Esta investigación está financiada por la AACR Henry Shepard cáncer de vejiga beca de investigación (09-60-30-LI).

Los procedimientos que implican temas de los animales han sido aprobados por el Cuidado de Animales institucional y el empleo (IACUC) de la Universidad de California en San Francisco. 1. Preparación de la célula <ol><li> El cáncer de vejiga humano línea de células KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) se cultiva en medio RPMI-1640 suplementado con suero fetal bovino al 10% y 50 gentamicina g / ml. </li><li> Los cultivos se mantuvieron a 37 ° C, 5% de CO 2 con los medios cambia cada dos o tres días. </li><li> Las células fueron cosechadas por digestión con tripsina y se contaron utilizando un hemocitómetro. Una suspensión de células individuales de 2 x 10 6 células en 50 l se preparó en tampón fosfato salino y se mantuvieron en hielo. </li></ol> 2. Vesical ortotópica modelo de tumor <ol><li> Femeninos desnudos (nu / nu) ratones 8-10 semanas de edad (Simonsen Laboratorio, Gilory, CA) fueron utilizados. </li><li> Coloque un paño estéril sobre una almohadilla térmica para servir a unsa quirúrgica plataforma. Gel lubricante estéril, de 24 idiomáticas catéteres con agujas estilete, solución de nitrato de plata, y el agua estéril, se preparan. </li><li> La anestesia general se induce en los ratones con isoflurano inhalado (3% para la inducción, 2.1% para el mantenimiento) y se mantiene a través de ojiva. Ungüento oftálmico estéril se aplica a los ojos del animal, y una almohadilla de calor se utiliza para mantener el calor del cuerpo. </li><li> Sitúa el cursor en la posición supina. </li><li> Agarre suavemente la vulva del animal con una pinza para la estabilización. </li><li> El uso de un catéter intravenoso calibre 24 con el estilete de la aguja eliminado, la uretra del ratón se sondaje. Lubricación amplio debe ser utilizado. Con el ratón en posición supina, la uretra se encuentra inmediatamente por detrás de los pliegues de la vulva y anterior de la vagina. Un ángulo de 45 grados que se haya determinado inicialmente con el fin de canalizar la uretra y pasar por debajo del hueso púbico. Un ángulo más superficial se lleva luego a pasar a través de la uretra hasta la vejiga. Suave presión pélvicaAsegúrese de enderezar la uretra a menudo puede ayudar. Se debe tener cuidado de no empujar demasiado fuerte contra la resistencia, ya que esto puede conducir a la perforación uretral o vesical. La orina visto dentro del catéter confirma su ubicación dentro de la luz vesical. </li><li> Aspirar la orina desde el lumen del catéter y de la vejiga con la aguja del estilete. La aguja del estilete se utiliza para todas las aspiraciones y las inyecciones posteriores, dejando el catéter en su lugar. Esto permite la inyección de volúmenes precisos, eliminando el espacio muerto-dentro del catéter, y hace que cateterizaciones repetidas innecesarias. </li><li> Para poner en peligro la capa de glucosaminoglucano de la vejiga urotelio, 10 l de nitrato de plata 0,5 M se inyecta y se dejó que habitan durante 10 segundos. Esto puede formar un precipitado blanco con orina restante. </li><li> Aspirar y desechar el contenido de precipitado y de la vejiga usando una aguja de estilete. </li><li> Lave la vejiga mediante la inyección de 100 l de agua estéril. Aspirar y desechar el agua. Repea lavado con agua estéril 3 veces para un total de 4 lavados. </li><li> Colocar una corbata de seda 4-0 alrededor del meato uretral para ocluir la uretra, en preparación para la retirada del catéter. </li><li> Inyectar previamente preparado 2 x 10 6 KU-7-luc2-GFP células en 50 l usando la aguja estilete. </li><li> Simultáneamente retirar el catéter y la aguja estilete, dejando la uretra ocluido por la corbata de seda. </li><li> El ratón se mantendrá bajo anestesia general durante 2 horas antes de la corbata de seda se retira y el animal se despierta. </li></ol> 3. Ultrasonido <ol><li> La anestesia general se indujo con isoflurano vaporizado y se mantiene a través de cono de la nariz. </li><li> Un VisualSonics Vevo 770 in vivo de alta resolución de Micro-Imaging System (VisualSonics Inc., Toronto, Ontario, Canadá) con la RMV-704 de la sonda de 40 MHz se usó. </li><li> Sitúa el cursor en la posición supina sobre la plataforma de ultrasonido. </li><li> Asegure las patas traseras del animalcon cinta para evitar un movimiento excesivo durante la manipulación de la sonda de ultrasonidos. </li><li> Aplicar