Cette recherche est soutenue par un financement du programme intra-muros des NIH.

Préparation des cellules I. <ol><li> Les cellules sont cultivées jusqu&#39;à confluence dans du DMEM avec 10% FBS environ un 80% de confluence 225 cm 3 ballon pendant toutes les 5 souris pour être implantés. </li><li> Aspirer les médias hors des cellules, laver avec 10 ml de PBS sans calcium ni magnésium. Aspirer PBS cellules off. </li><li> Trypsiniser cellules avec 3 ml de trypsine, attendez 10-15 minutes, et de neutraliser la trypsine avec 15 ml de milieu. </li><li> Pipeter toutes les cellules et les médias dans une forme conique de 50 ml. Deux grands flacons entrer dans une forme conique de 50 ml. </li><li> Spin cellules à 1600 rpm pendant 7 min à 4 ° C. Aspirer complètement le surnageant. Remettre en suspension les cellules dans 10 ml de PBS froid, et rincez délicatement les cellules par aspiration et refoulement. Assurez-vous que le culot cellulaire est mélangé uniformément avec le PBS. La suspension doit être couvert. </li><li> A cette époque une numération cellulaire est effectuée à l&#39;aide d&#39;un compteur Coulter. Les cellules sont centrifugées à nouveau à 1600 rpm pendant 7 min à 4 ° C. </li><li> Après la centrifugation 2 ème des cellules, en tantpirate tout PBS et remettre en suspension dans 1 ml de PBS. </li><li> Les cellules sont filées dans la 1ml de PBS à 3000 rpm pendant 10 min à 4 ° C. Le PBS est aspiré, et le PBS est ajouté pour ajuster la concentration finale à 1x10 6 pour 5 microlitres U87 cellules, d&#39;autres lignées cellulaires peuvent nécessiter un nombre différent de cellules et la concentration de la croissance tumorale optimale. </li><li> Après le volume final et la concentration est obtenue, les cellules sont conservés dans la glace dans un tube Eppendorf. </li></ol> II. Xénogreffes <ol class="ualpha"><li> Préparation de la souris <ol><li> Les souris sont anesthésiées en utilisant un mélange d&#39;injection de kétamine / xylazine / Saline. Le mélange est de 120 mg / kg de kétamine, 16 mg / kg de Rompun et Saline. Les souris sont pesées individuellement et donné 0,1 ml par 10 g en fonction de leur poids. L&#39;anesthésie est donnée avec une aiguille 26G attaché à 1 ml seringue IP. Il faudra environ 15 minutes pour les souris à atteindre une phase de sommeil sans réponse à la douleur. Vous pouvez vérifier la réponse à la douleur de l&#39;souris en pinçant leur coussinet plantaire. </li><li> Après la souris est entièrement anesthésié, pommade ophtalmique est administré à leurs yeux pour les empêcher de se dessécher. </li><li> La souris est placée sur le lit de la stéréotaxique avec les dents supérieures de l&#39;encoche sur la barre bouche. Le lit est ensuite placé dans l&#39;stéréotaxique. </li><li> Le garde nez est placé sur le nez de la souris pour empêcher un mouvement de la tête, et les barres d&#39;oreilles sont doucement placer dans les conduits auditifs de la souris. Soyez prudent de ne pas faire de placement, soit de la barre de nez ou barres d&#39;oreilles trop serrés, que le crâne peut se fissurer et / ou empêcher la souris de la respiration. </li><li> Assurez-vous que la tête de la souris est de niveau en utilisant les graduations sur le stéréotaxique est utile au cours de ce processus. </li><li> Pour de préparation de l&#39;iode de la souris, l&#39;alcool et providine est appliqué généreusement sur la tête de la souris en alternance, pour un total de 3 demandes, les demandes d&#39;alcool et 3 de l&#39;iode. Une application finale d&#39;un tampon imbibé d&#39;alcool à 70% est appliqué àla tête. </li></ol></li><li> La procédure chirurgicale <ol><li> Tous les instruments de la procédure doivent être stérilisés soit par autoclavage ou tremper les instruments dans l&#39;éthanol à 70% avant de commencer la procédure. </li><li> Une incision est faite sur le dessus du crâne derrière l&#39;œil droit en diagonale sur la partie postérieure gauche du crâne. L&#39;incision doit être d&#39;environ 10 mm à 15 mm de long. </li><li> L&#39;utilisation d&#39;un Q-tip, lentement séparer l&#39;incision pour exposer le crâne et essuyer la membrane qui est entre le crâne et la peau. Utilisez un coton-tige pour effacer toute trace de sang et de regarder pour un saignement excessif. </li><li> Exposer le sommet du crâne pour visualiser le bregma. En utilisant le point de seringue Hamilton, positionnez-la sur le bregma. La position correcte du trou à percer dans le crâne est de 2 mm à droite et 1 mm antérieure de bregma (figure 2). Mèche feutre de chirurgien marqueur, qui est achetée stérile, est utilisé pour marquer l&#39;emplacement du trou à percer. S&#39;il vous plaît noter que le trou est situé à proximité de l&#39;uneligne de suture Intérieur. </li><li> Ensuite, percez un petit trou dans le crâne à la position marquée en utilisant la pointe d&#39;une aiguille 22G dans un vase broches ou alimenté par une aiguille 18G. </li><li> Chargez le 10 ul de verre Hamilton seringue avec 5 pi de cellules. La seringue