Nous sommes reconnaissants à MM. Diane Palmieri et Patricia Steeg de NCI pour nous fournir des cellules MDA-MB231Br. Nous remercions le Dr Ralph Mason, M. Jason Reneau et Mme Ramona Lopes pour le support technique et collégiale. Ce travail a été financé en partie par l&#39;IDEA DOD Prix W81XWH-08-1-0583 et W81XWH-12-1-0317. Expériences d&#39;IRM ont été réalisées dans le Centre d&#39;imagerie de recherche avancée, une installation NIH BTRP # P41-RR02584, et l&#39;injection intracardiaque guidée par échographie a été réalisée avec VisualSonics Vevo 770 sous 1S10RR02564801.

Toutes les procédures d&#39;animaux ont été approuvés par le Comité institutionnel de protection et d&#39;utilisation des animaux de l&#39;Université du Texas Southwestern Centre médical. Une. Préparation des cellules MDA-MB231/Br-GFP <ol><li> Récupérer et culture des cellules MDA-MB231/Br-GFP (aimablement fournies par les Drs. Palmieri et Steeg, NCI) dans du milieu DMEM contenant 10% de FBS, 1% de glutamine et 1% de pénicilline / streptomycine. </li><li> Observer l&#39;état des cellules et la couleur du support, et de le remplacer milieu tous les 2-3 jours. </li><li> Trypsiniser et recueillir les cellules lorsque 80% de confluence est atteinte, comme indiqué dans les étapes 1.3.1-1.3.5: <ol><li> Retirer l&#39;ancien milieu complètement et ajouter 5 ml de PBS (1x) pour laver en douceur les cellules. Retirez le PBS dans le plat. </li><li> Ajouter 1,5 ml de trypsine dans le plat, incliner le plat doucement pour assurer toutes les cellules sont couverts par la trypsine. Mettre le plat de nouveau à l&#39;incubateur de cellules et le maintenir à 37 ° C pendant 1 min. </li><li> Sortez le plat du incubateur et observerles cellules sous le microscope optique pour s&#39;assurer que les cellules se détachent de l&#39;antenne. </li><li> Ajouter 3 ml de milieu pour arrêter l&#39;effet de la trypsine. Recueillir le mélange de cellules dans un tube de centrifugation. Centrifuger le mélange à 2000 tpm pendant 5 min. </li><li> Retirer soigneusement le milieu et remettre en suspension les cellules dans un milieu exempt de 5 ml de sérum jusqu&#39;à homogénéité. </li></ol></li><li> Compter le nombre approprié de cellules et remettre en suspension dans du milieu DMEM sans sérum avec une concentration finale de 1,75 x 10 5 cellules dans un volume de 100 ul. <ol><li> Prenez mélange de cellules pi 50 et ajouter 50 ul bleu Trypan. Après le mélange, prendre 10 mélange de pi et soigneusement ajouter à la diapositive de comptage de cellules. Utilisez plusieurs tests afin de s&#39;assurer précise le nombre de cellules estimation. </li><li> Coller à la lame de comptage des cellules, une extrémité à la fois et le compteur de cellules commence à compter automatiquement. Prendre la moyenne de tous les résultats de comptage et calculer le nombre total de cellules. </li><li> Centrifuger les cellules et remettre en suspension à nouveau dans lesle volume approprié de milieu sans sérum pour aboutir à la concentration finale de 1,75 x 10 5 cellules/100 ul volume. </li></ol></li><li> La place des cellules sur la glace avant injection intracardiaque. </li></ol> 2. Ultrasound Imaging guidée injection intracardiaque <ol><li> Utilisez la souris nu féminin (BALB / c nu / nu) entre 6-8 semaines. </li><li> Connectez-vous au système d&#39;imagerie. Initialiser le transducteur 704 (40 MHz). </li><li> Lancer une nouvelle étude et remplir les informations. </li><li> Anesthésier (3% isoflurane/100% O 2 dans une chambre d&#39;induction) et de maintenir les animaux avec de l&#39;isoflurane (2%) à 100% de O 2 (1 dm 3 / min) pendant l&#39;ensemble de la procédure par l&#39;intermédiaire d&#39;un cône de nez. L&#39;anesthésie est confirmé en l&#39;absence de réflexe de retrait est observé avec pincement de l&#39;orteil. </li><li> Régler la température de la table de formation d&#39;image à 37 ° C. Collez la souris anesthésiée à la table d&#39;imagerie chauffée en position couchée. </li><li> Gardez le gel à ultrasons à 37 ° C avant