Authors thank Jeffrey Sellers to assist orthotopic pancreatic cancer mouse modeling. This work was supported by Research Initiative Pilot Awards from the Department of Radiology at UAB and NIH grants 2P30CA013148 and P50CA101955.

Toutes les procédures ont été approuvées par le soin et l&#39;utilisation des animaux Commission institutionnelle à l&#39;Université d&#39;Alabama à Birmingham. Modélisation 1. Orthotopique pancréatique tumeurs Souris <ol><li> Culture des lignées cellulaires cancéreuses humaines pancréatiques-standard dans du milieu de Eagle modifié par Dulbecco (DMEM) supplémenté avec du sérum bovin fœtal à 10%. Maintenir toutes les cultures à 37 ° C dans une atmosphère humidifiée avec 5% de CO 2. </li><li> Utilisez 8-10 souris femelles graves semaine-vieux combinés immunodéficients. Placez les cages des animaux à 12 heures de lumière et le cycle de l&#39;obscurité de 12 heures à température ambiante (21 ± 2 ° C) et 60% d&#39;humidité. </li><li> Anesthésier les animaux en utilisant une ventilation avec 2% d&#39;isoflurane mélangé avec de l&#39;oxygène (2 L / min) pendant toute la chirurgie. Confirmer la profondeur de l&#39;anesthésie par pincement de l&#39;orteil réflexe. La place des animaux sur un coussin chauffant (37 ° C) pour maintenir la température du corps. Appliquer une pommade ophtalmique vétérinaire sur les yeux pour prévenir la sécheresse sous anesthésie. &amp;# 160; </li><li> Enlever les poils dans le quadrant supérieur gauche de l&#39;abdomen de chaque souris, et de donner un médicament analgésique (carpofen, 5 mg / kg de poids corporel sous-cutanée) dans la région. Appliquer une solution de bétadine sur la peau exposée. Préparer instruments chirurgicaux autoclave. </li><li> Faire une incision de 1 cm dans la peau et du péritoine en utilisant l&#39;iris ciseaux droits. Retirez délicatement le pancréas de l&#39;abdomen en utilisant des pinces chirurgicales. </li><li> Introduire 28 G aiguille d&#39;une seringue 0,5 ml d&#39;insuline dans la queue du pancréas et infuser ensuite lentement une solution de 2,5 millions de cellules cancéreuses pancréatiques humaines dans 30 pl de DMEM. Assurez-vous que une petite cloque se forme dans la tête du pancréas par la solution. </li><li> Placez délicatement le pancréas dans l&#39;abdomen en utilisant des pinces chirurgicales. Fermez le péritoine et la peau dans une couche avec deux interrompu 5-0 sutures Prolene, puis mettre fin à l&#39;anesthésie. Ne retournez pas un animal qui a subi une intervention chirurgicale à la compagnie d&#39;autres animaux jusqu&#39;à guérison complète.60; Retirer sutures à la chirurgie de poste 7 ~ 10 jours. </li><li> Donner une autre dose du médicament analgésique (carpofen, 5 mg / kg de poids corporel sous-cutanée) à 24 h après la chirurgie. </li><li> Vérifiez la taille de la tumeur en palpant la zone de la chirurgie en utilisant deux doigts. Les tumeurs se sentent généralement plus dense et plus bosselée que les tissus et organes environnants. Habituellement, cela prend ~ 1-2 semaines pour commencer à se sentir une tumeur. </li><li> De surveiller les animaux quotidiennement les signes de la maladie. Lorsque les animaux apparaissent (comportements de toilettage et d&#39;évitement normales manque) malades, nous les résilié par dislocation cervicale sous anesthésie. </li></ol> 2. imagerie par résonance magnétique <ol><li> Appliquer IRM lorsque la taille de la tumeur est d&#39;environ 