Cette étude est financée en partie par le DOD sein prix IDEA cancer W81XWH-08-1-0583 et le NIH / NCI CA141348-01A1 (DZ) et d&#39;attribution FAMRI scientifique clinique (DS). L&#39;infrastructure d&#39;imagerie est fournie par des animaux sud-ouest de petit programme de recherche d&#39;imagerie soutenu en partie par U24 CA126608 et Simmons Cancer Center (P30 CA142543) et NIH 1S10RR024757-01.

Toutes les procédures d&#39;animaux ont été approuvés par le soin des animaux et du Comité institutionnel Utilisation de l&#39;Université du Texas Southwestern Medical Centre. 1. Dosage in vitro de HIF-1α bioluminescence <ol><li> Matériel et méthodes: <ul><li> Homme de ligne métastatique des cellules de cancer du sein MDA-MB231 transfectées avec un gène rapporteur HIF-1-dépendante roman, 5HRE-ODD-Luc a été générée par le Dr Harada. </li><li> En condition d&#39;hypoxie, l&#39;expression accrue de la dégradation de domaine dépendant de l&#39;oxygène (ODD)-luciférase protéine de fusion est entraîné par 5 exemplaires de l&#39;hypoxie-réponse de l&#39;élément (5HRE). La présence d&#39;ODD provoque la dégradation de ODD-Luc protéines résultant de très faible activité de fond luciférase dans des conditions de normoxie. Par conséquent, ce nouveau système peut être utilisé pour détecter les régions hypoxiques dans une tumeur par imagerie en temps réel. La construction de ce vecteur d&#39;expression 5HRE-ODD-Luc a été rapporté par Harada et al 1,2. </li></ul></li><li> Cellules Culture sous normoxie ou d&#39;hypoxie: <ul><li> Maintenir le recombinant cellules MDA-MB231 de 10% de sérum de veau fœtal (SVF)-milieu DMEM contenant 1% de glutamine, des antibiotiques de 400 pg / ml de G418 et 1% de pénicilline / streptomycine. </li><li> Pour le dosage in vitro de HIF-1α bioluminescence, la plaque 3 x 10 5 cellules MDA-MB231 exprimant 5HRE-ODD-Luc vecteur dans chaque puits de deux six plat bien. </li><li> Laisser les cellules à attacher au mur plat après une nuit d&#39;incubation et ensuite transférer un plat dans une chambre d&#39;hypoxie (Billups-Rothenberg, Inc Del Mar, CA) pour les études de l&#39;hypoxie, tandis que l&#39;autre plat maintenir en condition normoxique (21% O 2). </li><li> Remontez la chambre et la chambre à gaz avec 0,1% d&#39;O 2 en connectant le tube orifice d&#39;entrée avec une bouteille de gaz. Remarque: Les deux brides d&#39;entrée et de sortie du port doit être ouvert pendant cette procédure. </li><li> Déconnecter la source de gaz après la chambre a été purgé et chambre d&#39;étanchéité par la fermeture de pinces en plastique. </li><li> Placez la chambre dans un incubateur à 37 ° C avec 5% de CO 2. Note: La chambre doit être humidifié pour éviter l&#39;évaporation excessive des cultures. Ceci peut être accompli en plaçant 10 - 20 ml d&#39;eau stérile dans la chambre. </li></ul></li><li> Dosage de bioluminescence: <ul><li> Après 24 heures d&#39;incubation, éliminer le milieu et laver les cellules rapidement avec PBS glacé (2X). </li><li> Ajouter 1 ml de PBS froid avec 100 pi de luciférine (Biotechnologie Or, St Louis, MO). </li><li> Acquérir BL imagerie (système Spectrum IVIS, Caliper Life Sciences, Hopkinton, MA) avec des durées d&#39;exposition différentes (1, 30, 60, 180 s). </li><li> Mesurer l&#39;intensité lumineuse dans chaque puits en utilisant le logiciel image vivante (Caliper Life Sciences). </li></ul></li></ol> 2. Création d&#39;un modèle de cancer du sein métastase cérébrale <ol><li> Préparation des cellules MDA-MB231/5HRE-ODD-luc <ul><li> Récupérer et la culture des cellules dans un milieu de DEME MDA-MB231/5HRE-ODD-luc contenant 10% de FBS, 1 glutamine% et 1% de pénicilline / streptomycine. </li><li> Remplacer moyenne tous les 2-3 jours. Tripsinize et laver les cellules quand elles atteignent 80% de confluence. </li><li> Compter le nombre approprié de cellules et de les remettre en suspension dans un milieu de DEME sans sérum de Matrigel 25% (BD Biosciences, San Jose, CA) avec une concentration finale de 10 5 cellules dans le volume 4 pl. </li><li> Cellules de lieu sur la glace avant l&#39;injection intracrânienne. </li></ul></li><li> L&#39;implantation chirurgicale <ul><li> Souris nude femelles (BALB / c nu / nu; National Cancer Institute, Bethesda, MD) à 4-6 semaines d&#39;âge sont utilisés dans cette étude. </li><li> Anesthetize (3% d&#39;isoflurane / O2 dans une chambre d&#39;induction; isoflurane de Baxter International Inc, Deerfield, IL, Etats-Unis) et de maintenir les animaux avec de l&#39;isoflurane (1%) dans de l&#39;oxygène (1 dm 3 / min) pendant l&#39;intervention chirurgicale 3. </li><li> La peau doit être pariétal droit préparé avec la bétadine et ensuite 70% d&#39;alcool avant l&#39;incision. </li><li> En utilisant une perceuse à haute vitesse, sans bavures un trou de 1 mm dans l&#39;hémisphère droit du crâne, 1mm antérieur à la suture coronale et 2 mm latéralement à la suture sagittale. </li><li> Draw 4 mélange de cellules ul (10 5 cellules) en utilisant une seringue Hamilton de 10 ul (Hamilton Company, Reno, NV). Injecter des cellules directement dans le droit caudale diencéphale, 1,5 mm sous la dure-mère avec un sur-mesure 32G Hamilton aiguille. Gardez l&#39;aiguille en place pendant environ 30 s avant le retrait. Utilisation d&#39;une aiguille 32G fines minimise les dommages tissulaires. </li><li> Remplissez