l&#39;imvieillissement. Appliquer le gel sur la poitrine de la souris. </li><li> Montez la sonde dans le support. Abaissez le transducteur jusqu&#39;à la profondeur d&#39;imagerie désirée est atteinte. Déplacez la scène jusqu&#39;à ce que le ventricule gauche est identifié avec l&#39;aorte ascendante comme le point de repère (figure 1A). Verrouiller le stade où une vision claire de ventricule gauche est visualisée. </li><li> Tracer 100 ul de mélange de cellules dans une seringue de 1 ml avec une aiguille 22 G. Placer et fixer la seringue sur le montage de seringue. </li><li> Déplacez la seringue vers l&#39;avant vers la poitrine de la souris et déplacer avec précaution côté à l&#39;autre jusqu&#39;à ce que la pointe de l&#39;aiguille est dans le champ de vision d&#39;imagerie (avant d&#39;entrer dans la souris). </li><li> Réglez la hauteur de l&#39;aiguille et de l&#39;angle de viser vers le bas le ventricule gauche. </li><li> Pénétrer l&#39;aiguille de la seringue dans l&#39;espace intercostale rapidement à travers les couches de la peau et des muscles dans le ventricule gauche, sous la direction de l&#39;imagerie par ultrasons. Une indication de l&#39;insertion réussie de l&#39;aiguille dans le ventricule gauchecle est le reflux de sang artériel frais (de couleur rose à la différence de sang veineux rouge sombre) dans la seringue, </li><li> Injecter le mélange de cellules lentement (figure 1B). </li><li> À la fin, retirer l&#39;aiguille, soulever le transducteur, nettoyer le gel à ultrasons avec de la gaze humide et enlever le ruban. </li><li> Préparer une cage propre avec un tampon préchauffé. </li><li> Placez l&#39;animal sur le pavé et observer l&#39;animal jusqu&#39;à guérison complète. </li><li> Surveiller le comportement de l&#39;animal toutes les 24 heures pendant deux jours. </li><li> Conditions générales et les signes neurologiques de complications chez les souris expérimentales sont régulièrement contrôlés. </li></ol> 3. Surveillance IRM de la tumeur intracrânienne développement <ol><li> Utilisez un aimant Tesla 9.4 pour suivre le développement intracrânienne des métastases cérébrales. </li><li> Initier IRM deux semaines après implantation de la tumeur et répéter une fois par semaine pendant trois semaines. </li><li> Endormir les animaux avec 3% d&#39;isoflurane et de les maintenir sous gèneanesthésie ral (1,5% de l&#39;isoflurane). </li><li> Surveiller et maintenir la température du corps de l&#39;animal et de la respiration constante tout au long de l&#39;expérience. </li><li> Résolution multislice élevé (14 tranches avec 1 mm d&#39;épaisseur, pas d&#39;écart) T 1 - et T 2 pondéré images coronales, couvrant la région du lobe frontal à la fosse postérieure, sont acquises avec les paramètres suivants: T images 1-pondérés: écho de spin tranche multiple (SEMS), TR / TE = 400 ms msec/20, matrice: 256 x 256, FOV 20 x 20 mm, en résolution du plan: 78 x 78 um 2. T images 2-pondérés: écho de spin rapide tranche multiple (FSEMS) des séquences, TR / TE = 2500 msec/48 ms, 8 trains d&#39;écho, matrice: 256 x 256, FOV 20 x 20 mm, en résolution du plan: 78 x 78 um 28,9. lésions tumorales semblent plus lumineux que les tissus cérébraux normaux sur des T-images 2-pondérés. </li><li> taille de la tumeur est déterminée sur T images 2-pondérés en décrivant manuellement le enhanpartie de la masse cement sur ​​chaque image à l&#39;aide de programmes MATLAB écrites par nous 8. Compte tenu de la majeure partie de la tumeur diamètres sont plus petits que l&#39;épaisseur des tranches (1 mm), la taille de la tumeur est présenté comme dans la zone de plan plutôt que le volume. </li></ol> 4. H &amp; E coloration Confirmant les métastases <ol><li> Sacrifier les souris immédiatement après le dernier MR scanner, disséquer les cerveaux porteurs de tumeurs et l&#39;intégrer dans un milieu PTOM et figé dans -80 ° C. </li><li> Section d&#39;une série de 10 um d&#39;épaisseur cerveau coronale spécimens avec cryostat. </li><li> Effectuer coloration H &amp; E de sections de cerveau. </li></ol>

