Ce travail a été généreusement financé par des fonds de la Fondation du cancer (Baltimore, MD), Brandon Lee Carrington Foundation (Silver Spring, MD), DOD (W81XWH-10-1-0137) et le NIH pour enfants (R01CA108641). Nous tenons à remercier le Dr Anton Wellstein et les membres de son laboratoire pour partager leur expérience et nous aider à optimiser les méthodes présentées.

1. Préparation <ol><li> Toutes les mesures doivent être effectuées dans des conditions stériles dans une hotte de culture de tissus. </li><li> La station de xCELLigence est placé dans un incubateur à 37 ° C en présence de 5% de CO 2. </li><li> Utilisation des cellules HUVEC de passage faible, de préférence pas plus de passage 6. Aussi, assurez-vous que les cellules ne sont plus que 75% de confluence au moment de la récolte. </li><li> Les cellules HUVEC sont cultivées dans des EGM-2 milieux reconstitués avec kit EGM-2 balles (Lonza Biosciences) contenant des facteurs de croissance, des suppléments et 5% de sérum de veau fœtal. </li><li> Toutes les traces de trypsine doivent être retirés de suspensions cellulaires par les cellules filature à 200xg, lave une fois avec PBS, et leur remise en suspension à des densités cellulaires indiquées. </li><li> Manteau xCELLigence E-plaque 16 avec 0,1% de gélatine pendant 1 heure à 37 ° C ou une nuit à 4 ° C. Laver la plaque avec du PBS et ajouter 100 ul EGM-2 reconstituée médias. Effectuer une lecture à blanc sur le système de xCELLigence pour mesurer fondimpédance en l&#39;absence de cellules. </li></ol> 2. Génération HUVEC monocouche <ol><li> Récolter un flacon sous-confluentes de cellules HUVEC par la trypsine. Remettre les cellules en reconstituées EGM-2 médias à une densité finale de 2,5 x 10 5 cellules / ml. </li><li> Pour chaque puits d&#39;un fond calibré E-plaque contenant 100 ul EGM-2 médias, ajouter 100 pi de la suspension de cellules HUVEC (2,5 x 10 4 cellules HUVEC). </li><li> Installer immédiatement E-Plate dans l&#39;instrument de xCELLigence. </li><li> logiciel de xCELLigence de programme pour effectuer des lectures d&#39;impédance à 10 minutes d&#39;intervalle. </li><li> Laissez les cellules endothéliales se développent pour 18-21 heures jusqu&#39;à ce qu&#39;ils forment une monocouche, comme en témoigne l&#39;aplatissement de l&#39;indice de la cellule (figure 2). </li></ol> 3. Addition des cellules et la normalisation des données d&#39;invasion. <ol><li> Une fois une monocouche HUVEC stable s&#39;est formée, récolte les cellules tumorales par la trypsine et remettre en suspension à une finale tanièreslité de 1 x 10 5 cellules / ml dans les médias utilisés pour cultiver des cellules tumorales (comme RPMI ou médias DMEM contenant 10% de FBS) </li><li> lectures d&#39;impédance de pause sur le logiciel de xCELLigence et supprimer E-Plate de la gare. </li><li> Retirer EGM-2 médias par aspiration. Ajouter 100 ul de la suspension de cellules tumorales contenant 1 x 10 4 cellules. En règle générale, un ratio 1:2,5 des cellules tumorales: cellules endothéliales ensemencées, qui fonctionne le mieux pour le dosage. Toutefois, le ratio peut être optimisé pour des lignées de cellules individuelles. </li><li> Placer le E-plaque sur le système de xCELLigence dans