Diese Arbeit wurde durch Mittel aus Kinder-Krebs-Stiftung (Baltimore, MD), Brandon Lee Carrington Foundation (Silver Spring, MD), DOD (W81XWH-10-1-0137) und NIH (R01CA108641) unterstützt. Wir möchten Dr. Anton Wellstein und Mitglieder seines Labors für den Austausch ihrer Erfahrungen und helfen uns, präsentiert Methoden optimieren danken.

1. Vorbereitung <ol><li> Alle Schritte sollten unter sterilen Bedingungen in einer Gewebekultur Haube erfolgen. </li><li> Die xCELLigence Station in einem 37 ° C Inkubator in Gegenwart von 5% CO 2 gestellt. </li><li> Verwenden Sie einen niedrigen Durchgang HUVEC Zellen, vorzugsweise nicht mehr als Durchgang 6. Stellen Sie außerdem sicher, dass die Zellen nicht mehr als 75% konfluent zum Zeitpunkt der Ernte sind. </li><li> HUVEC-Zellen werden in EGM-2-Medien rekonstituiert mit EGM-2 Kugel Kit (Lonza Biosciences), enthaltend Wachstumsfaktoren, Ergänzungen und 5% fötalem Rinderserum. </li><li> Alle Spuren von Trypsin sollte aus Zellsuspensionen durch Spinnen Zellen bei 200 × g, einmaligem Waschen mit PBS und Resuspendieren sie angezeigt Zelldichten entfernt werden. </li><li> Coat xCELLigence E-16 mit 0,1% Gelatine für 1 Stunde bei 37 ° C oder über Nacht bei 4 ° C. Waschen Platte mit PBS und fügen Sie 100 ul rekonstituierten EGM-2-Medien. Führen Sie eine leere Lesung zu dem xCELLigence System Hintergrund messenImpedanz in Abwesenheit von Zellen. </li></ol> 2. Generieren HUVEC Monolayer <ol><li> Ernte ein subkonfluenten Flasche HUVEC Zellen durch Trypsin. Die Zellen in rekonstituierten EGM-2-Medien zu einer endgültigen Dichte von 2,5 x 10 5 Zellen / ml. </li><li> Zu jeder Vertiefung einer kalibrierten Hintergrund E-Platte mit 100 ul EGM-2-Medien, fügen 100 ul der HUVEC Zellsuspension (2,5 x 10 4 HUVEC-Zellen). </li><li> Installieren Sie sofort E-Platte xCELLigence Instrument. </li><li> Programm xCELLigence Software Impedanz Messungen in 10-minütigen Abständen durchzuführen. </li><li> Lassen Sie die Endothelzellen wachsen für 18-21 Stunden, bis sie eine Monoschicht bilden, wie durch eine Abflachung der Zelle Index (Abbildung 2) zeigt. </li></ol> 3. Die Zugabe von eindringenden Zellen und Daten Normalisierung. <ol><li> Sobald ein stabiler HUVEC Monolayer gebildet hat, Ernte Tumorzellen durch Trypsinisierung und resuspendieren zu einer endgültigen denskeit von 1 x 10 5 Zellen / ml in Medium verwendet werden, um Tumorzellen (zB RPMI oder DMEM-Medium, enthaltend 10% FBS) wachsen </li><li> Pause Impedanz Lesungen auf dem xCELLigence Software und entfernen Sie E-Platte vom Bahnhof entfernt. </li><li> Entfernen EGM-2-Medien durch Aspiration. In 100 ul von Tumor-Zell-Suspension mit 1 x 10 4 Zellen. Im Allgemeinen wird ein 1:2,5 Verhältnis von Tumorzellen: funktioniert Endothelzellen ausgesät, am besten für den Test. Jedoch kann das Verhältnis der einzelnen Zelllinien optimiert werden. </li><li> Legen Sie die E-Platte auf dem xCELLigence System in den Inkubator und weiter