We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

Déclaration éthique Toutes les études sur les animaux ont été approuvés par le Comité institutionnel de protection des animaux et l&#39;utilisation (IACUC) du Hunter College de la City University de New York. Remarque: Huit souris BALB / c ont été utilisées dans ce mode opératoire expérimental. Cinq souris BALB / c ont été implantés avec 5 x 10 6 cellules de carcinome hépatocellulaire BNL 1ME A.7R.1 souris pour produire les tumeurs primaires. Trois souris BALB / c avec aucun implantations ont été utilisés comme témoins. BNL 1ME A.7R.1 est une lignée cellulaire de carcinome hépatocellulaire murin qui a été dérivée par transformation chimique de la non-tumorigène lignée cellulaire d&#39;hépatocytes murins BNL CL.2. BNL 1ME A.7R.1 a été obtenu à partir de la American Type Culture Collection (ATCC). 1. Pré-chirurgie <ol><li> Veiller à ce que les souris pour toutes les chirurgies de survie sont en bonne santé. Pour assurer que les souris sont en bonne santé, d&#39;inspecter des signes de déshydratation par tentes de la peau. Ne pas utiliser la souris avec aucune preuve de maladie ou distress dans ces expériences. </li><li> Peser souris pour vous assurer qu&#39;ils ne sont pas d&#39;insuffisance pondérale. Assurez-vous que le poids est d&#39;au moins 16-20 grammes. </li><li> Préparer 5 × 10 6 BNL 1ME A.7R.1 cellules HCC de la souris pour l&#39;implantation par souris: <ol><li> Aspirer le milieu de 10 ml 60% - plat de 70% de la culture de cellules confluentes de cellules HCC A.7R.1 de souris BNL 1ME. Laver deux fois avec 5 ml de PBS. Ajouter 2 ml de trypsine à l&#39;antenne et incuber à 37 ° C dans un incubateur humidifié avec de l&#39;air et 5% de CO 2 pendant 5 min. </li><li> Par la suite, pipette 10 ul trypsinisées cellules dans un hémocytomètre manuel pour le comptage des cellules au microscope à un grossissement de 10X. Comptez le nombre total de cellules présentes dans les quatre grands carrés de coin et multiplier le facteur de dilution par le nombre total de cellules. Ensuite, on divise par le nombre de carrés comptés et multiplier par 6 10 pour la concentration de cellules (cellules par ml). </li><li> En utilisant le calcul, de recueillir 5 millions de cellules par souris dans stérile1,5 ml microtubes. Étiqueter 1,5 ml microtubes. </li></ol></li><li> Remettre en suspension les 5 × 10 6 cellules BNL 1ME A.7R.1 HCC souris dans 100 ul de solution saline tamponnée au phosphate (PBS). Gardez ces cellules sur de la glace jusqu&#39;à l&#39;implantation en chirurgie. </li><li> Effectuer des activités nécessaires à la préparation de la souris pour la chirurgie, tels que induction de l&#39;anesthésie et les cheveux écrêtage loin de l&#39;équipement et du matériel stérile pour éviter la contamination. </li><li> Induire une anesthésie générale chez les souris par inhalation d&#39;isoflurane l&#39;aide d&#39;un vaporisateur isoflurane avec l&#39;oxygène à un débit constant de concentration de 1 litre par minute (env. 2,1%). Remarque: Le système est autonome, et comprend un vaporisateur de précision, une alimentation en oxygène et un cartouche de charbon actif qui est relié à un circuit de respiration nécessaire pour le gaz / anesthésie balayage. </li><li> Assurez-vous que l&#39;anesthésie est suffisante pour empêcher l&#39;animal de subir la douleur pendant la chirurgie. Effectuer toe-pincement et d&#39;observer l&#39;animal en cas de non-responsiveness pour confirmer l&#39;anesthésie. </li><li> A cette époque, passer la ligne de soupape, joint à la boîte et ouverte sur le cône de nez. </li><li> Dès que la souris est anesthésiée, appliquer une petite quantité d&#39;une pommade ophtalmique à la cornée comme une mesure préventive contre le dessèchement et les dommages. </li><li> Par la suite, déplacer l&#39;animal à la zone de préparation, avec le cône de nez correctement placé et connecté (pour entretien de l&#39;anesthésie). Préparer le site de l&#39;incision chirurgicale pour la chirurgie par épilation à cette zone de préparation. </li><li> Assurez-vous que le domaine de la chirurgie est à un emplacement libre de toute autre activité. </li><li> Assurez-vous que la surface de travail est robuste et convenablement désinfectés, non sali et avant la chirurgie sans encombrement. Placez un morceau de linge stérile ou plateau chirurgical sur elle. </li><li> Assurez-vous que le champ opératoire est seulement disponible pour les personnes qui effectuent la chirurgie. Assurez-vous que la pièce chirurgicale est nettoyé et balayé avec un nettoyant approprié et pulvérisé avec un disinfe appropriéectant. </li><li> Assurez-vous qu&#39;il est également positionné loin des portes et fenêtres que les courants d&#39;air peut se propager la poussière conduisant à la contamination du site. </li><li> Effectuer la chirurgie de survie avec des instruments stériles. Soit faire tremper les instruments dans un désinfectant selon la recommandation du fabricant