Cette recherche est financée par l&#39;AACR Henry Shepard vessie Cancer Research Grant (09-60-30-LI).

Procédures portant sur des sujets animaux ont été approuvés par le soin des animaux et le Comité institutionnel d&#39;utilisation (IACUC) à l&#39;Université de Californie, San Francisco. 1. Préparation des cellules <ol><li> La ligne de cellules humaines de cancer de la vessie KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) est cultivé dans un milieu RPMI-1640 supplémenté avec 10% sérum de veau foetal et 50 pg / ml de gentamicine. </li><li> Les cultures sont maintenues à 37 ° C, 5% de CO 2 avec les médias change tous les deux à trois jours. </li><li> Les cellules ont été récoltées par digestion à la trypsine et comptées à l&#39;aide d&#39;un hémocytomètre. Une suspension cellulaire unique de 2 x 10 6 cellules dans 50 l a été préparé dans une solution saline tamponnée au phosphate et maintenu sur la glace. </li></ol> 2. Orthotopique Modèle tumeur de la vessie <ol><li> Femme nue (nu / nu) chez la souris âgées de 8-10 semaines (Simonsen Laboratoire, Gilory, CA) ont été utilisés. </li><li> Placez un champ stérile sur un coussin chauffant pour servir unsa plate-forme chirurgicale. Lubrifiante stérile gelée, 24-linguistiques cathéters avec des aiguilles stylet, la solution de nitrate d&#39;argent et de l&#39;eau stérile sont préparées. </li><li> L&#39;anesthésie générale est induite chez les souris avec de l&#39;isoflurane en inhalation (3% pour l&#39;induction, 1-2% pour la maintenance) et maintenu par l&#39;intermédiaire d&#39;ogive. Pommade ophtalmique stérile est appliquée pour les yeux de l&#39;animal, et un tampon de chaleur est utilisé pour maintenir la chaleur du corps. </li><li> Placez la souris en position couchée. </li><li> Saisissez délicatement la vulve de l&#39;animal avec des pinces pour la stabilisation. </li><li> L&#39;utilisation d&#39;un cathéter 24-gue par voie intraveineuse avec le stylet aiguille enlevée, l&#39;urètre de la souris est un cathéter. Ample lubrification doit être utilisé. Avec le dos de la souris, l&#39;urètre est situé immédiatement postérieure aux plis de la vulve et du vagin antérieures. Un angle de 45 degrés est d&#39;abord prises pour cathétériser l&#39;urètre et de passer sous l&#39;os pubien. Un angle plus faible profondeur est ensuite prise de passer à travers l&#39;urètre dans la vessie. Doux pression pelvienneAssurez-vous de redresser l&#39;urètre peut souvent aider. Des précautions doivent être prises pour éviter de pousser trop fort contre la résistance, car cela peut conduire à la perforation urétrale ou de la vessie. Urine vu l&#39;intérieur du cathéter confirme sa position dans la lumière de la vessie. </li><li> Aspirer l&#39;urine de la lumière du cathéter et de la vessie à l&#39;aide de l&#39;aiguille de stylet. L&#39;aiguille stylet est utilisé pour toutes les aspirations et les injections suivantes, en laissant le cathéter en place. Ceci permet l&#39;injection de volumes précis en éliminant l&#39;espace mort dans le cathéter-, et il est cathétérismes répétées inutiles. </li><li> Pour nuire à la couche de glycosaminoglycane de l&#39;urothélium de la vessie, 10 uL de 0,5 M de nitrate d&#39;argent est injecté et a permis d&#39;habiter pendant 10 secondes. Cela peut former un précipité blanc avec de l&#39;urine restant. </li><li> Aspirer et jeter le contenu précipiter et de la vessie à l&#39;aide d&#39;une aiguille stylet. </li><li> Laver la vessie par injection de 100 pi d&#39;eau stérile. Aspirer et jeter l&#39;eau. Repeà laver à l&#39;eau stérile 3 fois pour un total de 4 lavages. </li><li> Placez une cravate de soie 4-0 autour du méat urétral pour occlure l&#39;urètre en préparation pour le retrait du cathéter. </li><li> Injecter préalablement préparée 2 x 10 6 KU-7-luc2-GFP cellules dans 50 pL à l&#39;aide de l&#39;aiguille de stylet. </li><li> Enlever simultanément le cathéter et l&#39;aiguille de stylet, laissant l&#39;urètre occlus par la cravate en soie. </li><li> La souris sera maintenu sous anesthésie générale pendant 2 heures avant la cravate en soie est retiré et l&#39;animal est éveillé. </li></ol> 3. Ultrason <ol><li> L&#39;anesthésie générale est induite par l&#39;isoflurane vaporisé et maintenus par cône de nez. </li><li> Un VisualSonics Vevo 770 In Vivo à haute résolution de micro-système d&#39;imagerie (VisualSonics Inc, Toronto, Ontario, Canada) avec la sonde RMV-704 40 MHz a été utilisé. </li><li> Placez la souris en position couchée sur la plate-forme ultra-sons. </li><li> Fixez pattes postérieures de l&#39;animalavec du ruban adhésif afin d&#39;éviter un mouvement excessif lors de la manipulation