Impedanz Messungen bei 10 Minuten-Takt. </li><li> Invasion können in Echtzeit über die nächsten 6-12 Stunden werden wie ein Tropfen in Zelle Index durch das Zurückziehen des endothelial Kreuzungen und das Eindringen von eindringenden Tumorzellen überwacht. </li><li> Normalisieren Ergebnisse der Zeitpunkt der Zugabe des eindringenden Tumorzellen. Die Software erlaubt die Normalisierung xCELLigence zu jedem Zeitpunkt und Ergebnissekönnen direkt in der Software-Fenster angezeigt werden. </li></ol> 4. Repräsentative Ergebnisse: Ein Beispiel für HUVEC Monoschicht Invasion durch metastatischen und nicht-metastatischen Zellen ist in Fig. 3 gezeigt. K7M2 ist ein metastasierendem Osteosarkom-Zelllinie. Diese Zellen exprimieren hohe Mengen des Zytoskeletts Linkerprotein Ezrin, welche Konten für die invasive Eigenschaften dieser Zelllinie 13,14. Wenn HUVEC Zellen mit K7M2 Zellen sind herausgefordert, es ist ein Tropfen im elektrischen Widerstand innerhalb von 6 Stunden. Dieser Rückgang des elektrischen Widerstandes stellt die Invasion von HUVEC Monolayer von den eindringenden Tumorzellen. Die K12-Zelllinie, auf der anderen Seite, drückt deutlich geringeren Ezrin und ist somit weniger metastasierendem 13. Die Anfechtung der HUVEC Monolayer mit K12-Zellen führt zu einem weniger starken Rückgang der Widerstand, wenn die Zellen nicht in der Lage, um zu haften oder dringen durch die HUVEC Monolayer (Abbildung 3). <imgsrc = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot; alt = &quot;1&quot; /&gt; Abbildung 1. Schematische Darstellung der Workflow-Prozess für die Invasion von Endothelzellen durch Tumorzellen. HUVEC-Zellen auf mit Gelatine beschichtete E-Platten ausplattiert und eine Monolayer bilden. Bösartige Zellen werden hinzugefügt sobald eine Monolage gebildet hat und Invasion wird in Echtzeit überwacht, wie die Zelle Index ändert aufgrund des Eindringens der endothelialen Monolayer. <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="Abbildung 2"/> Abbildung 2. HUVEC Monolayerbildung. Veränderungen in der Zelle als Index HUVEC Zellen zu befestigen und zu verbreiten auf Gelatine-beschichteten Gold-Elektroden. Die Bildung einer konfluenten Monoschicht durch eine Stabilisierung des Zell-Index nach 18 Stunden dargestellt. <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Invasion von HUVEC Zellen durch K7M2 und K12 Osteosarkomzellen. Ein steiler Rückgang in cell-index erfolgt innerhalb von ein paar Stunden von der Einführung des hoch metastatischen K7M2 Zellen. K12-Zellen, auf der anderen Seite, sind weniger metastatischen und nicht imstande sind, die endothelialen Monoschicht so effizient wie K7M2 Zellen eindringen. Dies wird durch eine weniger steile Abnahme in der Zell-Index, wenn der HUVEC Monoschicht mit K12-Zellen in Frage gestellt wird dargestellt. Steuerelement darstellt Impedanz HUVEC Monoschicht in Abwesenheit von Krebszellen. Die Experimente wurden in dreifacher Ausführung durchgeführt.