gel de ultrasonido de la pelvis del ratón. </li><li> Baje un RMV-704 (40 MHz) sonda de ultrasonido en la pelvis del ratón para obtener imágenes transversales o longitudinales. </li><li> Si se encuentra un tumor, las imágenes se pueden guardar para la medición de las dimensiones del tumor. </li></ol> 4. Bioluminiscente de imágenes <ol><li> Realizar la inyección intraperitoneal de 200 l (150 mg / kg) sustrato luciferina. </li><li> Inducir la anestesia general con isoflurano vaporizado y mantener a través de cono de la nariz. </li><li> Coloque los ratones en posición de decúbito supino en el reproductor de imágenes de bioluminiscencia. </li><li> Cinco minutos después de la inyección luciferina, medir la bioluminiscencia del animal en fotones por segundo. </li></ol> 5. SARNA Preparación <ol><li> Sintetizar ARN oligonucleótidos a una escala de 50 a 100 mmol. </li><li> Recocido complementarias oligonucleótidos de cadena sencilla into dúplex saRNAs. </li><li> Lípidos nanopartículas (LNP)-SARNA se preparan con el lípido ionizable 1,2-dilinoleyl-4-(2 - dimetilaminoetil) - [1,3]-dioxolano (DLinKC2-DMA), disteroylphosphatidyl colina, colesterol, y PEG-DMG utilizando una formación espontánea de vesículas procedimiento de formulación que se describió anteriormente. 1 </li></ol> 6. Administración intravesical de SARNA Formulado <ol><li> Después de establecer el modelo de tumor de vejiga ortotópica, los ratones son tratados con activador intravesical pequeño ARN (Sarna) formulado en nanopartículas lipídicas (LNPs) (Tabla 1). </li><li> La anestesia general se establece y mantiene con isoflurano vaporizado, y lubricante oftálmico estéril se aplica. </li><li> Colocar el animal en decúbito supino. </li><li> Realizar un cateterismo uretral como se describió anteriormente. </li><li> Coloque una corbata de seda 4-0 alrededor de la uretra y el catéter. </li><li> Aspirar toda la orina desde el catéter yvejiga mediante una jeringa vacía y la aguja del estilete. </li><li> Inyectar LNP-SARNA intravesicalmente para una dosis total de 50 l (3 mg / kg). Simultáneamente retirar el catéter y la aguja estilete, dejando la uretra ocluido por la corbata de seda. </li><li> El ratón seguirá siendo anestesiados con la uretra ocluido durante 2 horas. </li><li> El tratamiento con intravesical LNP-SARNA se inició el día 4 después de la implantación del tumor. Los ratones fueron tratados en serie, cada 3 días, para un total de 14 tratamientos. </li></ol> 7. Los resultados representativos Los tumores de vejiga derivados de KU-7-luc2-GFP células pueden ser controlados por la luciferasa de imagen bioluminiscente (Figura 1A y C) y el ultrasonido vesical (Figura 1B y D). Los tumores suelen ser detectables dentro de 3-5 días después de la inoculación de células. Hemos encontrado éxito de la implantación del tumor de vejiga en más del 90% de los ratones. Como el tratamiento con dsRNA intravesical se produce, la progresión del tumor se puede controlar wiª de estas modalidades. Después de que el animal es sacrificado, el crecimiento del tumor también puede ser confirmado mediante la fluorescencia de GFP. En general, la media de bioluminiscencia correlacionada con dimensiones medias de ultrasonido tumores de vejiga, como se ve visualmente en las figuras 1C y 1D. Sin embargo, después los animales fueron sacrificados y el tumor fue confirmada por GFP, hemos encontrado que la bioluminiscencia correlaciona más estrechamente con la presencia de tumor viable que hizo mediciones de ultrasonido. Esto era especialmente evidente entre los ratones tratados con Sarna (en oposición a los controles) porque el tejido cicatricial residual en los ratones tratados fue visualizado por ultrasonido a menudo, pero no pudo ser diferenciada de tumor en vivo. <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="Figura 1" /> Figura 1. La cinética de crecimiento del tumor de los tumores de vejiga. (A) imágenes de bioluminiscencia mide la actividad de la luciferasa en los tumores de la vejiga para confirmar su ubicación y el crecimiento. (B) En ultrasoun vejiga in vivod utilizando una sonda de 40 MHz produce las imágenes del tumor cuyas dimensiones pueden ser medidos con precisión. (C) gráficos que ilustran aumento progresivo del tumor en las dimensiones de bioluminiscencia y el ultrasonido. Los valores representan la media (+ / - desviación estándar) de medición de 6 animales. <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> .