Hamilton est de 10 pi avec un bout arrondi pour 30G 33G aiguille fixe qui s&#39;y rattachent. Assurez-vous de mélanger les cellules avant de les charger, soit en les pipette ou en utilisant une seringue de 1 ml avec une aiguille 25G attaché. Après la seringue Hamilton est chargé, le replacer dans le bras pour l&#39;aligner avec le trou dans le crâne. </li><li> Abaisser l&#39;extrémité de l&#39;aiguille carie à la surface du crâne, une fois l&#39;aiguille est à niveau avec le crâne; abaisser l&#39;aiguille jusqu&#39;à ce qu&#39;il soit à la profondeur de 3 mm au-dessous de la surface du crâne. </li><li> Après l&#39;aiguille est abaissée, elle doit y rester pendant 2 minutes pour éviter tout traumatisme majeur de l&#39;aiguille qui touche le cerveau. L&#39;implantation des cellules est à un taux de 1 pl par minute pour un total de 6-8 minutes, faire attention à toute backflow. Après la dernière des cellules ont été implantées, laisser l&#39;aiguille en place pendant encore 2 minutes, pour permettre à toutes les cellules de régler po </li><li> Retirez l&#39;aiguille du cerveau, et nettoyer le trou avec un Q-tip. Procéder au nettoyage de l&#39;aiguille et une seringue à 50% Actone, EtOH 100%, et le PBS, 3x dans chaque solution et dans cet ordre. </li><li> La peau sur le crâne est prêt à être suturée à ce point. Utilisation de 5-0 PBS refermer l&#39;incision avec environ 3 points de suture. </li><li> Laissez la souris de se réveiller sur un coussin chauffant, et réappliquer la pommade oculaire en cas de besoin. La souris peut être retourné à la cage après montre le mouvement de sa propre initiative (c.-à-levage jusqu&#39;à la tête ou déplacer les épaules). </li></ol></li></ol> III. Traitement de l&#39;implant IC par rayonnement et des drogues <ol class="ualpha"><li> La radiothérapie <ol><li> Selon la lignée cellulaire, le traitement des souris peut avoir lieu 5 à 7 jours post-implantation. Notre département utilise une unité de radiothérapie pour traiter orthovoltage rayonnementment. </li><li> D&#39;imagerie comme BLI (imagerie par bioluminescence), l&#39;IRM ou la TDM peuvent être utilisés pour visualiser la tumeur avant la randomisation (figure 3A). </li><li> Les souris sont anesthésiées avec le cocktail kétamine / xylazine / solution saline comme avant, et une pommade ophtalmique est appliqué à leurs yeux. </li><li> Les souris sont placées dans un gabarit de rayonnement fait sur mesure qui est configuré comme une roue à tarte, dans laquelle les chefs des souris pointer vers le centre. Les corps des souris sont protégées contre le rayonnement par le plomb. </li><li> Le traitement médicamenteux ne peut être donnée soit avant, soit un rayonnement à travers les itinéraires de livraison différents, à savoir la SQ, IP et IV. </li></ol></li></ol> IV. Résultats La souris doit se réveiller de la procédure au sein de 45-60 minutes après l&#39;injection d&#39;anesthésie. Si la souris a difficilement se réveiller après la procédure et le temps a dépassé 1 ½ h après l&#39;intervention, la souris peut ne pas récupérer et l&#39;euthanasie peut être une alternative efficace. Si eoici un saignement excessif ou le crâne devient fissurée à n&#39;importe quel moment au cours de la procédure de l&#39;euthanasie est conseillé. Si tout va bien et en fonction de la lignée cellulaire, une tumeur doit commencer à se former dans les 2-4 semaines après l&#39;intervention a lieu. Lors de l&#39;utilisation des modèles de xénogreffes intracrâniennes, l&#39;efficacité thérapeutique est habituellement mesurée par la survie post-implantation 5. Toutefois, puisqu&#39;il s&#39;agit d&#39;un des signes et symptômes de modèle IC aura lieu avant le décès. Certains des signes physiques de la souris peuvent présenter sont une posture voûtée, la perte de poids corporel, et l&#39;inactivité. Certains des signes neurologiques peuvent être saisies, tête inclinée, et la perte d&#39;équilibre. La figure 3B montre une tache de SE d&#39;une tumeur sur la mortalité. Une courbe de Kaplan-Meier est utilisé pour évaluer les données (figure 4). Le nombre de jours de survie à 50% sont calculés et comparés à la commande. Si le groupe combinaison vécu plus longtemps que l&#39;addition du rayonnement individuel et les bras de médicaments, la combinaison est était réussie. <img alt = &quot;Figure 1&quot; src = &quot;/ files/ftp_upload/3397/3397fig1.jpg&quot; /&gt; Figure 1. L&#39;équipement Stoelting stéréotaxique. <img alt="Figure 2" src="/files/ftp_upload/3397/3397fig2.jpg" /> Figure 2. Un diagramme de l&#39;emplacement du trou de forage dans le crâne de la souris. <img alt="Figure 3A" src="/files/ftp_upload/3397/3397fig3A.jpg" /> La figure 3A. BLI imagerie après l&#39;implantation GBM. <img alt="Figure 3B" src="/files/ftp_upload/3397/3397fig3B.jpg" /> La figure 3B. H et E de la tumeur GBM à la mortalité. <img alt="Figure 4" src="/files/ftp_upload/3397/3397fig4.jpg" /> Figure 4. Résultats des implants IC U87 injectés avec l&#39;inhibiteur PARP 7 après la chirurgie jours.