<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>

Die Publikation kostet für dieses Manuskripts wurde uns freundlicherweise von Roche Applied Science zur Verfügung gestellt.

Die Echtzeit-HUVEC Invasion Assay ist eine einfache, aber leistungsfähige Methode zur Überwachung Invasion. Es liefert Ergebnisse in Echtzeit, was ein wesentlicher Vorteil gegenüber anderen herkömmlichen Techniken, die auf Endpunktanalyse verlassen. Der Assay kann auf Testverbindungen, Antikörper, Liganden und Proteine ​​als Inhibitoren oder Stimulatoren der Metastasierung modifiziert werden. Die Wirkung verschiedener Mittel auf die endotheliale / Krebszelle cross-talk kann auch beurteilt werden. Bei der Einführung von exogener Mittel, wie Wachstumsfaktoren und Medikamenten in den Kulturmedien, ist es wichtig sicherzustellen, dass sie keinen Einfluss auf die Integrität der HUVEC Monoschicht. Die Unterbrechung der HUVEC Monoschicht durch Einführen der exogene Substanz in Abwesenheit von eindringenden Zellen, und sicherzustellen, dass es keine Veränderung in der Zelle Index geprüft werden. Daher sollten geeignete Kontrollen als Teil des experimentellen Designs enthalten sein. Verschiedene Zelllinien zeigen unterschiedliche Zell-Index-Kurve, wie sie eine conf bildenluent Monoschicht. Die Form der Kurve, sowie die maximale Zell-Index erreicht ist Zelltyp-spezifisch. HUVEC-Zellen zeigen eine charakteristische transiente Abflachung der Zelle Index 4-6 Stunden nach dem Aussäen von einem anderen Stabilisierung bei einer höheren Zelle Index nach 16-18 Stunden. Daher ist es wichtig, damit die Zellen bilden eine konfluente Monoschicht für mindestens 18 Stunden vor der Zugabe von Tumorzellen. Da die Zelle Index ist abhängig von der ionischen Umgebung an der Elektrode / Zelle Interphase Einfluss verschiedener Faktoren wie zum Beispiel Zell-Morphologie und die Qualität der Zell-Zell-Adhäsion der Zellen-Index, zusätzlich zu dem Bereich der Schachtboden bedeckt. Somit ist die Abnahme in der Zell-Index, der auf Tumorzellinvasion auftritt, eine Funktion verschiedener Faktoren, die das Zurückziehen der endothelialen Verbindungen und anschließende Tumorzellinvasion umfassen. Das xCELLigence Instrument ist in drei verschiedenen Konfigurationen hergestellt: Echtzeit Zellanalysator(RTCA) SP, MP und RTCA RTCA DP. Die RTCA SP Instrument hält eine 96-well E-Platte, während die RTCA MP Instrument kann bis zu sechs 96-Loch-E-Platten gleichzeitig. Dies ermöglicht für das Hochdurchsatz-Screening von Medikamenten und Verbindungen. Die Experimente in diesem Artikel werden unter Verwendung des RTCA DP Instrument, das drei 16-Well-Platten E-halten kann. Jede E-Platte Haltestation können unabhängig voneinander betrieben werden, was ermöglicht mehreren Benutzern die Experimente gleichzeitig laufen. Die RTCA DP Instrument kann auch verwendet werden, um die Migration mit CIM-Platten zu untersuchen. Dies sind 16-Well-Platten mit oberen und unteren Kammern durch eine Polyethylenterephthalat (PET)-Membran mit einer mittleren Porengröße von 8um getrennt. Die elektronischen Sensoren sind an der Unterseite der porösen Membran von der oberen Kammer angeordnet ist. Die untere Kammer als Reservoir für Lockstoff für Zellen in der oberen Kammer. Allerdings ist ein großer Vorteil der HUVEC Invasionsassay über die CIM-Platten, die die Tumorzelle Invasion in der ehemaligen is nicht durch die Porengröße beschränkt, wie Endothelzellen Invasion hängt von Übersprechen zwischen den Tumorzellen und Endothelzellen. Die HUVEC Invasionsassay können auch auf ECIS Z Instrument durchgeführt werden, hergestellt von Applied Biophysik (Troy, NY). Die ECIS Z Instrument nutzt eine Technologie ähnlich dem xCELLigence Instrument, mit Unterschieden in der Array-und Elektroden-Konfigurationen. Wir haben erfolgreich HUVEC Invasionsassays mit den 8-well ECIS Arrays durchgeführt. Es ist wichtig zu beachten, dass die gut Bodenfläche größer in der ECIS Arrays, die eine größere Anzahl von Endothel und Tumorzellen erfordert plattiert werden soll: 2,5 x 10 5 und 1 x 10 5 Zellen sind.

