Unterstützt von NIH Zuschüsse P30 CA051008 und T32 CA009686 (ATR).

1. Erzeugung von Spheroids <ol><li> Vorbereitung Einzelzellsuspension für hängende Tropfen Kulturen durch Ablösen haftKrebsZellKulturen von ~ 70% Konfluenz unter Verwendung eines PBS-Wasch durch Belichtung mit 0,05% Trypsin-EDTA-Lösung. <ol><li> Neutralisieren die Trypsin-Lösung mit Zellkulturmedien und zählen Sie die Zellen unter Verwendung eines Aliquots der Zellsuspension. Anmerkung: Die spezifische Zellkulturmedien werden auf der Zelllinie abhängig getestet. Folgen ATCC Medienempfehlungen, wobei die Zellkulturmedien typischerweise DMEM + 10% FBS. Weitere Informationen finden Sie in der Werkstoff-Tabelle gefunden werden. </li><li> Führen eine Verdünnung für die Aussaat von 500-1000 Zellen pro 20 ul Tropfen der Zellkulturmedien ermöglichen. </li></ol></li><li> Erwerben Sie 10 cm Schüssel und 5 ml sterilem PBS auf den Grund. Hinweis: Dieser Schritt schützt den hängenden Tropfen aus der Verdampfung. Kostengünstigere &quot;bakteriologischen grade&quot; Gerichte können an Stelle der Zellkultur Geschirr verwendet werden, wie Zellen nicht conthandeln die Kulturfläche. </li><li> Verwenden einer Mehrkanal-Pipette 20 ul Tropfen der verdünnten Zellsuspension auf die innere Oberfläche des Deckels übertragen. <ol><li> Pipette 40 Tropfen (5 Reihen von 8 Tropfen) auf dem Deckel des 10 cm-Schale. </li></ol></li><li> Kehren Sie die Deckel und Platz über der Kulturschale. Der hängende Tropfen Kulturen bei 37 ° C inkubieren 72 h bis Sphäroide zu erzeugen. <ol><li> Klappen Sie den Deckel in eine selbstbewusste, dennoch kontrolliert. Hinweis: Invertierung des Deckels zu schnell oder zu langsam, kann dazu führen, die Tröpfchen zu verschieben. Einige Zelllinien können Sphäroiden in 48 Stunden oder weniger zu bilden, während andere erfordern mehr als 72 Stunden, um kompakte Aggregate zu erzeugen. </li></ol></li></ol> 2. Einbetten von Sphäroiden in 3D-Matrix <ol><li> Auftauen eines Aliquots Wachstumsfaktor reduziert Basalmembran Stoffe bei 4 ° C über Nacht vor dem Einbetten der Sphäroide. <ol><li> Optional: Layer Vertiefungen mit ECM im voraus, um mögliche Sphäroid Wechselwirkung mit dem Gewebe zu verhindern cultuwieder auftauchen. <ol><li> Jeweils 200 ul ECM pro Well auf einer 24-Well-Platte. </li><li> Kippen Sie die Platte, um sicherzustellen, dass das ECM umfasst die gesamte Erdoberfläche. </li><li> Überschüssige ECM Entfernen Sie vorsichtig mit einer Pipette. </li><li> Die Platte, bis die Brunnen sind trocken: 3 Stunden bei Raumtemperatur oder über Nacht bei 4 ° C. </li></ol></li></ol></li><li> Sammeln der Sphäroide durch Kippen des Deckels der Gewebekulturschale und das Poolen der Medien. Übertragen Sie die Medien mit den Sphäroiden in ein 1,5 ml Mikrozentrifugenröhrchen. </li><li> Gestatten 10 min für die Sphäroide, sich am Boden des Mikrozentrifugenröhrchens zu regeln. Sphäroiden sollte mit dem Auge sichtbar sein. </li><li> Mischungs 100 ul der Basalmembran Materialien mit 100 ul kaltem (4 ° C) Kollagen vom Typ I in einem separaten vorgekühlte Röhrchen. Halten der Mischung bei 4 ° C entweder Gel verfestigt vorzeitig zu verhindern. <ol><li> Verwenden Sie vorgekühlte Pipettenspitzen, wenn die Übertragung kleiner Volumina. </li><li> Geben Sie