<ol>
	<li>Bleau, A. M., Holland, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+the+mouse+genome+to+hunt+human+alternations.">Trapping the mouse genome to hunt human alternations.</a> Proc. Nat. Acad. Sci. U.S.A. 104, 7737-7738 (2007).</li><li>McGrady, B. J., McCormick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+intracranial+invasion:+morphological+observations+on+central+nervous+system+invasion+by+melanoma+cells.">A murine model of intracranial invasion: morphological observations on central nervous system invasion by melanoma cells.</a> Clin. Exp. Metastasis. 10, 387-393 (1992).</li><li>Fei, X., Zhang, Q., Dong, J., Diao, y., Wang, Z., Li, R., Wu, Z., Wang, A., Lan, Q., Zhang, S., Huang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+clinically+relevant+orthotopic+xenograft+mouse+model+of+metastatic+lung+cancer+and+glioblastoma+through+surgical+tumor+tissues+injection+with+trocar.">Development of clinically relevant orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma through surgical tumor tissues injection with trocar.</a> Journal of Experimental &#38; Clinical Cancer Research. 29, 84-84 (2010).</li><li>Fujita, M., Zhu, X., Sasaki, K., Ueda, R., Low, K., Pollack, I., Okada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+STAT3+Promotes+the+Efficacy+of+Adoptive+Transfer+Therapy+Using+Type-1+CTLs+by+Modulation+of+the+Immunological+Microenvironment+in+a+Murine+Intracranial+Glioma.">Inhibition of STAT3 Promotes the Efficacy of Adoptive Transfer Therapy Using Type-1 CTLs by Modulation of the Immunological Microenvironment in a Murine Intracranial Glioma.</a> J. Immunol. 180, 2089-2098 (2008).</li><li>Candolfi, M., Curtin, J. F., Nichols, W. S., Muhammad, A. K. M. .. . G., King, G., Pluhar, G. D., McNiel, G. E., Ohlfest, E. A., Freese, J. R., Moore, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathlogical+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathlogical characterization and tumor progression.</a> J. Neurooncol. 85, 133-148 (2007).</li><li>Kaye, A., Morstyn, G., Gardner, I., Pyke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+xenograft+glioma+model+in+mouse+brain.">Development of a xenograft glioma model in mouse brain.</a> Cancer Research. 46, 1367-1373 (1986).</li><li>Zhao, Y., Xiao, A., diPierro, C., Carpenter, J., Abdel-Fattah, R., Redpath, G., Lopez, M., Hussaini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Extensive+Invasive+Intracranial+Human+Glioblastoma+Xenograft+Model.">An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model.</a> American Journal of Pathology. 176, 3032-3049 (2010).</li><li>Learn, C., Grossi, P., Schmittling, R., Xie, W., Mitchell, D., Karikari, I., Wei, Z., Dressman, H., Sampson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Analysis+of+Intracranial+Tumors+in+a+Murine+Model+of+Glioma+Demonstrate+a+Shift+in+Gene+Expression+in+Response+to+Host+Immunity.">Genetic Analysis of Intracranial Tumors in a Murine Model of Glioma Demonstrate a Shift in Gene Expression in Response to Host Immunity.</a> J. Neuroimmunol. 182, 63-72 (2007).</li><li>Martens, T., Laabs, Y., Günther, H., Kemming, D., Zhu, Z., Witte, L., Hagel, C., Westphal, M., Lamszus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Glioblastoma+Growth+in+a+Highly+Invasive+Nude+Mouse+Model+Can+Be+Achieved+by+Targeting+Epidermal+Growth+Factor+Receptor+but+not+Vascular+Endothelial+Growth+Factor+Receptor-2.">Inhibition of Glioblastoma Growth in a Highly Invasive Nude Mouse Model Can Be Achieved by Targeting Epidermal Growth Factor Receptor but not Vascular Endothelial Growth Factor Receptor-2.</a> Clin. Cancer Res. 14, 5447-5458 (2008).</li><li>Purow, B., Schiff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+genetics+of+glioblastoma:+are+we+reaching+critical+mass.">Advances in the genetics of glioblastoma: are we reaching critical mass.</a> Nat. Rev. Neurol. 5, 419-426 (2009).</li></ol>