<ol>
	<li>Schouten, L. J., 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=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., 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=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. F., 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=The+biology+of+brain+metastases-translation+to+new+therapies.">The biology of brain metastases-translation to new therapies.</a> Nat. Rev. Clin. Oncol. , (2011).</li><li>Lockman, P. R., 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=Heterogeneous+blood-tumor+barrier+permeability+determines+drug+efficacy+in+experimental+brain+metastases+of+breast+cancer.">Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer.</a> Clin. Cancer Res. 16, 5664-5678 (2010).</li><li>Yoneda, T., 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+bone-seeking+clone+exhibits+different+biological+properties+from+the+MDA-MB-231+parental+human+breast+cancer+cells+and+a+brain-seeking+clone+in+vivo+and+in.">A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in.</a> 16, 1486-1495 (2001).</li><li>Palmieri, D., 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=Her-2+overexpression+increases+the+metastatic+outgrowth+of+breast+cancer+cells+in+the+brain.">Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.</a> Cancer Res. 67, 4190-4198 (2007).</li><li>Percy, D. B., 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=In+vivo+characterization+of+changing+blood-tumor+barrier+permeability+in+a+mouse+model+of+breast+cancer+metastasis:+a+complementary+magnetic+resonance+imaging+approach.">In vivo characterization of changing blood-tumor barrier permeability in a mouse model of breast cancer metastasis: a complementary magnetic resonance imaging approach.</a> Invest. Radiol. 46, 718-725 (2011).</li><li>Zhou, H., 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=Longitudinal+MRI+evaluation+of+intracranial+development+and+vascular+characteristics+of+breast+cancer+brain+metastases+in+a+mouse+model.">Longitudinal MRI evaluation of intracranial development and vascular characteristics of breast cancer brain metastases in a mouse model.</a> PLoS One. 8, (2013).</li><li>Zhou, H., 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=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, (2009).</li><li>Zhang, R. 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Aucun conflit d&#39;intérêt déclaré.

Dans la présente étude, nous avons démontré que l&#39;injection ventriculaire gauche imagerie guidée ultrasons garantit la précision de sorte que tous les animaux de cette étude développé des métastases cérébrales et aucune mort de l&#39;animal a été observé. La beauté de l&#39;injection guidée par l&#39;image, c&#39;est que le chemin de la pénétration de l&#39;aiguille de la peau et enfin dans le ventricule gauche peut être contrôlé et ajusté sous l&#39;image, qui est distincte de la procédure m...

métastases du cerveau est la tumeur maligne la plus fréquente intracrânienne chez les adultes. Le pronostic est très mauvais, avec une médiane de survie de 4-6 mois, même avec un traitement agressif. Le cancer du sein est l&#39;un des trois principaux cancers primaires avec une morbidité élevé de métastases cérébrales 1-3. Plusieurs lignées de cancer du sein cerveau-tropique sont capables de développer des métastases cérébrales après injection intracardiaque ou intracarotidienne 4. L&#39;injection directe de cellules tumorales dans le ventricule gauche peut contourner le lit capillaire pulmonaire et d&#39;augmenter ainsi l&#39;incidence de la formation de métastases cérébrales, tout en minimisant les métastases viscérales. La ligne MDA-MB231Br est l&#39;une des lignées de cancer du sein le plus largement utilisé droits pour développer des métastases cérébrales dans des modèles rongeurs 5,6. Comme de nombreuses autres études 4,7, nous avons effectué une procédure manuelle de l&#39;injection intra-cardiaque dans nos études précédentes. Cependant, seulement 50% de taux de succès a été obtenu avec l&#39;emploiinjection et une fraction de souris sont mortes des procédures invasives répétées si les essais antérieurs ont échoué. Ici, nous présentons l&#39;utilisation d&#39;une procédure d&#39;imagerie guidée pour garantir l&#39;injection de cellules de cancer du sein cerveau cherchant dans le ventricule gauche de souris athymiques. Longitudinal IRM à haute résolution est appliqué à suivre le développement intracrânienne des métastases cérébrales.