vorsichtig, wenn SieMischen der Basalmembran Materialien und Kollagen Typ I, um Luftblasenbildung zu verhindern. Anmerkung: Luftblasen können Abbildungs ​​später in dem Protokoll zu behindern, wenn sie in dem Gel eingebettet werden. </li></ol></li><li> Absaugen Sphäroide aus der 40 ul Bodenabschnitt des Mikrozentrifugenröhrchens, und verbinden sich mit der Basalmembran Stoffe / Kollagen Typ I-Gemisch. Seien Sie vorsichtig, um Luftblasenbildung beim Mischen zu verhindern. Anmerkung: Die Lösung, die von 100 ul Basalmembran Materialien 100 ul Kollagen Typ I, und 40 ul der Sphäroide genügend Material für 4 unabhängige 3D Kulturen zu schaffen. Dies kann nach oben oder unten skaliert werden. </li><li> Pipette 40 ul Tropfen der viskosen Mischung in die Zentren der Vertiefungen auf einer Platte mit 24 Vertiefungen. Halten Sie die Platte Ebene, um die Mischung aus Laufen in die Seite des Bohrlochs zu verhindern. Hinweis: Eine einzelne Spalte auf der 24-Well-Platte - 4 Brunnen - wird empfohlen, für jede Bedingung getestet werden. </li><li> Legen Sie die Platte intoa 37 ° C-Inkubator und lassen Sie ungestört für 30 Minuten. Die 3D-Kultur wird in diesem Zeitraum zu polymerisieren. </li><li> Langsam tauchen die 3D-Kulturen in 1 ml Zellkulturmedien. <ol><li> Stellen Sie sicher, dass die Medien ist warm, wenn Sie die Ware in die Vertiefungen zur weiteren Förderung Gelpolymerisation. </li><li> Wichtiger Schritt: die Medien, um die 3D-Kulturen hinzuzufügen vorsichtig. Pipettieren der Medien zu schnell in die Vertiefungen können zur Ablösung der 3D-Kulturen aus dem Gewebekulturoberfläche führen. </li></ol></li></ol> 3. Überwachung und Analyse der Spheroid Invasion <ol><li> Bild Invasion aus der Sphäroide in das umgebende 3D-Matrix bei vom Forscher beschlossen Zeitpunkten. Erwerben Fotografien mit einem inversen Mikroskop mit 20-facher Objektivlinse. Anmerkung: Ideal Zeitpunkten in Abhängigkeit von der Zelllinie getestet abweichen. Invasiveren Zellinien ihre Austritt aus den Sphäroiden kurz nach der Plattierung zu beginnen, und daher nehmen anfängliche Fotografies innerhalb von ein paar Stunden nach der Plattierung. Im Allgemeinen werden Fotografien bei 0 h (nach der Beschichtung), 24 h und 48 h entnommen. Invasion, schließt in der Regel 24 bis 48 Stunden nach der Plattierung. </li><li> Quantifizieren invasive Möglichkeit, anhand der Bildanalyse-Software wie Bild J. <ol><li> Analyse Invasion als der Zellenabstand von der Kante des Sphäroids. <ol><li> Verwenden Sie den Straight Draw-Tool, um den Radius und / oder maximale invasive Entfernung zu markieren. </li><li> Klicken Sie auf &quot;Analysieren&quot; in der oberen Menüleiste und klicken Sie auf &quot;Messen&quot;, um die Längenmessung anzuzeigen. </li></ol></li><li> Analysieren Invasion als der Gesamt invasive Bereich außerhalb der Sphäroid. <ol><li> Verwenden Sie die Freihand-Zeichenwerkzeug, um die Grenze des Sphäroids und / oder Gesamt invasive Bereich zu verfolgen. </li><li> Klicken Sie auf &quot;Analysieren&quot; in der oberen Menüleiste und klicken Sie auf &quot;Messen&quot;, um die Flächenmessung anzuzeigen. </li></ol></li><li> Verwenden Sie ein Plugin wie ROI Manager Tools, um Messungen für einen Stapel von Bildern erstellen. </li></ol>
	</li>
</ol>