de la sonde d&#39;échographie. </li><li> Appliquer le gel des ultrasons pour le bassin de la souris. </li><li> Abaisser une sonde à ultrasons RMV-704 (40 MHz) sur le bassin de la souris pour obtenir des images transversales ou longitudinales. </li><li> Si la tumeur est trouvée, les images peuvent être sauvegardées pour la mesure des dimensions tumorales. </li></ol> 4. Bioluminescent Imaging <ol><li> Effectuer une injection intrapéritonéale de 200 pi (150 mg / kg) substrat luciférine. </li><li> Provoquer une anesthésie générale à l&#39;isoflurane vaporisé et de maintenir par cône de nez. </li><li> Placez la souris en position couchée dans l&#39;imageur bioluminescence. </li><li> Cinq minutes après l&#39;injection de luciférine, de mesurer la bioluminescence de l&#39;animal en photons par seconde. </li></ol> 5. Préparation SARNA <ol><li> Synthétiser l&#39;ARN oligonucléotides à une échelle de 50-100 umol. </li><li> Recuit complémentaires oligonucléotides simple brin into saRNAs duplex. </li><li> Lipid-nanoparticule (TNL)-SARNA sont préparés avec le lipide ionisable 1,2-dilinoleyl-4-(2 - diméthylaminoéthyl) - [1,3]-dioxolane (DLinKC2-DMA), disteroylphosphatidyl choline, de cholestérol, et le PEG-DMG en utilisant une procédure vésicule spontanée formation formulation comme décrit précédemment. 1 </li></ol> 6. L&#39;administration intravésicale de Sarna Formulé <ol><li> Après avoir établi le modèle de la vessie orthotopique de tumeur, les souris sont traitées avec intravésicale d&#39;activation petit ARN (SARNA) formulé dans des nanoparticules lipidiques (LNPs) (Tableau 1). </li><li> L&#39;anesthésie générale est établie et maintenue avec l&#39;isoflurane vaporisé, et le lubrifiant ophtalmique stérile est appliqué. </li><li> Placez l&#39;animal en position couchée. </li><li> Effectuer un cathétérisme urétral tel que décrit précédemment. </li><li> Placez une cravate de soie 4-0 autour de l&#39;urètre et le cathéter. </li><li> Aspirer toute l&#39;urine à partir du cathéter etvessie à l&#39;aide d&#39;une seringue et une aiguille vide stylet. </li><li> Injecter TNL-SARNA intravésicale pour une dose totale de 50 ul (3 mg / kg). Enlever simultanément le cathéter et l&#39;aiguille de stylet, laissant l&#39;urètre occlus par la cravate en soie. </li><li> La souris restera anesthésiés avec de l&#39;urètre sous pansement occlusif pendant 2 heures. </li><li> Le traitement par voie intravésicale TNL-SARNA a commencé le jour 4 après implantation de la tumeur. Les souris ont été traitées en série, tous les 3 jours, pour un total de 14 traitements. </li></ol> 7. Les résultats représentatifs Les tumeurs de vessie découlant de la KU-7-luc2-cellules GFP peut être contrôlé par la luciférase bioluminescente imagerie (figure 1A et C) et l&#39;échographie de la vessie (figure 1B et D). Les tumeurs sont souvent détectable dans les 3-5 jours après l&#39;inoculation des cellules. Nous avons trouvé avec succès l&#39;implantation tumeur de la vessie dans plus de 90% des souris. Comme le traitement par voie intravésicale ARNdb s&#39;ensuit, la progression de la tumeur peut être surveillé wie ces modalités. Après l&#39;animal est sacrifié, la croissance tumorale peut également être confirmée par fluorescence de la GFP. En général, signifie bioluminescence en corrélation avec les dimensions moyennes à ultrasons pour les tumeurs de la vessie, comme on le voit visuellement dans figures 1C et 1D. Cependant, après que les animaux ont été sacrifiés et la tumeur a été confirmée par la GFP, nous avons constaté que la bioluminescence corrélation plus étroite avec la présence de tumeur viable que fait mesures échographiques. Cela était particulièrement évident chez les souris traitées avec Sarna (par opposition aux contrôles) parce que les tissus cicatrice résiduelle chez les souris traitées était souvent visualisée par échographie, mais n&#39;a pas pu être différenciées de la tumeur en direct. <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="Figure 1" /> Figure 1. La cinétique de croissance tumorale de tumeurs de la vessie. (A) l&#39;imagerie par bioluminescence mesure l&#39;activité luciférase dans les tumeurs de la vessie afin de confirmer leur emplacement et de la croissance. (B) Dans la vessie ultrasoun vivod en utilisant une sonde 40MHz produit des images tumorales dont les dimensions peuvent être mesurées avec précision. (C) Graphiques illustrant l&#39;augmentation progressive de la tumeur dans les dimensions de bioluminescence et l&#39;échographie. Les valeurs représentent la moyenne (+ / - écart-type) des mesures à partir de 6 animaux. <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> .