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			<td height="21" style="height:21px;width:413px;">DMEM</td>
			<td style="width:136px;">HyClone</td>
			<td style="width:87px;">SH30022.01</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Trypsin</td>
			<td style="width:136px;">Mediatech</td>
			<td style="width:87px;">25-051-C1</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Automatic cell counter</td>
			<td style="width:136px;">Biorad</td>
			<td style="width:87px;">TC10</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
			<td style="width:136px;">Baxter International Inc.</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:147px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
			<td></td>
			<td style="width:147px;"> </td>
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		<tr height="21">
			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
			<td style="width:87px;"> </td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Sakura Finetek USA</td>
			<td style="width:87px;">4583</td>
			<td style="width:147px;"> </td>
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ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

cerveau du cancer du sein métastatique, survenant dans 30% des patients atteints de cancer du sein au stade IV, est associée à une mortalité élevée. La médiane de survie est de seulement 6 mois. Il est essentiel d&#39;avoir des modèles animaux appropriés pour imiter la propagation de l&#39;hémodynamique des cellules métastatiques dans le scénario clinique. Ici, nous présentons l&#39;utilisation de l&#39;imagerie du petit animal ultrasons pour guider une injection précise des cellules cancéreuses du sein tropicale du cerveau dans le ventricule gauche de la souris nude athymiques. IRM longitudinal est utilisée pour l&#39;évaluation de l&#39;initiation et de la croissance intra-crânienne de métastases cérébrales. Injection intracardiaque guidée par échographie assure non seulement une injection précise et présente un taux de réussite plus élevé, mais a également diminué de manière significative le taux de mortalité, par rapport à notre manuel de procédure précédente. In vivo IRM à haute résolution permet la visualisation des lésions multifocales hyperintenses, aussi petit que 310 um de diamètre sur T 2 images pondéré à 3 semaines après l&#39;injection. Follow-up IRM révèle une croissance de tumeur intracrânienne et l&#39;augmentation du nombre de métastases qui distribuent tout au long de l&#39;ensemble du cerveau.

Avec la haute résolution spatiale de l&#39;IRM (78 um dans la résolution de l&#39;avion), les lésions hyperintenses peuvent être identifiés aussi petit que 310 m de diamètre (figure 2). Depuis les métastases dans cette étude sont très faibles et le développement de la nécrose et de l&#39;œdème est minime, la lésion hyperintense en T 2 images pondérées représentait vraiment la masse de la tumeur. Des études longitudinales permettent d&#39;IRM ...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

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

Un modèle murin de cancer du cerveau de métastase est établi par échographie injection intracardiaque guidée par imagerie de cellules MDA-MB231/Br-GFP. Développement des métastases intracrâniennes multifocales a été suivie longitudinalement en utilisant à haute résolution de 9,4 T IRM.

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 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.6K vues.  University of Texas Southwestern Medical Center.  Un modèle murin de cancer du cerveau de métastase est établi par échographie injection intracardiaque guidée par imagerie de cellules MDA-MB231/Br-GFP. Développement des métastases intracrâniennes multifocales a été suivie longitudinalement en utilisant à haute résolution de 9,4 T IRM. 

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

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 ...

Recherche

Médecine

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Guidée par IRM rupture de la barrière hémato-encéphalique en utilisant transcrânienne d&#39;ultrasons focalisés dans un modèle de rat

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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 ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantification du cancer du sein cellulaire envahissement aide d&#39;un (3D) du modèle en trois dimensions

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Aiguë traumatisme cérébral chez la souris Suivi par longitudinale à deux photons imagerie

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Un préclinique modèle murin de métastases hépatiques

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injection Orthotopique des cellules du cancer du sein dans le Fat Pad mammaire de souris pour étudier la croissance de la tumeur.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm développement sur ​​les organismes étrangères médicalement pertinents dans une souris modèle sous-cutanée Suivi par bioluminescence Imaging