5-7 mm de diamètre en général à 2 ~ 4 semaines après l&#39;implantation des cellules. Utilisez un scanner RM dédiée à l&#39;imagerie du petit animal ou un scanner RM clinique équipée d&#39;une bobine spécialisé pour l&#39;imagerie du petit animal. NOTE: Nous avons utilisé un petit animal MR scanner 9.4T avec une combinaison d&#39;un volume résonateur 1 H/ Émetteur et un récepteur de bobine de surface (30 mm de diamètre) (Bruker BioSpin Corp., Billerica, MA). Une bobine de surface offre une meilleure rapport (SNR) 14 signal-sur-bruit. </li><li> Préparer un agent de contraste IRM à base de gadolinium à injecter ~ de 0,1 à 0,2 mmol / kg à chaque animal dans ~ 0,1 au 0,2 ml de PBS (tampon phosphate salin). NOTE: Nous avons utilisé gadotéridol, et injecté 0,2 mmol / kg dans 0,15 ml de PBS sur une période de 15 sec (0,1 ml / sec). </li><li> Préparer un tube micro-polyéthylène (longueur: 7,62 mm, diamètre intérieur: 0,28 mm, diamètre extérieur: 0,64 mm). Insérez une aiguille 30 G (longueur de 12,7 mm) dans une extrémité du tube, et une aiguille de 30 G émoussée de pointe (9,5 mm de longueur) dans l&#39;autre extrémité. Connecter une seringue de 1 ml contenant MR agent de contraste à l&#39;aiguille de pointe émoussée, et poussez lentement la seringue pour remplir le tube entier avec l&#39;agent de contraste MR. </li><li> Anesthésier des animaux à l&#39;aide de la ventilation avec ~ 1 - 2% d&#39;isoflurane mélangé avec de l&#39;oxygène (2 L / min) tout au long de la préparation et de l&#39;imagerie. Confirmer la profondeur de anesthesia par pincement de l&#39;orteil réflexe. Appliquer une pommade ophtalmique vétérinaire sur les yeux pour prévenir la sécheresse sous anesthésie. Dilater la veine caudale en utilisant une lampe de chaleur avant l&#39;insertion de l&#39;aiguille. Prenez le milieu de l&#39;aiguille de 30 G en utilisant une pince Kelly, et l&#39;insérer dans la veine de la queue. Bande fois la queue et le tube sur un morceau de plastique ou de papier de carton (10 mm de largeur x 100 mm de longueur) pour garder la queue droite. </li><li> Placez l&#39;animal en position couchée dans un lit de l&#39;animal équipé d&#39;une circulation d&#39;eau chaude (ou air chaud) pour réguler la température du corps pendant l&#39;imagerie. Régler la température sur le lit à 37 ° C. Insérer une sonde de température rectale à surveiller la température du corps lors de l&#39;imagerie. </li><li> Appliquer une carte en plastique orthogonalement plié dans la région abdominale. Assurez-vous que la tumeur est située derrière l&#39;extrémité supérieure de la planche, puis déroulez le bord légèrement (~ 2 mm) pour assurer la tumeur est pris par le conseil. Collez le conseil au lit animal fermement. </li><li> Tape à transduce pad de la respirationr (SA Instrument, Inc., Stony Brook, NY) sur la région de la poitrine de surveiller la respiration de l&#39;animal lors de l&#39;imagerie. Placez une bobine de surface sur le dessus de la région de la tumeur, et la bande au lit animal fermement. Appuyez sur le lit animal dans le scanner MR de placer la région de la tumeur à l&#39;axe de la bobine de volume (diamètre intérieur: 72 mm). </li><li> Effectuer adaptation et d&#39;accord à la fois pour l&#39;émetteur et le récepteur, suivi par calage. </li><li> Commencer avec une séquence d&#39;MR anatomique pour localiser la tumeur. Utilisez une séquence d&#39;écho de spin (T2) turbo T2 pour obtenir des