ou un autoclave. Utilisez fournitures stériles de base qui sont prêts à l&#39;emploi. </li><li> Instruments distincts selon leur fonction pour aider à prévenir la contamination des zones stériles. </li><li> Raser environ 1 pouce autour du site d&#39;incision (incision médiane) pour accéder à la foie de l&#39;animal. Enlever les poils de l&#39;animal en dehors du site de l&#39;intervention chirurgicale. </li><li> Après le rasage, frotter la zone avec un désinfectant savonneuse (bétadine chirurgicale de gommage) et laver l&#39;excès de désinfectant loin avec une solution stérile PBS. Effectuez cette opération plusieurs fois, avec le processus de gommage toute durée d&#39;environ 5 min. Retirer et jeté les gants usagés. </li><li> Ensuite, changer de vêtements de protection comme Surgicrobe al, haut de gommage et le masque de visage. Ensuite, désinfecter les mains avec une solution appropriée de gommage avant de mettre des gants chirurgicaux. Note: Ceci est une étape nécessaire pour empêcher une chute et de contaminer le site chirurgical avec des contaminants potentiels. </li></ol> 2. Chirurgie <ol><li> Fournir une source de chaleur externe pour plus de chaleur pendant toute la durée de l&#39;anesthésie. L&#39;animal peut éprouver une certaine hypothermie, car l&#39;anesthésie provoque des perturbations de thermorégulation; par conséquent, de maintenir la température du corps. </li><li> Posez la souris sur son dos tout au long de la chirurgie. </li><li> Fixer les membres antérieurs, membres postérieurs et la queue de la souris à la table d&#39;opération avec des morceaux de rubans chirurgicaux. </li><li> Faire une incision droite immédiatement inférieure à la xyphisternum. </li><li> Utiliser un écarteur chirurgical autostatique stérile pour exposer le foie. </li><li> Avec une seringue ayant une aiguille de 20G, lentement et avec précaution, injecter 100 ml de PBS contenant 5 × 10 6 BNL 1ME A.7R.1cellules dans le lobe droit du foie de souris HCC. </li><li> Après avoir retiré l&#39;aiguille, appliquer un tampon de coton stérile pendant au moins 1 min d&#39;arrêter toute hémorragie potentielle raison de la forte vascularisation du foie. </li><li> Fermez le tissu sous-cutané avec une suture absorbable suivie d&#39;une fermeture de la peau en utilisant des agrafes chirurgicales. Par la suite, nettoyer la plaie avec de la bétadine nouveau gommage. Remarque: Cela permettra d&#39;assurer la fermeture de la plaie chirurgicale. </li><li> Donner en sous-cutané 100 pi de solution saline normale. </li></ol> 3. post-chirurgie <ol><li> Immédiatement après la chirurgie, transférer la souris dans une cage propre avec une carte d&#39;identité post-chirurgie pour informer les gardiens de la condition de la souris. Fournir de la nourriture et de l&#39;eau. Remarque: Dans notre expérience, une souris qui vient de subir une intervention chirurgicale sera meilleur tarif si logé seul avec la nourriture humide et de l&#39;eau pour la période de 24 heures après la chirurgie initiale. </li><li> Gardez l&#39;animal au chaud avec une lampe de chaleur qui génère de la chaleur dépassant pas 38 ºC. USE thermomètre pour mesurer la température jusqu&#39;à ce que la souris est entièrement récupéré. </li><li> Maison de l&#39;animal dans une cage de préférence avec de la literie de papier pour assurer le confort. </li><li> Après la récupération initiale, surveiller l&#39;animal pour des signes d&#39;inconfort, saignement, douleur et la détresse. Donnez buprénorphine (0,05 -0,1 mg / kg) pour soulager la douleur. Surveiller de près l&#39;activité, le comportement et l&#39;apparence, ainsi que l&#39;eau et la consommation alimentaire pendant environ 2 heures. Si ambulatoires, déplacer l&#39;animal revenir à la zone de logement régulière normale de souris et continuer à surveiller à intervalles de 6-12 h pour 24 h post-chirurgie. Effectuer un contrôle de poids, si désiré. </li><li> Toutefois, si l&#39;animal a des problèmes récupérer, prendre des mesures de soins de soutien appropriés. Si les souris sont incapables de récupérer, euthanasier humainement souris par asphyxie au CO2 et dislocation cervicale. Suivez les directives institutionnels approuvés pour l&#39;euthanasie. </li><li> Post-chirurgie, vérifier les preuves cliniques de HCC qui est habituellement Observable réduction que significative du score d&#39;état corporel. Remarque: À ce stade, une tumeur primaire a probablement développé dans le foie et les dépôts métastatiques ont probablement développé dans les poumons. </li><li> A ce point, sacrifier toutes les souris (expérimentales et de contrôle) impliqués dans l&#39;expérience par euthanasie. Soumettre les souris à une asphyxie dioxyde de carbone: libérer lentement du dioxyde de carbone dans la chambre sur une période de 5 à 10 min. Cela entraînera un arrêt respiratoire. </li></ol>

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The authors declare that they have no competing financial interests.