<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

Metastatischen Verbreitung von bösartigen Zellen erfordert Abbau Basalmembran Befestigung von Tumorzellen an Gefäßendothel, Einziehen der endothelialen Kreuzungen und schließlich Invasion und Migration von Tumorzellen durch das Endothel in die Blutbahn als Transportmittel geben zu entfernten Stellen in dem Host 1-3. Einmal in das Kreislaufsystem, sich Krebszellen Wände Kapillare und extravasieren des umliegenden Gewebes zu bilden Metastasen 4,5. Die verschiedenen Komponenten der Tumorzellen Endothelzellinteraktion kann in vitro durch die Herausforderung eine Monoschicht von menschlichen Nabelschnur-Endothelzellen (HUVEC) mit Krebszellen repliziert werden. Studien mit Elektronen-und Phasenkontrast-Mikroskopie durchgeführt vermuten, dass die in vitro Ablauf getreu die in vivo Metastasierungsprozess 6. Hier beschreiben wir eine elektrische Impedanz-basierte Technik, die quantifiziert und überwacht in Echtzeit den INVASion von Endothelzellen von malignen Tumorzellen. Giaever und Keese zuerst beschrieben eine Technik zum Messen von Schwankungen in der Impedanz, wenn eine Population von Zellen auf der Oberfläche der Elektroden 7,8 wachsen. Die xCELLigence Instrument, hergestellt von Roche, verwendet eine ähnliche Technik, um Änderungen in der elektrischen Impedanz zu messen, wie Zellen befestigen und in eine Kulturschale mit einer Gold-Mikroelektroden-Anordnung, die etwa 80% der Fläche bedeckt auf dem Boden eines Wells bedeckt verbreiten. Als Zellen anlagern und ausbreiten auf der Elektrodenoberfläche, es führt zu einer Erhöhung in der elektrischen Impedanz 9-12. Die Impedanz wird als dimensionslose Parameter bezeichnet Zell-Index, der direkt proportional zu der Gesamtfläche von Gewebekulturen, die von Zellen bedeckt ist, angezeigt. Daher kann die Zell-Index verwendet werden, um die Zelladhäsion, Streichen, Morphologie und die Zelldichte zu überwachen. Die Invasion Assay in diesem Artikel beschriebenen Änderungen auf der Grundlagein elektrische Impedanz an der Elektrode / Zelle Zwischenphase, als eine Population von bösartigen Zellen eindringen durch eine HUVEC Monolayer (Abbildung 1). Die Störung der endothelialen Kreuzungen, Rückzug der endothelialen Monolayer und Ersatz von Tumorzellen führen zu großen Veränderungen in der Impedanz. Diese Veränderungen korrelieren direkt mit der Kapazität von invasiven Tumorzellen, dh Invasion durch hoch aggressive Zellen zu großen Veränderungen in der Zelle Impedanz und umgekehrt führen. Diese Technik bietet einen zweifachen Vorteil gegenüber bestehenden Methoden zur Messung der Invasion, wie Boyden-Kammer und Matrigel-Tests: 1) die Endothelzellen-Tumor-Zell-Wechselwirkung mehr ahmt die in vivo-Verfahren, und 2) die Daten in Echtzeit erhalten Zeit und ist leicht zu quantifizieren, um Endpunktanalyse für andere Verfahren entgegen.

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

Dieser Artikel beschreibt ein<em&gt; In vitro</em&gt; Verfahren zur Überwachung Krebszellen Invasion durch eine Monoschicht aus Endothelzellen. Die Daten werden in Echtzeit in Abhängigkeit von Änderungen der Impedanz auf der Oberfläche der Elektroden, die an der Unterseite auch erworben.

Eine Echtzeit-Elektrische Impedanz basierte Technik zur Invasion von Endothelzellen Monolayer von Krebs Zellen zu messen

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 Aufrufe.  Georgetown University. Dieser Artikel beschreibt ein In vitro Verfahren zur Überwachung Krebszellen Invasion durch eine Monoschicht aus Endothelzellen. Die Daten werden in Echtzeit in Abhängigkeit von Änderungen der Impedanz auf der Oberfläche der Elektroden, die an der Unterseite auch erworben.

Serie: 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 von Nanopartikel-Aufnahme in Tumoren in Echtzeit über 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 ...

Forschung

Medizin

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-Photonen-Imaging der Tumorzellinvasion in einem orthotopen Mausmodell des oralen Plattenepithelkarzinoms

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.

Spheroid Assay zur TGF-β-induzierte Invasion Measure

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.

Modeling and Imaging 3-Dimensional Collective Zellinvasion

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.

Ein Multiplex-Luciferase-basierte Screening-Plattform zur Abfrage Krebs-assoziierte Signaltransduktion in kultivierten Zellen

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.

Liquor microRNA Profiling mittels quantitativer 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 ...

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.

Eine modifizierte<em&gt; In-vitro-</em&gt; Invasion Assay, um die mögliche Rolle von Hormonen, Cytokinen und / oder Wachstumsfaktoren bei der Vermittlung der Invasion von Krebszellen fest,

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.

Beurteilung von Eierstockkrebs Sphäroid Bindung und Invasion von Mesothelzellen in Echtzeit

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.

Dreidimensionale (3D) Tumor Spheroid Invasionsassay

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.

Isolation of Human Lymphatic Endothelzellen durch Multiparameter-Fluoreszenz-aktivierten Zellsortierung

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