images axiales avec les paramètres d&#39;acquisition suivantes. temps de répétition (TR) / temps d&#39;écho (TE) = 3000/34 ms, 128 x 128 matrice, le terrain de 30 x 30 mm de vue, nombre de moyennes = 1, écho longueur du train = 4, et 20 contiguës 1 mm tranches épaisses dans un mode entrelacé pour couvrir toute la région de la tumeur (durée totale du balayage: 1,6 min). Remarque: Comme les tumeurs pancréatiques orthotopique sont plus difficiles à se trouver que celles sous-cutanées, locali conventionnelzer images ayant une résolution inférieure peuvent ne pas être utile. </li><li> Acquérir (en T1) images pondérées en T1 avec différents angles bascules pour récupérer T1 carte. A cet effet, utiliser un écho de gradient approche angle multiflip avec les paramètres suivants: temps de répétition (TR) / temps d&#39;écho (TE) = 115/3 ms, 128 x 128 matrice, un terrain de 30 x 30 mm de vue, nombre de moyennes = 4, ~ 5-7 contigus 1 mm d&#39;épaisseur des tranches en mode entrelacé pour couvrir la région de la tumeur, et sept angles de bascule de 10, 20, 30, 40, 50, 60, et 70 (temps total de balayage par angle de bascule: 1 min). NOTE: Cependant, l&#39;approche de l&#39;angle multiflip ne est efficace que lorsque le champ B1 homogénéité est élevé. Si non, cartes T1 peut être obtenu avec l&#39;approche de TR multiples au lieu de 15. </li><li> Acquérir des images en T1 avant et après injection de contraste MR à base de gadolinium. Utilisez les mêmes paramètres d&#39;acquisition et de la géométrie pour la cartographie de T1, mais avec l&#39;angle de bascule fixe de 30. Utiliser le codage linéaire pour assurer l&#39;état d&#39;équilibre lorsque le centre de l&#39;espace k est obtenu, especially lors d&#39;un court TR et un angle de bascule de faible degré sont utilisés. Acquérir cinq images de référence avant l&#39;injection de contraste. Puis acquérir 40 images après injection de contraste (durée totale du balayage: 45 min). Utilisation d&#39;une pompe à seringue pour injecter l&#39;agent de contraste à une vitesse constante (0,01 ml / sec). </li><li> Contrôler la respiration animale continue, et ajuster la concentration de l&#39;isoflurane pour maintenir le taux respiratoire de 50 à 100 respirations par minute. Surveiller la température du corps des animaux tout au long de l&#39;imagerie. </li><li> Après avoir terminé DCE-MRI, enlever l&#39;aiguille et d&#39;autres sondes, et placer l&#39;animal dans une cage vide lits avec des serviettes en papier. Masser doucement la zone inférieure de l&#39;abdomen. La cage doit être placée moitié sous une lampe chauffante pour permettre à l&#39;animal de se déplacer dans et hors du gradient thermique tel qu&#39;il récupère. Ne pas laisser un animal sans surveillance tant qu&#39;il a repris conscience suffisante pour maintenir décubitus sternal. </li></ol> 3. Traitement et analyse d&#39;images <ol><li> REGI Segment tumorales<html> dans les images T2. Dans images pondérées en T2, l&#39;intensité du signal dans la région de la tumeur est plus brillante que celle de tissus environnants, de sorte que la limite de la tumeur peut être délimitée manuellement. REMARQUE: techniques de segmentation semi-automatiques comme seuil global ou contour actif peuvent être utilisés 16,17, mais l&#39;intensité de fond inégale doivent être corrigées surtout quand une bobine de surface est utilisé. </li><li> Créez des cartes T1 et densité de proton. En pondération T1 