Dans cet article, une description approfondie de la méthode est donnée qui a été récemment rapporté par Ogunwobi et ses collègues de mise en place réussie d&#39;un modèle de souris syngénique orthotopique du HCC métastases (figure 2) 3. Le taux de participation des tumeurs de cette procédure est généralement élevé. Nous avons déjà observé un taux de participation de la tumeur de 100% 3. Cependant, le taux d&#39;utilisation peut être variable en fonction de la co...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table border="0">
	<tbody>
		<tr>
			<td>Micro Dissecting Tweezer&#160;</td>
			<td>Roboz</td>
			<td>(RS-5040)</td>
			<td>Tip .20 x .12mm</td>
		</tr>
		<tr>
			<td>Graefe Micro Dissecting Forceps, serrated curved tip</td>
			<td>Roboz</td>
			<td>(RS-5111)</td>
			<td>1 X 2 teeth, curved tip width 0.6mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6501)</td>
			<td>Blunt 3 X 3 prongs, depth 4mm, spread 25mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6503)</td>
			<td>Blunt, 3 X 3 prongs, depth 4mm, spread 19mm</td>
		</tr>
		<tr>
			<td>Jameson Caliper (Measures tumors)</td>
			<td>Roboz</td>
			<td>(RS-6466)</td>
			<td>80mm/3 inch scale, chrome plated&#160;</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td>
			<td>Roboz</td>
			<td>(RS-5852)</td>
			<td>23mm blades, length 4 inches, flat shanks and large rings</td>
		</tr>
		<tr>
			<td>Scalpel with blades, for delicate dissecting procedures</td>
			<td>Roboz</td>
			<td>(RS-9861-36)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle&#160;</td>
			<td>Roboz</td>
			<td>(RS-9884)</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>Littauer Stitch Sissors</td>
			<td>Roboz</td>
			<td>(RS-7074)</td>
			<td>Length 4.5 inches</td>
		</tr>
		<tr>
			<td>Brown needle holder, for easy suture tying</td>
			<td>Roboz</td>
			<td>(RS-7960)</td>
			<td>Convex jaw, fine serrations</td>
		</tr>
		<tr>
			<td>Reflex 7MM wound clips with reflex 7 clip applier</td>
			<td>Roboz</td>
			<td>(RS-9262)</td>
			<td>safe, secure alternative method of wound closure</td>
		</tr>
		<tr>
			<td>Instrument tray and lid</td>
			<td>Roboz</td>
			<td>RT-1350S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-clipper with detachable blade</td>
			<td>Roboz</td>
			<td>(RS-5903)</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td>
			<td>Roboz</td>
			<td>Ds-400, Ds-401, DS-501</td>
			<td>For fast decontamination of micro-dissecting instruments.&#160;&#160; Instruments decontaminate within 15 seconds.&#160;</td>
		</tr>
		<tr>
			<td>Microinjection needles</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
	</tbody>
</table>

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.

Les foies de souris Balb / c ont été implantés avec 5 × 10 6 cellules HCC de la souris BNL 1ME A.7R.1. Des preuves cliniques du développement HCC était observable 63 jours post-chirurgie. Par conséquent, les souris ont été euthanasiés. L&#39;autopsie a été effectuée sur des souris euthanasiées. Les poumons et le foie ont été réséquées et un examen macroscopique attention a été réalisée pour identifier les tumeurs macroscopiques. Dans une souris, une tumeur superficielle a été observé...

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

La dissémination métastatique du cancer est la principale cause de décès liés au cancer. Nous fournissons une description en profondeur de notre méthodologie de chirurgie de survie pour établir un système orthotopique &quot;patient comme&quot; modèle de souris syngénique pour étudier les mécanismes de métastases dans les tumeurs d&#39;organes solides.

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 ...

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

12.1K vues.  Hunter College of the City University of New York.  La dissémination métastatique du cancer est la principale cause de décès liés au cancer. Nous fournissons une description en profondeur de notre méthodologie de chirurgie de survie pour établir un système orthotopique "patient comme" modèle de souris syngénique pour étudier les mécanismes de métastases dans les tumeurs d'organes solides. 

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

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 ...

Recherche

Médecine

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 ...

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 ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Génération de sous-cutanée et intra-hépatique droits de carcinome hépatocellulaire xénogreffes chez la souris immunodéficientes

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

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  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Initiation de carcinome du sein métastatique en ciblant de l&#39;épithélium canalaire avec adénovirus Cre: un modèle de souris transgénique roman du cancer du sein

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

La<em&gt; En Ovo</em&gt; Chick chorioallantoïque membrane (CAM) Assay comme un modèle de xénogreffe efficace de carcinome hépatocellulaire

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

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.

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 ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

Orthotopique Implantation et périphérique des cellules immunitaires surveillance dans le modèle II-45 Syngénique Rat mésothéliome

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...