l&#39;incubateur et continuer lectures d&#39;impédance à intervalles de 10 minutes. </li><li> Invasion peut être suivie en temps réel au cours des prochaines 6-12 heures par une chute de l&#39;indice de la cellule en raison de la rétraction des jonctions endothéliales et la pénétration de l&#39;invasion des cellules tumorales. </li><li> Normaliser les résultats au moment de l&#39;addition d&#39;envahir les cellules tumorales. Le logiciel permet xCELLigence normalisation à n&#39;importe quel point du temps et des résultatspeut être directement visualisé dans la fenêtre de logiciel. </li></ol> 4. Les résultats représentatifs: Un exemple de HUVEC monocouche invasion par les cellules métastatiques et non métastatiques est illustré à la figure 3. K7M2 est une lignée de cellules d&#39;ostéosarcome métastatique. Ces cellules expriment des niveaux élevés de l&#39;Ezrin de protéines de liaison du cytosquelette, ce qui explique les propriétés invasives de cette lignée cellulaire 13,14. Lorsque les cellules HUVEC sont mis au défi avec des cellules K7M2, il ya une baisse de la résistance électrique dans les 6 heures. Cette baisse de résistance électrique représente l&#39;invasion de la monocouche HUVEC par les cellules tumorales envahissantes. La lignée cellulaire K12, d&#39;autre part, exprime des niveaux beaucoup plus faibles de Ezrin et est par conséquent moins métastatique 13. Contestation de la monocouche HUVEC avec K12 cellules entraîne une baisse moins forte de la résistance que les cellules sont incapables d&#39;adhérer ou envahir par la monocouche HUVEC (Figure 3). <imgsrc = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot; alt = &quot;Figure 1&quot; /&gt; Figure 1. Schéma de processus de flux de travail pour l&#39;invasion des cellules endothéliales par les cellules tumorales. Les cellules HUVEC sont étalées sur E-plaques recouvertes de gélatine et le droit de former une monocouche confluente. Les cellules malignes se sont ajoutés une fois une monocouche a formé et invasion est surveillé en temps réel comme l&#39;indice de la cellule change en raison de l&#39;invasion de la monocouche endothéliale. <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="Figure 2"/> Figure 2. Formation de la monocouche HUVEC. Les variations de l&#39;indice de la cellule que les cellules HUVEC s&#39;attacher et se propagent sur des électrodes d&#39;or recouvertes de gélatine. La formation d&#39;une monocouche confluente est représentée par une stabilisation de l&#39;indice de cellules après 18 heures. <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="Figure 3"/> Figure 3. Invasion des cellules HUVEC par K7M2 et K12 cellules d&#39;ostéosarcome. Une forte diminution de cell-indice survient en quelques heures de la mise en place de cellules hautement métastatiques K7M2. Cellules K12, en revanche, sont moins métastatique et sont incapables de pénétrer la monocouche endothéliale aussi efficacement que les cellules K7M2. Ceci est représenté par une moins forte baisse de l&#39;indice de la cellule lorsque la monocouche HUVEC est contestée par des cellules K12. Contrôle représente l&#39;impédance de monocouche HUVEC en l&#39;absence de cellules cancéreuses. Des expériences ont été réalisées en triple.