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU international. 100, 1377-1384 (2007).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), e2523 (2011).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

LCL es un inventor llamado de solicitudes de patentes pendientes relacionadas con SARNA que han sido presentadas por la Universidad de California en San Francisco y con licencia para Alnylam Pharmaceuticals.

Se presenta un protocolo para el establecimiento de un modelo de ratón del tumor vesical ortotópico xenoinjertos seguido por el tratamiento intravesical de los tumores con SARNA LNP formulado que se dirige el promotor de p21 CIP1/WAF1 (p21) para la activación transcripcional. 2, 3 Los tumores resultantes pueden ser confirmación y seguimiento con imágenes bioluminiscentes y la ecografía de vejiga. Modelos ortotópico puede ser superior a intraperitoneal, subcutánea, intravenosa o modelos, pro...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> KU-7-luc-GFP células </td><td> Caliper </td><td> 128091 </td><td></td></tr><tr><td> tímicas ratones desnudos (nu / nu) </td><td> Simonsen Laboratorios </td><td> 6-7 semanas de edad </td><td> Sim: (NCR) nu / nu FISOL </td></tr><tr><td> Ultrasonido unidad </td><td> Visualsonics </td><td> Vevo 770 </td><td> RMV-704 de la sonda </td></tr><tr><td> La bioluminiscencia unidad </td><td> Caliper </td><td> IVIS Spectrum </td><td></td></tr><tr><td> Exel safelet catéter </td><td> Pescador </td><td> 14-841-21 </td><td> 24G x ¾ &quot; </td></tr><tr><td> Nitrato de plata </td><td> Sigma </td><td> S8157 </td><td></td></tr><tr><td> Hamilton jeringa </td><td> Hamilton company </td><td> 80301 </td><td></td></tr><tr><td> LNP-SARNA </td><td> Alnylam Pharmaceuticals </td><td></td><td></td></tr></tbody></table> Tabla 1. Reactivos específicos y equipos.

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

Se presenta un nuevo método para el tratamiento de cáncer de vejiga con intravesical entregado activación pequeño ARN (Sarna) en un modelo de xenoinjerto de tumor vesical ortotópico del ratón. El modelo de ratón se establece mediante cateterismo uretral por debajo inhalado anestesia general. Quemadura química se introduce entonces a la mucosa de la vejiga utilizando solución intravesical nitrato de plata para perturbar la capa de glicosaminoglicano vejiga y permite a las células para unir. Tras varios lavados con agua estéril, cáncer de vejiga humano KU-7-luc2-GFP células son inculcados a través del catéter en la vejiga para vivir durante 2 horas. Posterior crecimiento de los tumores de vejiga se confirma y monitoreado por ultrasonido de vejiga en vivo e imágenes bioluminiscentes. Los tumores se tratan a continuación intravesical con SARNA formulado en nanopartículas lipídicas (LNPs). El crecimiento del tumor se controla con el ultrasonido y la bioluminiscencia. Todos los pasos de este procedimiento se demuestra en el vídeo adjunto.

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

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

El establecimiento de un modelo de tumor de vejiga ortotópica para evaluar los efectos antitumorales de SARNA intravesical entrega y el control del crecimiento tumoral mediante ecografía y bioluminiscente.

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-bladder-tumor-model-evaluation-intravesical-sarna

Research

JoVE Journal

Medicine

14.6K vistas.  University of California, San Francisco. El establecimiento de un modelo de tumor de vejiga ortotópica para evaluar los efectos antitumorales de SARNA intravesical entrega y el control del crecimiento tumoral mediante ecografía y bioluminiscente.

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

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 ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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

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

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

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

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 ...

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  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Mínimamente Invasiva Establecimiento de murino ortotópico xenoinjertos vejiga

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Evaluación de tumor infiltrante de leucocitos subconjuntos en un modelo de tumor subcutáneo

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Evaluación de la eficacia de la<em&gt; H. pylori</em&gt; Proteína HP-PAN como herramienta terapéutica para el tratamiento de cáncer de vejiga en un modelo murino ortotópico

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Un &quot;paciente-Like&quot; ortotópico Singénico modelo de ratón de carcinoma hepatocelular Metástasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...