le trou de trépan avec de la cire d&#39;os et de fermer le cuir chevelu avec des sutures résorbables. </li><li> Préparer la région incision avec 70% d&#39;alcool. </li><li> Appliquer la buprénorphine analgésie toutes les 12 heures pendant deux jours. </li></ul></li></ol> 3. Dans l&#39;imagerie par bioluminescence in vivo de tumeurs dynamiques hypoxie dans les métastases cérébrales du cancer du sein <ul><li> Initier l&#39;imagerie par bioluminescence longitudinale (système Spectrum IVIS) deux semaines après l&#39;implantation intracrânienne et de répéter une fois par semaine pendant 8-10 Semaines. </li><li> Anesthetize trois souris à un moment (3% d&#39;isoflurane / O 2 dans une chambre d&#39;induction) </li><li> Administrer une solution de D-luciférine (120 mg / kg dans du PBS dans un volume total de 80 ul; Biotechnologie or) sous-cutanée dans la région du cou de chaque souris comme décrit en détail précédemment 4. </li></ul> D-luciférine est non-toxique et a été montré pour être capable de pénétrer intacte barrière hémato-encéphalique (BHE) et des membranes cellulaires 3,5. <ul><li> Placez la souris 3 dans la chambre de l&#39;imagerie et maintenue avec l&#39;isoflurane (1%) dans de l&#39;oxygène (1 dm 3 / min) pendant l&#39;imagerie. </li><li> Cinq minutes après l&#39;injection luciférine, acquérir BL imagerie avec un tableau de temps d&#39;exposition différents (1, 30, 60, 180 s). </li></ul> Nos observations montrent que la durée d&#39;émission de lumière est maximale à environ 5 minutes après l&#39;administration sous-cutanée de luciférine dans la région de 3,4 cou. <ul><li> Analyser les données avec le logiciel d&#39;imagerie habitable (Caliper Life Sciences) en utilisant le nombre absolu de photons (photons / s) dans une région d&#39;intérêt (ROI), tracées manuellement de décrire le signal BLI du cerveau. </li><li> Bien sûr la courbe Tracer le temps des comptages de photons pour indiquer la dynamique hypoxie tumorale. </li><li> Immédiatement après la dernière BLI, administrer pimonidazole, le marqueur de l&#39;hypoxie et une heure plus tard, le sacrifice de la souris et de disséquer le cerveau. Intégrer le cerveau entier dans la température de coupe optimale (PTOM) à moyen et gel -80 ° C au congélateur. Après coloration histologique violet de crésyl et la coloration immunohistochimique contre la luciférase, un marqueur d&#39;hypoxie pimonidazole, et HIF-1α effectués pour valider les observations d&#39;imagerie. </li></ul> 4. Les résultats représentatifs: Comme le montre la figure 1, sensiblement plus élevé de signal BLI a été observée après incubation des cellules transfectées dans la chambre hypoxique (1% O 2) pendant 24 heures. Émission de lumière faible a été observée à partir des cellules de contrôle en vertu normoxie. Cela peut résulter de la population de cellules surpeuplées après 24 h de culture de 3 x 10 5 cellules, ce qui induit un signal de stress pour les cellules pour surexprimer HIF-1α. Néanmoins, dans les résultats in vivo BLI ont été validés par la coloration immunohistochimique montrant une colocalisation entre hypoxie tumorale et expression de la luciférase. Un tableau automatisé des temps d&#39;exposition permet d&#39;acquisitions continue d&#39;une série d&#39;images, ce qui facilite la capture d&#39;un signal faible avec le temps d&#39;exposition plus long, et des signaux forts en utilisant un temps plus court, sans saturer le CCD. <img alt="Figure 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> Figure 1 test in vitro avec HIF-1α bioluminescence Rangée du haut:. 3 x 10 5 cellules MDA-MB231/5HRE-ODD-luc incubées dans chaque puit d&#39;une 6-bien-plat dans une chambre d&#39;hypoxie (0,1% O 2) pour 24 heure avant le milieu a été enlevé, lavé et remplacé par 1 ml de PBS. Immédiatement après 100 luciférine ul a été ajouté dans chaque puits, BLI a été acquise avec un tableau de durées d&#39;exposition (1, 30, 60, 180 s). Forte luminescence a été observée à partir de puits représentatifs Rangée du bas:. Comme contrôle, 3 x 10 5 cellules incubées sous normoxie (21% O 2) émis faible lumière. <img alt="Figure 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> Figure 2 Dans l&#39;imagerie par bioluminescence in vivo de la dynamique de l&#39;hypoxie tumorale. A) Un signal faible lumière du côté droit du cerveau de souris a été visualisée 5 semaines après l&#39;implantation intracrânienne d&#39;MDA-MB231/5HRE-ODD-luc cellules cancéreuses du sein. Augmentation du signal optique a été observée au cours supplémentaire de 6 semaines, indiquant l&#39;hypoxie tumorale accrue. B) L&#39;intrigue a montré la courbe bien sûr le temps d&#39;quantitative compte de photons de signal lumineux. <img alt="Figure 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> Figure 3 Colocaliztion de la luciférase et l&#39;hypoxie détecté par immunohistochimie-chimiques de coloration. Un cerveau de souris congelées portant une métastase d&#39;un cancer du sein MDA-MB231/5HRE-ODD-luc embarqué dans de l&#39;OCT a été sectionnée. A l&#39;article 10 um immunocolorés avec marqueur d&#39;hypoxie, pimonidazole, a révélé une hétérogénéité intratumorale de l&#39;hypoxie, qui a été trouvé pour corréler spatialement avec expression de la luciférase détectés par les anti-luciférase coloration. La barre d&#39;échelle, 100 um.

