha"><li> Tratamiento de radiación <ol><li> Dependiendo de la línea celular, el tratamiento de los ratones puede tener lugar de 5 a 7 días después del implante. Nuestro departamento utiliza una unidad de radioterapia para el tratamiento de radiación ortovoltajeambiente. </li><li> Imágenes como BLI (imágenes de bioluminiscencia), resonancia magnética o tomografía computarizada se puede utilizar para visualizar el tumor antes de la aleatorización (figura 3A). </li><li> Los ratones se anestesiaron con el cóctel de ketamina / xilazina / solución salina como antes, y ungüento para los ojos se aplica a sus ojos. </li><li> Los ratones se colocan en una plantilla de radiación por encargo que se configura como una rueda circular, en el que las cabezas de los ratones apuntan hacia el centro. Los cuerpos de los ratones están protegidos de la radiación con el plomo. </li><li> El tratamiento farmacológico se puede dar antes o radiación a través de las rutas de distribución diferentes, es decir, SQ, IP y IV. </li></ol></li></ol> IV. Resultados El ratón debe despertar del procedimiento dentro de 45-60 minutos después de la inyección de anestesia. Si el ratón ha difícilmente despertarse después del procedimiento y el tiempo ha superado una media hora después del procedimiento, el ratón no se recupere y la eutanasia puede ser una alternativa eficaz. Si ªantes que es el sangrado excesivo o se convierte en el cráneo roto en cualquier momento durante el procedimiento de la eutanasia se aconseja. Si todo va bien y en función de la línea celular, un tumor debe empezar a formar dentro de 2-4 semanas después del procedimiento tiene lugar. Cuando se utilizan modelos de xenoinjerto intracraneales, la eficacia terapéutica se mide generalmente como la supervivencia después de la implantación 5. Sin embargo, ya que este es un modelo de signos y síntomas de IC se producirá antes de la muerte. Algunos de los signos físicos del ratón se pueden presentar una postura encorvada, pérdida de peso corporal y la inactividad. Algunos de los signos neurológicos pueden ser convulsiones, inclinación de la cabeza y pérdida del equilibrio. La Figura 3B muestra una coloración de HE de un tumor en la mortalidad. Un gráfico de Kaplan-Meier se utilizó para evaluar los datos (Figura 4). El número de días de supervivencia del 50% se calculan y se compararon con el control. Si el grupo de combinación vivieron más que la adición de la radiación individual y brazos de drogas la combinación se tuvo éxito. <img alt = &quot;Figura 1&quot; src = &quot;/ files/ftp_upload/3397/3397fig1.jpg&quot; /&gt; Figura 1. El equipo Stoelting estereotáxica. <img alt="Figura 2" src="/files/ftp_upload/3397/3397fig2.jpg" /> Figura 2. Un diagrama de la ubicación del orificio en el cráneo del ratón. <img alt="Figura 3A" src="/files/ftp_upload/3397/3397fig3A.jpg" /> Figura 3. BLI imagen después de la implantación de GBM. <img alt="Figura 3B" src="/files/ftp_upload/3397/3397fig3B.jpg" /> Figura 3B. H y E de tumor GBM en la mortalidad. <img alt="Figura 4" src="/files/ftp_upload/3397/3397fig4.jpg" /> Figura 4. Resultados de los implantes U87 IC inyectados con inhibidor de la PARP 7 días después de la cirugía.

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No tenemos nada que revelar.

Los gliomas malignos, tales como el glioblastoma multiforme, representan los tumores cerebrales primarios más comunes y tienen un pronóstico sombrío 4. La supervivencia de los pacientes afectados por el GBM se ha mantenido prácticamente sin cambios durante la última década (es decir, 9-12 meses después del diagnóstico) a pesar de los avances en la cirugía, la radioterapia y la quimioterapia 5. Las características de un modelo apropiado para el estudio del tratamiento del glioma debe inclu...