images acquises avec une séquence d&#39;écho de gradient, en supposant que le temps d&#39;écho (TE) est bien inférieure à la valeur T2 *, la valeur de pixel est déterminée par <img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq1.jpg" /> où S 0 est la densité de protons, T 1 T est une constante de temps de relaxation, TR est le temps de répétition, et θ est un angle de bascule. L&#39;équation (1) peut être réécrite à 641eq2.jpg &quot;/&gt; lorsque S (θ) / sin est remplacée par Y et S (θ) / tanθ est remplacé par X. L&#39;équation (2) est une équation linéaire, et sa pente et l&#39;interception peut être utilisé pour récupérer T1 et S 0 valeurs, respectivement. </li><li> Calculer M. concentration de contraste des images DCE-MR. Lorsque le gadolinium à base d&#39;agents MR de contraste est injecté, T 1 de relaxation constante de temps est modifié au fil du temps. Donc, l&#39;équation (1) peut être réécrit pour <img alt="Équation 3" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq3.jpg" /> T 1 (t) est en relation avec la concentration de contraste RM, C (t), comme suit, <img alt="Equation 4" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq4.jpg" /> où r 1 est la relaxivité longitudinale d&#39;agent de contraste RM. Ainsi, en combinant les équations (3) et (4), MR concentration de contraste est déterminée par <img aLT = &quot;équation 5&quot; fo: contenu-width = &quot;3in&quot; src = &quot;/ files / ftp_upload / 52641 / 52641eq5.jpg&quot; /&gt; </li><li> Quantifier les paramètres pharmacocinétiques de l&#39;agent de contraste MR. C p (t) présente MR concentration de contraste dans le plasma sanguin à l&#39;instant t après le début de l&#39;injection de contraste. C p (t) est appelée fonction d&#39;entrée artérielle (AIF). Si AIF est disponible, les paramètres pharmacocinétiques de l&#39;agent de contraste RM peuvent être calculées par <img alt="Equation 6" fo:content-width="4in" src="/files/ftp_upload/52641/52641eq6.jpg" /> où C t (t) est la concentration de contraste MR dans un tissu cible, p v est le volume de plasma sanguin fractionnée, V e est le volume fractionnel extravasculaire extracellulaire, et K est trans constante de transfert de volume. Flux constante de vitesse, k ep, est égale à K trans divisé par v e. Si AIF ne est pas disponible, alors le modèle de région de référence peut être utilisé à la place 18,19. Le modèle de zone de référence est basé sur le modèle Kety écoulement limité 20 et utilise concentration de contraste dans une région de référence pour supprimer la nécessité d&#39;AIF comme suit, <img alt="Equation 7" fo:content-width="5in" src="/files/ftp_upload/52641/52641eq7.jpg" /> où C t, ROI (t), K trans, ROI, et v e, ROI sont la concentration de contraste, le transfert de volume constant, et le volume de extravasculaire extracellulaire fractionnée, respectivement, dans la région d&#39;intérêt (ROI), tandis que C t, RR (t), K trans, RR, et v e, RR sont ceux de la région de référence. Musculaire paravertébrale est souvent choisi comme région de référence, et v e, RR dans murin paravertebral musculaire est supposé être constant à 0,08 21. Nous avons utilisé le modèle de région de référence. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+true+orthotopic+gastric+cancer+murine+model+using+electrocoagulation.">A true orthotopic gastric cancer murine model using electrocoagulation.</a> J Am Coll Surg. 217 (1), 64-70 (2013).</li></ol>