<ol>
	<li>Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+hypoxia-response+enhancers+and+an+oxygen-dependent+proteolytic+motif+enables+real-time+imaging+of+absolute+HIF-1+activity+in+tumor+xenografts.">The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.</a> Biochem Biophys Res Commun. 360, 791-796 (2007).</li><li>Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+HIF-1+imaging+for+determining+optimal+timing+of+combining+bevacizumab+and+radiotherapy.">Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy.</a> Int J Radiat Oncol Biol Phys. 75, 463-467 (2009).</li><li>Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, e8051-e8051 (2009).</li><li>Contero, A., Richer, E., Gondim, A., Mason, R. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+effects+of+HIF-1+blockade+on+tumor+radiosensitivity.">Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity.</a> Cancer Cell. 8, 99-110 (2005).</li><li>Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dynamics+and+distinct+functions+of+hypoxia+in+primary+tumor+growth+and+organotropic+metastasis+of+breast+cancer.">In vivo dynamics and distinct functions of hypoxia in primary tumor growth and organotropic metastasis of breast cancer.</a> Cancer Res. 70, 3905-3914 (2010).</li><li>Zhao, D., Chang, C. H., Kim, J. G., Liu, H., Mason, R. 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Métastases cérébrales du cancer du sein survient dans 30% des patients atteints de cancer du sein au stade IV. Elle est associée à une morbidité et une mortalité élevées et a une survie médiane de 13 mois 6. Il est nécessaire d&#39;avoir des modèles animaux appropriés pour imiter cette maladie dévastatrice sur le plan clinique, afin de faciliter notre compréhension de son initiation et la progression intracrânienne ainsi que des profils physiopathologiques. Ici, nous avons développé un cance...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> D-luciférine </td><td> Or la biotechnologie </td><td> L-123 </td><td> 120 mg / kg dans du PBS dans un volume total de 80 ul pour l&#39;étude in vivo </td></tr><tr><td> L&#39;isoflurane </td><td> Baxter International Inc </td><td> 1001936060 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td></td></tr><tr><td> Seringue Hamilton </td><td> Hamilton Company </td><td> 1701 </td><td></td></tr><tr><td> 32G Hamilton aiguille </td><td> Hamilton Company </td><td> 7803-04 </td><td></td></tr><tr><td> Chambre de l&#39;hypoxie </td><td> Billups-Rothenberg, Inc </td><td> MIC-101 </td><td></td></tr><tr><td> Système d&#39;imagerie par bioluminescence </td><td> Caliper Life Sciences </td><td> Système Spectrum IVIS </td><td></td></tr><tr><td> G418 </td><td> Fisher Scientific </td><td> SV3006901 </td><td></td></tr></tbody></table>