<ol>
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The authors declare that they have no competing financial interests.

In dieser Studie wurde die Leistung einer Reihe von Krebszelllinien in einem Sphäroid Invasionstest (Tabelle 1). Im Allgemeinen finden wir, dass Sphäroidbildung ist in den mehr epitheliale gerichteten Zelllinien, bei denen die Anwesenheit von Zell-Zell-Verbindungen fördert die Bildung von einem Sphäroid-like-Architektur verbessert. Bekannten Zelllinien, welche eine epitheliale-mesenchymale Transition erfahren haben, wie MDA-MB-231-Zellen nicht Sphäroide im Drop Kultur wahrscheinlichste hängenden b...

Wird festgestellt, dass Krebszellbeweglichkeit prädiktiv ist metastatischen Potential, da ein solches Verhalten erleichtert Invasion durch die Basalmembran und den Eintritt in das Kreislaufsystem 1. Die meiste Forschung auf Motilität hat, wie sich Zellen in einem zweidimensionalen (2D) Einstellung, obwohl es immer weitgehend anerkannt, dass die Bewegung von Zellen in einer dreidimensionalen (3D) Matrix ist repräsentativ dafür, wie diese selben Zellen tatsächlich fokussiert verhalten in vivo 2. 3D-Kultursysteme werden zunehmend eingesetzt, um zelluläre Verhaltensweisen, die von Zellmorphologie reichen, um das Wachstum Kinetik und Arzneimittelempfindlichkeit 3 studieren. Der Wunsch, die Invasion von Krebszellen im Rahmen einer 3D Milieu überwacht wurde zur Synthese der zuvor festgelegten Verfahren, bei denen die Erzeugung von 3D-Zellaggregate (Sphäroide) über den hängenden Tropfen Kulturverfahren 4, gefolgt von Einbettung dieser Sphäroide in ein 3D extrazellulärem führten Matrix (ECM) von Kollagen und Basalmembran Materialien 5 zusammengesetzt. Diese Methode dient, auf diese vorher festgelegten Techniken durch Bereitstellung eines optimierten Ansatz, die leicht verwendet werden kann, um das Eindringen unter einer Vielzahl von experimentellen Bedingungen zu vergleichen verbessern. Weitere traditionelle Wege, um Zellbeweglichkeit in vitro zu beurteilen sind die kratz gewickelt Assay und die Transwell Assay 6. Erstere Assay zeigt Zellmotilität in einem 2D-Einstellung und ist daher unabhängig von einer Vielzahl von Funktionen kritisch für die in vivo-Invasion, zum Beispiel Proteaseaktivität 7. Die Transwell-Assay kann besseres Modell Zellinvasion, wenn die auch Einsätze mit ECM Substrate jedoch nur einen einzelnen Parameter beschichtet, dh das Auftreten von Zellen, die auf der gegenüberliegenden Membranoberfläche gemessen wird, und viele Nuancen der Zellinvasion sind daher nicht leicht beobachtbar. Im Gegensatz zu diesen Techniken ist die Krebszelle Sphäroid Invasionsassay (Abbildung 1 &lt;/ strong&gt;) ermöglicht die Echtzeit-Überwachung der Zellinvasion in einer Umgebung, die nicht nur physiologisch relevant, sondern erlaubt auch die wichtigen Zelllinie spezifische Merkmale sichtbar zu machen, wie einzelne vs. kollektiven Zellmigration 8. Dieses Verfahren bietet auch Vorteile gegenüber Standard-3D-Kulturwachstums-Assays. Die Erzeugung von Zellaggregaten über die Hängetropfenmethode zunächst schränkt Zellbewegung, so dass Zellen Anreize zu erobern, nachdem diese Einschränkung aufgehoben werden. Darüber hinaus einmal, dass Zwang angehoben wird, wird Zell Egress gehen in eine einheitliche Richtung, die dann bequem quantitativ bestimmt werden kann. Die für die Krebszelle Sphäroiden Assays verwendet beliebtesten ECM Materialien sind Matrigel und Kollagen Typ I, wobei jede dieser Komponenten hat wichtige und unterschiedliche Rollen bei der Beeinflussung metastatischen Verhalten. Matrigel ist eine sekretierte Proteingemisch von Engelbreth-Holm-Swarm-Maus Sarcomazellen erzeugt und in baseme angereichertnt Membranproteine ​​wie Laminin, Entactin und Kollagen Typ IV 9. Aus diesem Grund Matrigel wird fortan als &quot;Basalmembran Materialien.&quot; Diese Basalmembran Materialien liefern wichtige Liganden für Integrin Haftung während Invasion von Krebszellen 10 zusätzlich zu vielen anderen Proteinen, die eine Reihe von Auswirkungen auf das Verhalten der Zelle 11 auszuüben brauchte. Im Vergleich Kollagen Typ I, die gemeinhin aus sauren Aufschlüssen von Sehnen und anderen dichten Kollagenstrukturen 12 hergestellt wird, ist eine viel einfachere Matrixmaterial, das als Hauptstrukturelement des Bindegewebes und Stroma Stützgewebe und Organen des Körpers dient. Es hat sich gezeigt, dass die physikalischen Eigenschaften von Kollagen können eine Reihe von Merkmalen der Zellmotilität regeln; zum Beispiel die Ausrichtung der Kollagenfasern in den Tumor-Stroma-Schnittstelle ermöglicht die Krebszellen zu migrieren anschließend entlang dieser Fibrillen, wenn eindringende in das Stroma 13. In dem Assay hier präsentierten sowohl Typ I-Kollagen und Basalmembran-Materialien werden als Werkzeuge, um 3D-Zell-Stroma-Interaktionen zu untersuchen nutzt. Die Wirkung der Inhibierung oder Stimulierung von Bahnen, die Invasion überwacht, nachdem die Zellen in die 3D-Matrix eingebettet werden, zu steuern. Zellen können während des Wachstums in den hängenden Tropfen oder bei Übertragung der 3D-Kultur, abhängig davon, ob eine längere Behandlung erforderlich, um Invasion modulieren vorbehandelt werden. Für kürzere Behandlungs, ist es empfehlenswert, dass das Medikament mit dem Sphäroid Suspension nach der Erfassung, sowie die Medien, die den 3D-Kulturen umgeben wird, um eine angemessene Arzneimittelexposition zu den Zellen erleichtern gemischt werden. Als nächstes kann normal oder Tumor-assoziierten Stromazellen mit dem Matrixmaterial vermischt werden, um ihre Rolle bei der Modulation der Tumorzellinvasion zu bewerten, oder um festzustellen, wie parakrine und autokrine Signalisierung Einflüsse Zellverhalten. Diese Idee wurde in einer Studie gezeigt, wo die Co-Kultur von colüber Krebs und Endothelzellen in hängenden Tropfen zu einer Gefäßnetz innerhalb der Sphäroide 14. Schließlich kann das ECM Komponenten auch verändert werden, da Invasion von Krebszellen wird durch verschiedene Substrate 15 belastet. Die unten dargestellten Verfahren wird somit ein Rahmen für die Beurteilung Invasion von Krebszellen unter einer Vielzahl von Bedingungen. Allgemein wurde festgestellt, dass nicht alle Zelllinien Sphäroide in den hängenden Tropfen zu erstellen und epithelialen gerichteten Zelllinien bilden in der Regel regelmäßige Sphären.