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	<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 est un inventeur nommé dans l&#39;attente des demandes de brevet liées à SARNA qui ont été déposés par l&#39;Université de Californie à San Francisco et autorisé à Alnylam Pharmaceuticals.

Nous présentons un protocole pour la mise en place d&#39;une souris modèle de xénogreffes tumorales de la vessie orthotopique suivie d&#39;un traitement par voie intravésicale des tumeurs avec SARNA TNL formulé qui cible le promoteur de p21 CIP1/WAF1 (p21) pour l&#39;activation transcriptionnelle. 2, 3 Les tumeurs qui en résultent peuvent être confirmée et surveillée par imagerie bioluminescente et l&#39;échographie de la vessie. Modèles orthotopiques peut être supérieure à intrapéri...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> KU-7-luc-cellules GFP </td><td> Caliper </td><td> 128091 </td><td></td></tr><tr><td> thymiques chez la souris nude (nu / nu) </td><td> Laboratoires Simonsen </td><td> 6-7 semaines </td><td> Sim: (RCN) nu / nu Fisol </td></tr><tr><td> Appareil à ultrasons </td><td> Visualsonics </td><td> Vevo 770 </td><td> RMV-704 de sonde </td></tr><tr><td> Unité de bioluminescence </td><td> Caliper </td><td> IVIS spectre </td><td></td></tr><tr><td> Exel safelet cathéter </td><td> Pêcheur </td><td> 14-841-21 </td><td> 24G X ¾ &quot; </td></tr><tr><td> Le nitrate d&#39;argent </td><td> Sigma </td><td> S8157 </td><td></td></tr><tr><td> Hamilton seringue </td><td> Hamilton company </td><td> 80301 </td><td></td></tr><tr><td> TNL-SARNA </td><td> Pharmaceuticals Alnylam </td><td></td><td></td></tr></tbody></table> Tableau 1. Réactifs et équipements spécifiques.

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

Nous présentons une nouvelle méthode pour traiter le cancer de la vessie avec intravésicale livré activation petit ARN (SARNA) dans un modèle de xénogreffe de souris tumeur orthotopique de la vessie. Le modèle de la souris est mis en place par cathétérisme urétral sous anesthésie générale par inhalation. Brûlure chimique est ensuite introduit à la muqueuse de la vessie à l&#39;aide intravésicale solution de nitrate d&#39;argent pour perturber la couche glycosaminoglycane de la vessie et permet aux cellules d&#39;attacher. Après plusieurs lavages à l&#39;eau stérile, cancer de la vessie humaine KU-7-luc2-GFP cellules sont inculquées à travers le cathéter dans la vessie pour habiter pendant 2 heures. La croissance ultérieure de tumeurs de la vessie est confirmée et surveillée par échographie de la vessie dans l&#39;imagerie in vivo et bioluminescente. Les tumeurs sont alors traitées par voie intravésicale avec SARNA formulée dans des nanoparticules lipidiques (LNPs). La croissance tumorale est contrôlée par échographie et la bioluminescence. Toutes les étapes de cette procédure sont mises en évidence dans la vidéo qui l&#39;accompagne.

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

L&#39;établissement d&#39;un modèle de la vessie orthotopique tumeur à évaluer les effets antitumoraux de Sarna par voie intravésicale livré et suivi de la croissance tumorale par échographie et l&#39;imagerie bioluminescente.

Un modèle de la vessie orthotopique tumorale et l&#39;évaluation de Sarna traitement intravésicale

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 vues.  University of California, San Francisco. L'établissement d'un modèle de la vessie orthotopique tumeur à évaluer les effets antitumoraux de Sarna par voie intravésicale livré et suivi de la croissance tumorale par échographie et l'imagerie bioluminescente.

Vidéo: 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 modèle orthotopique de cancer de la vessie murin

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

Recherche

Médecine

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-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

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.

La radiothérapie combinée à un modèle de tumeur de cerveau de souris orthotopique

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 modèle de souris orthotopique de cancer de la thyroïde anaplasique

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 modèle de cancer de la vessie orthotopique d&#39;études livraison Gene

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 modèle murin orthotopique de cancer de la prostate humain métastases

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.

Minimalement invasive Création d&#39;murin orthotopique de la vessie xénogreffes

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.

Évaluation des leucocytes infiltrant les tumeurs sous-ensembles dans un Modèle de tumeur sous-cutanée

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.

Évaluation de l&#39;efficacité de la<em&gt; H. pylori</em&gt; Protéines HP-NAP comme outil thérapeutique pour le traitement du cancer de la vessie dans un modèle murin orthotopique

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 «Patient-Like&quot; Orthotopique Syngénique modèle de souris de carcinome hépatocellulaire Métastases

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 ...