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Les gliomes malins, tels que le glioblastome multiforme, représentent les tumeurs les plus fréquentes du cerveau primaire et ont un pronostic sombre 4. La survie des patients touchés par GBM est resté pratiquement inchangé au cours de la dernière décennie (c.-à-9-12 mois après le diagnostic), malgré les progrès de la chirurgie, la radiothérapie et la chimiothérapie 5. Les caractéristiques d&#39;un modèle approprié pour l&#39;étude du traitement gliome devrait inclure une localisati...

<table border="1"><tbody><tr><td> Lab standard stéréotaxique Instrutment </td><td> Stoelting souris et l&#39;adaptateur Rat </td></tr><tr><td> Betadine </td><td> EtOH à 100% </td></tr><tr><td> 70% EtOH </td><td> Acétone 50% </td></tr><tr><td> 6 &quot;Q-tips </td><td> Tampons imbibés d&#39;alcool </td></tr><tr><td> Hamilton seringue 80008 </td><td> Ethicon 5-0 PD </td></tr><tr><td> 1701 SN 31-33G / 1 &quot;/ PT3 aiguille </td><td> Des seringues de 1 ml </td></tr><tr><td> Aiguilles 25G </td><td> Coussin chauffant </td></tr><tr><td> Pommade artificielle larmes </td><td> Très belle pointe de marqueur permanent </td></tr></tbody></table>