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

a cancer cell spheroid assay to assess invasion in a 3d setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

Verwendung des Sphäroids Invasionsassay (Abbildung 1), eine Reihe von Krebszelllinien wurden auf ihre Fähigkeit zur Sphäroide als auch für die Höhe der Zelle Austritt nach der Implantation in einer 3D-Matrix gezeigt, die aus Basalmembran Materialien und Typ I-Kollagen zu bilden getestet (Tabelle 1). Diese Ergebnisse zeigen, dass nicht jeder Krebszelllinie wird wohlgeformt Sphäroiden, wo Zelllinien besitzen eine epitheliale Morphologie in vitro neigten dazu, regelmäßige ...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Eine Krebszelle Spheroid Assay zur Invasion in einem 3D-Rahmen Beurteilen

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

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Research

JoVE Journal

Medicine

32.3K Aufrufe.  Georgetown University. This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored. 

Serie: A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

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

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

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 ...

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 ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In-vitro-Assay Mesothelzellen Ausverkauf, dass die Modelle der frühen Schritte der Metastasierung Eierstockkrebs

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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.

Quantifizierung der Brustkrebs-Zelle Invasivität Mit einem Drei-dimensionale (3D) Modell

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

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 ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Fokus Formation: Ein Assay auf Zellbasis, um das onkogene Potential eines Gens zu bestimmen

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Halsschlagader Infusionen für pharmakokinetischen und pharmakodynamischen Analyse von Taxanen in Mäusen

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...