Authors do not have any conflicts of interest to declare.

Nous avons introduit les méthodes détaillées de la modélisation de tumeur pancréatique orthotopique utilisant des souris immunodéficientes, DCE-MRI des tumeurs abdominales chez la souris, et la quantification de ses paramètres cinétiques. Dans la modélisation de tumeur pancréatique orthotopique, il faut prendre soin lors de l&#39;insertion d&#39;une aiguille dans la queue du pancréas. En cas de succès, les cellules seront transférées à la tête du pancréas créer une petite bulle. Lors de l&#39;applicati...

L&#39;objectif global de cette méthode consiste à appliquer un contraste amélioré imagerie par résonance magnétique dynamique (DCE-MRI) pour des xénogreffes de tumeurs pancréatiques orthotopiques chez la souris. DCE-MRI est une méthode non invasive pour évaluer microvascularisation dans un tissu cible en contrôlant la variation de contraste MR sur une certaine période de temps après l&#39;injection. DCE-MRI a été utilisée pour diagnostiquer des tumeurs malignes et pour évaluer la réponse de la tumeur à divers traitements 4.1. Quantitative DCE-MRI a présenté une reproductibilité élevée 5. Pour quantifier les paramètres pharmacocinétiques d&#39;un agent de contraste pour MR dans un tissu cible, toutes les images DCE-RM acquises à différents points dans le temps et T1 carte obtenues avant l&#39;injection de contraste doivent être recalées 6. Toutefois, en raison de mouvements respiratoires et péristaltiques dans la région abdominale, quantitative DCE-MRI a eu une application limitée pour les tumeurs gastro-intestinales. Des modèles de tumeurs du pancréas orthotopique ont été utilisées pour évaluerréponse pancréatique-tumorale après des thérapies biologiques et chimiothérapies 7,8. Des modèles de tumeur orthotopique sont considérés comme supérieurs aux modèles sous-cutanés classiques, étant donné que le micro-environnement dans le site de la tumeur d&#39;origine est réfléchie et de ce fait la réponse tumorale à la thérapie humaine peut être prédit de façon plus précise. Cependant, le pancréas de souris se trouve dans le quadrant supérieur gauche de l&#39;abdomen, de sorte quantitative DCE-MRI de xénogreffes de tumeurs pancréatiques orthotopique chez la souris n&#39;a pas été mis en oeuvre facilement. Nous avons établi un protocole de DCE-MRI des tumeurs abdominales chez la souris en fixant les tumeurs en utilisant une carte en plastique orthogonalement plié pour empêcher le transfert de mouvement à partir de la région de la poitrine 9. La pression appliquée par ce forum a été localisé sur la zone abdominale, et n&#39;a pas donné lieu à des difficultés respiratoires. Une technique image coregistration automatisé a été validée pour DCE-MRI des organes abdominaux dans un mode libre-respiratoire, mais il effectue effectively seulement quand les régions cibles se déplacent lentement et régulièrement 10. La fréquence respiratoire des animaux est variable au cours de l&#39;imagerie, de retenue afin physique dans la région abdominale sera nécessaire de récupérer les paramètres pharmacocinétiques fiables dans les modèles de souris de tumeurs pancréatiques orthotopiques. Nous avons quantifié avec succès les paramètres pharmacocinétiques d&#39;un agent de contraste IRM dans les xénogreffes de tumeurs pancréatiques orthotopiques utilisant la carte en plastique orthogonalement plié dans DCE-MRI 11-13. Ici, nous présentons la procédure détaillée de la modélisation de la tumeur du pancréas orthotopique, DCE-MRI des xénogreffes de tumeurs chez la souris, et la quantification des paramètres pharmacocinétiques.

<table border="0" cellpadding="0" cellspacing="0" width="1275">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:712px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Harlan Laboratories</td>
			<td>BT-9501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td style="width:199px;">Purdue products</td>
			<td style="width:119px;">67618-153-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Prolene sutures</td>
			<td style="width:199px;">Ethicon</td>
			<td style="width:119px;">8720H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">9.4T MR scanner</td>
			<td>Bruker Biospin Corporation</td>
			<td style="width:119px;">BioSpec 94/20 USR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Gadoteridol</td>
			<td>Bracco Diagnostics Inc</td>
			<td style="width:119px;">NDC 0270-1111-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-polyethelene tube</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">#PE-10-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G blunt tip needle</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">89134-194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring and gating system</td>
			<td style="width:199px;">SA instruments, Inc</td>
			<td style="width:119px;">Model 1030</td>
			<td>This is an MR compatiable system to measure resiratory rating and body temperature of small animals at the same time.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump</td>
			<td>New Era Pump Systems, Inc.</td>
			<td>NE-1600</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic contrast enhanced magnetic resonance imaging of an orthotopic pancreatic cancer mouse model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

Des cellules tumorales pancréatiques humaines se développer avec succès dans le pancréas de souris créant une tumeur solide. La figure 1 représente des photographies de (A) un des pancréas normal où la solution de cellules tumorales est injecté, et (B) une souris représentant portant une xénogreffe de tumeur du pancréas orthotopique (MIA PACA-2 ). Tumeur est située dans le quadrant supérieur gauche de l&#39;abdomen, à côté de la rate. Il prend habituellement 2-4 semaines pour que les tu...

Watch this Scientific Journal Video about Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model at JoVE.com

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Contraste dynamique amélioré imagerie par résonance magnétique d&#39;un modèle orthotopique cancer du pancréas de souris

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

dynamic-contrast-enhanced-magnetic-resonance-imaging-an-orthotopic

Research

JoVE Journal

Medicine

15.0K vues.  University of Alabama at Birmingham. The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy. 

Vidéo: Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Imagerie navire Contraste rehaussé utilisant microCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Recherche

Médecine

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 ...

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modale d&#39;imagerie de l&#39;angiogenèse dans un modèle de rat nu des métastases osseuses du cancer du sein par imagerie par résonance magnétique, la tomodensitométrie et volumétrique ultrasons

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Modèle Orthotopique bioluminescente la progression du cancer du pancréas

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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  ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Imagerie simultanée PET / IRM cérébrale Pendant souris hypoxie ischémie

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...