combination radiotherapy in an orthotopic mouse brain tumor model

Le glioblastome multiforme (GBM) sont les plus communs et agressifs adultes tumeurs cérébrales primaires 1. Ces dernières années il ya eu des progrès substantiels dans la compréhension des mécanismes de l&#39;invasion tumorale, et l&#39;inoculation intracérébrale directe de la tumeur fournit l&#39;occasion d&#39;observer le processus invasif dans un environnement physiologique appropriée 2. En ce qui concerne les tumeurs cérébrales de l&#39;homme sont concernés, les modèles actuellement disponibles orthotopiques sont établies soit par injection stéréotaxique de suspensions cellulaires ou d&#39;implantation d&#39;un morceau de la tumeur par le biais d&#39;une procédure compliquée craniotomie 3. Dans notre technique, nous prélever des cellules de culture de tissus pour créer une suspension de cellules utilisée pour implanter directement dans le cerveau. La durée de la chirurgie est d&#39;environ 30 minutes, et que la souris doit être dans un plan constant chirurgicale, un anesthésique injectable est utilisé. La souris est placée dans un gabarit stéréotaxique par Stoetling (figure 1). À l&#39;arrièreer la zone chirurgicale est nettoyé et préparé, une incision est faite, et la bregma est situé pour déterminer l&#39;emplacement de la craniotomie. L&#39;emplacement de la craniotomie est de 2 mm vers la droite et 1 mm rostrale au bregma. La profondeur est de 3 mm de la surface du crâne, et les cellules sont injectés à un débit de 2 pi toutes les 2 minutes. La peau est suturée avec 5-0 PDS, et la souris est autorisée à se réveiller sur un coussin chauffant. D&#39;après notre expérience, en fonction de la lignée cellulaire, le traitement peut avoir lieu à partir de 7-10 jours après la chirurgie. De délivrance de médicament est dépendante de la composition de médicament. Pour des traitements de radiothérapie les souris sont anesthésiées, et mis dans un gabarit fait sur mesure. Le plomb couvre le corps de la souris et expose que le cerveau de la souris. L&#39;étude de la tumorigenèse et l&#39;évaluation de nouvelles thérapies pour GBM besoin précis et reproductibles modèles de tumeur de cerveau des animaux. Ainsi, nous utilisons ce modèle orthotopique du cerveau pour étudier l&#39;interaction du microenvironnement du cerveau et de la tumeur, afin de tester l&#39;EFFICACITÉcessus de différents agents thérapeutiques avec et sans rayonnement.

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

Le but de cet article est de décrire l&#39;utilisation d&#39;un modèle de glioblastome orthotopique pour les études de chimioradiothérapie. Cet article va passer si le traitement des cellules, l&#39;implantation, et de la radiothérapie de la souris en utilisant un modèle intracrânienne.

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 ...

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

Research

JoVE Journal

Medicine

16.3K vues.  National Cancer Institute. Le but de cet article est de décrire l'utilisation d'un modèle de glioblastome orthotopique pour les études de chimioradiothérapie. Cet article va passer si le traitement des cellules, l'implantation, et de la radiothérapie de la souris en utilisant un modèle intracrânienne.

Vidéo: 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 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 ...

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.

Dans l&#39;imagerie de bioluminescence in vivo de la tumeur Dynamics hypoxie des métastases cérébrales du cancer du sein dans un modèle murin

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 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 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 ...

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.

A Novel haute résolution<em&gt; In vivo</em&gt; Technique d&#39;imagerie pour étudier la réponse dynamique des structures intracrâniennes à la croissance tumorale et de thérapeutique

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 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  ...

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.

Un modèle de souris orthotopique glioblastome maintien contraintes physiques et parenchyme cérébral Convient pour intravitale deux microscopie biphotonique

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.

Tumeur traitement Field Therapy en association avec le bevacizumab pour le traitement de glioblastome récurrent

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 ...