<table border="1"><tbody><tr><td> Estándar de laboratorio estereotáxica Instrutment </td><td> Stoelting mouse y el adaptador de rata </td></tr><tr><td> Betadine </td><td> 100% de EtOH </td></tr><tr><td> 70% de EtOH </td><td> 50% de acetona </td></tr><tr><td> 6 &quot;Q-tips </td><td> Torundas con alcohol </td></tr><tr><td> Hamilton jeringa de 80008 </td><td> Ethicon 5-0 PD </td></tr><tr><td> 1701 SN 31-33G / 1 &quot;/ aguja PT3 </td><td> 1 ml jeringas </td></tr><tr><td> Agujas 25G </td><td> Almohadilla eléctrica </td></tr><tr><td> Las lágrimas artificiales Ungüento </td><td> Muy buen marcador permanente de punta </td></tr></tbody></table>

combination radiotherapy in an orthotopic mouse brain tumor model

El glioblastoma multiforme (GBM) son los más comunes y agresivos tumores cerebrales en adultos de primaria 1. En los últimos años ha habido un progreso sustancial en la comprensión de los mecanismos de invasión tumoral y la inoculación directa de un tumor intracerebral ofrece la oportunidad de observar el proceso invasivo en un entorno fisiológico de 2. En cuanto a los tumores del cerebro humano se refiere, los modelos disponibles en la actualidad ortotópico se establecen, ya sea por inyección estereotáxica de suspensiones celulares o el implante de una pieza sólida de tumor a través de un procedimiento de craneotomía complicada 3. En nuestra técnica se cosechan las células de cultivo de tejidos para crear una suspensión celular utilizada para implantar directamente en el cerebro. La duración de la cirugía es de aproximadamente 30 minutos, y como el ratón tiene que estar en un plano quirúrgico constante, un anestésico inyectable que se usa. El ratón se coloca en una plantilla estereotáxica hecha por Stoetling (figura 1). En popaer el área quirúrgica se limpia y se prepara, se hace una incisión; y el bregma está localizado para determinar la ubicación de la craneotomía. La localización de la craneotomía es de 2 mm a la derecha y 1 mm rostral al bregma. La profundidad es de 3 mm de la superficie del cráneo, y las células se inyectan a un ritmo de 2 l cada 2 minutos. La piel se sutura con 5-0 PDS, y el ratón se le permite despertar en una almohadilla térmica. Desde nuestra experiencia, en función de la línea celular, el tratamiento puede llevarse a cabo de 7-10 días después de la cirugía. Entrega fármaco depende de la composición del fármaco. Para el tratamiento de radiación que los ratones se anestesiaron, y poner en una plantilla de encargo. El plomo cubre el cuerpo del ratón y expone sólo el cerebro del ratón. El estudio de la tumorigénesis y la evaluación de nuevas terapias para MBG requieren exactos y reproducibles cerebrales modelos animales de tumores. Así que utilizamos este modelo ortotópico del cerebro para estudiar la interacción del microambiente del cerebro y el tumor, para comprobar la EFICACIAceso de diferentes agentes terapéuticos con o sin radiación.

Watch this Scientific Journal Video about Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model at JoVE.com

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

El propósito de este artículo es describir el uso de un modelo ortotópico de glioblastoma para los estudios de quimiorradioterapia. En este artículo se irá si el procesamiento de células, la implantación, y la radioterapia del ratón usando un modelo intracraneal.

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 ...

combination-radiotherapy-in-an-orthotopic-mouse-brain-tumor-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

16.3K vistas.  National Cancer Institute. El propósito de este artículo es describir el uso de un modelo ortotópico de glioblastoma para los estudios de quimiorradioterapia. En este artículo se irá si el procesamiento de células, la implantación, y la radioterapia del ratón usando un modelo intracraneal.

Video: Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Un modelo ortotópico de cáncer de vejiga murino

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Investigación

Medicina

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 ...

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 ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Un modelo de tumor vesical ortotópica y la Evaluación de tratamiento intravesical SARNA

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modelo de ratón ortotópico de carcinoma de tiroides anaplásico

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Un modelo ortotópico de cáncer de vejiga de Estudios de liberación de genes

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

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 ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un modelo murino de cáncer de próstata humano Metástasis ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

El mantenimiento de un modelo de ratón ortotópico Glioblastoma Restricciones cerebral parenquimatosa físicas y Conveniente para intravital microscopía de dos fotones

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

Tumor Tratamiento Terapia del Campo en combinación con bevacizumab para el tratamiento de glioblastoma recurrente

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...