<ol>
	<li>Klein, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer.+The+metastasis+cascade.">Cancer. The metastasis cascade.</a> Science. 321, 1785-1787 (2008).</li><li>Chiang, A. C., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+metastasis.">Molecular basis of metastasis.</a> N Engl J Med. 359, 2814-2823 (2008).</li><li>Orr, F. W., Wang, H. H., Lafrenie, R. M., Scherbarth, S., Nance, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+cancer+cells+and+the+endothelium+in+metastasis.">Interactions between cancer cells and the endothelium in metastasis.</a> J Pathol. 190, 310-329 (2000).</li><li>Bacac, M., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+cancer+cell.">Metastatic cancer cell.</a> Annu Rev Pathol. 3, 221-247 (2008).</li><li>Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+signals+from+the+invasive+front.">New signals from the invasive front.</a> Nature. 441, 444-450 (2006).</li><li>Kramer, R. H., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+tumor+cells+with+vascular+endothelial+cell+monolayers:+a+model+for+metastatic+invasion.">Interactions of tumor cells with vascular endothelial cell monolayers: a model for metastatic invasion.</a> Proc Natl Acad Sci U S A. 76, 5704-5708 (1979).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proc Natl Acad Sci U S A. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromotion+of+mammalian+cells+measured+electrically.">Micromotion of mammalian cells measured electrically.</a> Proc Natl Acad Sci U S A. 88, 7896-7900 (1991).</li><li>Tiruppathi, C., Malik, A. B., Del Vecchio, P. J., Keese, C. R., Giaever, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+method+for+detection+of+endothelial+cell+shape+change+in+real+time:+assessment+of+endothelial+barrier+function.">Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.</a> Proc Natl Acad Sci U S A. 89, 7919-7923 (1992).</li><li>Whipple, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+promotes+tubulin+Detyrosination+and+Microtentacles+that+enhance+endothelial+engagement.">Epithelial-to-mesenchymal transition promotes tubulin Detyrosination and Microtentacles that enhance endothelial engagement.</a> Cancer Res. 70, 8127-8137 (2010).</li><li>Boyd, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-microelectronic+sensing+technique+for+profiling+cytotoxicity+of+chemicals.">A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.</a> Anal Chim Acta. 615, 80-87 (2008).</li><li>Khanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane-cytoskeleton+linker+ezrin+is+necessary+for+osteosarcoma+metastasis.">The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.</a> Nat Med. 10, 182-186 (2004).</li><li>Ren, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin-cytoskeleton+linker+protein+ezrin+is+regulated+during+osteosarcoma+metastasis+by+PKC.">The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.</a> Oncogene. 28, 792-802 (2009).</li></ol>