in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model

Il est bien reconnu que l&#39;hypoxie tumorale joue un rôle important dans la promotion de la progression maligne et affectant négativement la réponse thérapeutique. Il ya peu de connaissances sur in situ, in vivo, l&#39;hypoxie tumorale pendant le développement intra-crânienne des tumeurs cérébrales malignes en raison du manque de moyens efficaces de le suivre dans ces tumeurs profondes orthotopique. L&#39;imagerie par bioluminescence (BLI), basé sur la détection de la lumière émise par les cellules vivantes exprimant un gène de la luciférase, a été rapidement adopté pour recherche sur le cancer, en particulier, pour évaluer la croissance des tumeurs ou des changements de taille de la tumeur en réponse au traitement dans les études animales précliniques. Par ailleurs, en exprimant un gène rapporteur sous le contrôle d&#39;une séquence promotrice, l&#39;expression de gènes spécifiques peuvent être surveillés de façon non invasive par BLI. Sous le stress hypoxique, les réponses de signalisation sont médiés principalement par les inductible factor-1α hypoxie (HIF-1α) pour conduire la transcription des gènes différents. Par conséquent, nous avons utilisé une construction de HIF-1α journaliste, 5HRE-ODD-Luc, transfectées de façon stable dans le cancer du sein humain MDA-MB231 cellules (MDA-MB231/5HRE-ODD-luc). Dosage in vitro de HIF-1α bioluminescence est effectuée par incuber les cellules transfectées dans une chambre hypoxique (0,1% O 2) pendant 24 heures avant le BLI, tandis que les cellules en normoxie (21% O 2) servir de contrôle. Significativement plus élevé de flux de photons observés pour les cellules en hypoxie suggère une augmentation de HIF-1α se liant à son promoteur (éléments HRE), comparativement à ceux qui en normoxie. Les cellules sont injectées directement dans le cerveau de la souris pour établir un cancer du sein, métastases cérébrales modèle. Dans l&#39;imagerie par bioluminescence in vivo de la dynamique de l&#39;hypoxie tumorale est initié deux semaines après l&#39;implantation et répétée une fois par semaine. BLI révèle l&#39;augmentation des signaux lumineux du cerveau comme la progression tumorale, indiquant augmenté hypoxie tumorale intracrânienne. Des études histologiques et immunohistochimiques sont utilisés pour confirmer les résultats de l&#39;imagerie in vivo. Ici, nous allons introduire des approches d&#39;analyse in vitro de HIF-1α bioluminescence, l&#39;établissement chirurgicale d&#39;une métastase cérébrale du cancer du sein chez une souris nude et l&#39;application de l&#39;imagerie par bioluminescence in vivo pour contrôler l&#39;hypoxie tumorale intracrânienne.

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

L&#39;imagerie par bioluminescence des inducible factor-1α activité hypoxie est appliqué pour surveiller le développement intracrânienne hypoxie tumorale dans un modèle de cancer du sein métastatique cerveau de souris.

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 ...

in-vivo-bioluminescence-imaging-tumor-hypoxia-dynamics-breast-cancer

Research

JoVE Journal

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

19.5K vues.  University of Texas Southwestern Medical Center. L'imagerie par bioluminescence des inducible factor-1α activité hypoxie est appliqué pour surveiller le développement intracrânienne hypoxie tumorale dans un modèle de cancer du sein métastatique cerveau de souris.

Vidéo: In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

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

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 ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Implantation stéréotaxique intracrânienne et<em&gt; In vivo</em&gt; Imagerie bioluminescente de xénogreffes tumorales dans un système de modèle de souris de glioblastome multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Vivez l&#39;imagerie des réponses de drogues dans le microenvironnement des tumeurs dans les modèles murins de cancer du sein

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Imagerie time-lapse de primaires prénéoplasiques cellules épithéliales mammaires issues de modèles de souris génétiquement modifiées de cancer du sein

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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 ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Ultrasound Imaging guidée injection intracardiaque de développer un modèle de souris de cancer du sein métastases cérébrales Suivi par IRM longitudinale

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

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 ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo L&#39;imagerie optique de tumeurs cérébrales et l&#39;arthrite Utilisation fluorescent SAPC-DOPS nanovésicules

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...