Le coût de publication de ce manuscrit a été aimablement fourni par Roche Applied Science.

Le dosage de l&#39;invasion HUVEC en temps réel est une méthode simple, mais puissant pour la surveillance invasion. Il fournit des résultats en temps réel, ce qui est un avantage considérable sur les autres techniques traditionnelles qui s&#39;appuient sur l&#39;analyse du point final. Le dosage peut être modifié pour tester des médicaments, des anticorps, des ligands et des protéines que des inhibiteurs ou des stimulateurs de métastases. L&#39;effet de différents agents sur l&#39;endothélium / cellules cancéreuses diaphonie peut être évaluée aussi bien. Lors de l&#39;introduction des agents exogènes, tels que les facteurs de croissance et des drogues dans les milieux de culture, il est important de veiller à ce qu&#39;ils n&#39;affectent pas l&#39;intégrité de la monocouche HUVEC. Perturbation de la monocouche HUVEC peut être testée en introduisant l&#39;agent exogène en l&#39;absence de cellules envahissant, et d&#39;assurer qu&#39;il n&#39;ya pas de changement de l&#39;indice de la cellule. Ainsi, des contrôles appropriés doivent être inclus dans le cadre de la conception expérimentale. Différentes lignées cellulaires montrent des courbes différentes cellules d&#39;indice car ils forment une confmonocouche luent. La forme de la courbe, ainsi que la cellule d&#39;indice maximale atteinte, est un type de cellule spécifique. Les cellules HUVEC montrent un aplatissement de l&#39;indice de la cellule transitoire caractéristique 4-6 heures après l&#39;ensemencement, suivie d&#39;une autre stabilisation à un indice de la cellule supérieure après 16-18 heures. Par conséquent, il est important de laisser les cellules forment une monocouche confluente pendant au moins 18 heures avant l&#39;addition des cellules tumorales. Etant donné que l&#39;indice de cellules est dépendant de l&#39;environnement ionique sur l&#39;électrode / cellule en interphase, différents facteurs tels que la morphologie cellulaire et la qualité des adhérences cellule-cellule d&#39;influencer la cellule d&#39;indice, en plus de la zone de fond bien couvert. Ainsi, la diminution de l&#39;indice de la cellule, qui se produit lors de l&#39;invasion des cellules tumorales, est une fonction de plusieurs facteurs, qui comprennent la rétraction des jonctions endothéliales et l&#39;invasion des cellules tumorales ultérieure. L&#39;instrument xCELLigence est fabriqué dans trois configurations différentes: Analyseur de cellules en temps réel(RTCA) SP, RTCA MP et RTCA DP. L&#39;instrument SP RTCA détient un E-plaque de 96 puits alors que l&#39;instrument MP RTCA peut contenir jusqu&#39;à 96 puits E-plaques simultanément six. Cela permet de criblage à haut débit de médicaments et de composés. Les expériences présentées dans cet article sont effectuées en utilisant l&#39;instrument DP RTCA, qui peut contenir trois 16 et E-plaques. Chaque station de maintien E-plaque peut être utilisé indépendamment, ce qui permet à plusieurs utilisateurs d&#39;exécuter simultanément des expériences. L&#39;instrument RTCA DP peut également être utilisé pour étudier la migration en utilisant CIM-plaques. Ce sont des plaques de 16 puits avec des chambres supérieure et inférieure séparées par un téréphtalate de polyéthylène (PET) de la membrane avec une taille de pore médian de 8um. Les capteurs électroniques sont situés sur la face inférieure de la membrane poreuse de la chambre supérieure. La chambre inférieure sert de réservoir pour la chimio-attractive pour les cellules dans la chambre supérieure. Cependant, un avantage majeur de l&#39;essai d&#39;invasion HUVEC sur les CIM-plaques est que l&#39;invasion des cellules tumorales dans l&#39;ex-in&#39;est pas limité par la taille des pores, comme l&#39;invasion des cellules endothéliales dépend de la diaphonie entre les cellules tumorales et endothéliales. Le dosage de l&#39;invasion HUVEC peut également être effectuée sur ECIS instrument Z, fabriqué par Applied Biophysique (Troy, NY). L&#39;instrument ECIS Z utilise une technologie semblable à celle de l&#39;instrument de xCELLigence, avec des différences dans les configurations de modules et de l&#39;électrode. Nous avons réalisé avec succès des essais d&#39;invasion HUVEC en utilisant les tableaux de ECIS 8 puits. Il est important de noter que la superficie bien inférieure est plus grande dans les tableaux ECIS, qui exige un plus grand nombre de cellules endothéliales et tumorales d&#39;être plaqué: 2,5 x 10 10 cellules de 5 et 1 x 5 respectivement.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr> 
 <tr>
 	<td>xCELLigence RTCA DP station</td>
 <td>Roche	</td>
 <td>05469759001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>RTCA control unit</td>
 <td>Roche</td>
 <td>05454417001</td>
 <td>Notebook with preinstalled RTCA software</td>
 </tr>
 <tr>
 	<td>E-Plate 16</td>
 <td>Roche</td>
 <td>05469830001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HUVEC cells</td>
 <td>Lonza</td>
 <td>CC-2517</td>
 <td></td>
 </tr>
 <tr>
 	<td>EGM-2 media</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>EGM-2 bullet kit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>0.1% Gelatin in 
sterile H2O
</td>
 <td>Millipore</td>
 <td>MCPROTO045</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells

La dissémination métastatique des cellules malignes nécessite une dégradation de la membrane basale, la fixation des cellules tumorales à l&#39;endothélium vasculaire, la rétraction des jonctions endothéliales et, enfin, l&#39;invasion et la migration des cellules tumorales dans la couche endothéliale à pénétrer dans le sang en tant que moyen de transport à des sites distants chez l&#39;hôte 1-3. Une fois dans le système circulatoire, les cellules cancéreuses adhèrent aux parois capillaires et s&#39;extravaser aux tissus environnants pour former des tumeurs métastatiques 4,5. Les différentes composantes de l&#39;interaction cellulaire des cellules endothéliales tumorales peuvent être reproduits in vitro en contestant une monocouche de cellules endothéliales de veine ombilicale humaine (HUVEC) avec des cellules cancéreuses. Les études réalisées avec la microscopie électronique et à contraste de phase suggèrent que la séquence dans in vitro des événements représentent fidèlement le processus métastatique in vivo 6. Ici, nous décrivons une technique basée électrique impédance qui surveille et quantifie en temps réel les invasionique des cellules endothéliales par les cellules tumorales malignes. Giaever et Keese décrit d&#39;abord une technique de mesure des variations de l&#39;impédance quand une population de cellules se développent à la surface des électrodes 7,8. L&#39;instrument de xCELLigence, fabriqué par Roche, utilise une technique similaire pour mesurer les changements d&#39;impédance électrique que les cellules se fixent et se répandent dans une boîte de culture recouvert d&#39;une matrice de microélectrodes d&#39;or qui couvre environ 80% de la surface au fond d&#39;un puits. Comme les cellules se fixent et se propagent à la surface de l&#39;électrode, elle conduit à une augmentation de l&#39;impédance électrique 9-12. L&#39;impédance est affiché en tant que paramètre appelé cellule d&#39;indice sans dimension, qui est directement proportionnelle à la surface totale de culture de tissu et qui est recouverte par les cellules. Ainsi, la cellule-index peut être utilisé pour contrôler l&#39;adhérence des cellules, d&#39;étalement, de la morphologie et de la densité des cellules. Le dosage de l&#39;invasion décrit dans cet article est basé sur des changementsl&#39;impédance électrique au niveau de l&#39;électrode / cellule en interphase, en tant que population de cellules malignes envahir par une monocouche HUVEC (Figure 1). La perturbation des jonctions endothéliales, la rétraction de monocouche endothéliale et le remplacement par des cellules tumorales conduit à de grandes variations d&#39;impédance. Ces modifications sont en corrélation directe avec la capacité invasive des cellules tumorales, à savoir, d&#39;une invasion par des cellules très agressifs conduisent à de grandes variations de l&#39;impédance de la cellule et vice versa. Cette technique permet un double avantage par rapport aux méthodes existantes de mesure invasion, tels que chambre de Boyden et analyses matrigel: 1) l&#39;interaction cellulaire des cellules tumorales endothéliales imite de plus près le procédé in vivo, et 2) les données sont obtenues en temps réel, temps et est plus facilement quantifiable, par opposition à l&#39;analyse des paramètres pour d&#39;autres méthodes.

Watch this Scientific Journal Video about A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells at JoVE.com

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Cet article décrit une<em&gt; In vitro</em&gt; Technique pour les cellules cancéreuses de surveillance d&#39;invasion par une monocouche de cellules endothéliales. Les données sont acquises en temps réel en fonction des variations d&#39;impédance sur la surface des électrodes en fond de puits.

Une technique basée Impédance électrique en temps réel pour mesurer Invasion de monocouche de cellules endothéliales par les cellules cancéreuses

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

a-real-time-electrical-impedance-based-technique-to-measure-invasion

Research

JoVE Journal

Medicine

27.2K vues.  Georgetown University. Cet article décrit une In vitro Technique pour les cellules cancéreuses de surveillance d'invasion par une monocouche de cellules endothéliales. Les données sont acquises en temps réel en fonction des variations d'impédance sur la surface des électrodes en fond de puits.

Vidéo: A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluation de la fixation des nanoparticules dans les tumeurs en temps réel par imagerie intravitale

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Recherche

Médecine

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Assay Sphéroïde pour mesurer le TGF-β induit Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modélisation et Imagerie en 3 dimensions l&#39;invasion des cellules collectives

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Une plateforme de criblage à base de luciférase multiplexé pour interroger associée au cancer transduction du signal dans des cellules cultivées

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

Liquide céphalo-rachidien profilage de microARN Utilisation quantitative PCR en temps réel

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Une modification<em&gt; In vitro</em&gt; Invasion Test pour déterminer le rôle potentiel des hormones, des cytokines et / ou facteurs de croissance dans le cancer Médiation invasion cellulaire

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Évaluation de Cancer de l&#39;ovaire Spheroid attachement et l&#39;invasion des cellules mésothéliales en temps réel

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionnelle (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolement des cellules endothéliales humaines lymphatique par multi-paramètres fluorescence tri cellulaire activé

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...