Wir möchten das Nikon Imaging Center an der Harvard Medical School, speziell Jennifer Waters, Lara und Wendy Petrak Lachs, für die Ausbildung und den Einsatz ihrer Zeitraffer Mikroskope danken. Wir möchten auch an Rosa Ng und Achim Besser für wertvolle Diskussionen danken. Diese Arbeit wurde vom NIH Grant 5695837 (an M. Iwanicki) und GM064346 zu GKI unterstützt; durch einen Zuschuss von Dr. Miriam und Sheldon G. Adelson Medical Research Foundation (bis GKI).

1. Eierstockkrebszelllinien Spheroid Formation <ol><li> RFP-exprimierenden Eierstockkrebs Zellen werden in 10% Base-Medium (ein benutzerdefiniertes Zellkulturmedium, das ein 50:50-Gemisch von 199 und MCDB105, 10% fötales Rinderserum und 1% Pen-Strep) kultiviert. Um RFP in unmarkierten Eierstockkrebs zu exprimieren, zu transfizieren der Zellen mit einem Plasmid, das RFP und wählen für Zellen RFP. Alternativ können virale Vektoren verwendet werden, um transient exprimieren fluoreszierende Proteine, oder Zellen können vor inkubieren mit einem roten Fluoreszenzfarbstoff Zelle Tracker-Farbstoff (Invitrogen). </li><li> Vor der Bildung von Eierstockkrebs Sphäroide, ist es notwendig, mit geringer Haftung 96 Well Rundboden Kulturschalen vorbereitet. Um die geringer Haftung Kultur Herstellung von Platten und 30μl Poly-HEMA (6 mg Polyhydroxyethylmethacrylat in 1 ml 95% EtOH)-Lösung in jede Vertiefung einer 96-Well Corning Zellkulturschale zugegeben. Die 96-Well-Platten werden in einem 37 ° C nicht-befeuchteten Inkubator, um das Ethanol, l verdunsten inkubierteaving eine Folie aus Poly-HEMA auf jeder Vertiefung. Diese Poly-HEMA-Film verhindert, dass Zellen Befestigung am Boden der Vertiefung und zwingt die Zellen in Suspension 18 wachsen. [Alternativ kann Ultra-Low Befestigung Kultur-Platten (Corning) anstelle von Poly-HEMA beschichteten Schalen verwendet werden.] </li><li> Nach dem niedrigen Kraftschlußbeiwert Kultur-Platten hergestellt werden, trypsinieren einen Teller mit Eierstockkrebs Zellen, Pellet die Zellen in einer Tischzentrifuge (Heraeus) bei 900 RCF für 3 Minuten, den Überstand aspirieren und in 10% Basismedium erneut zu suspendieren. </li><li> Zählen Sie die Zellen mit einer Zählkammer. </li><li> Die Konzentration der Zellen, so dass es 100 Zellen pro 50 ul von 10% Base-Medium. </li><li> In 50 ul der gleichmäßig suspendiert verdünnten Zellsuspension in jede Vertiefung der 96-Well Poly-HEMA beschichteten Kulturschale. </li><li> Inkubieren der Platte mit 96 Vertiefungen bei 37 ° C Zellkulturbrutschrank für 16 Stunden (der Betrag der Zeit zu erhöhen oder abhängig von der Menge an Zeit, die verringert werdeneine bestimmte Zelllinie zu mehrzelligen Sphäroiden oder gewünschten experimentellen Bedingungen) bilden, um die Eierstockkrebszellen zu Cluster zusammen zu ermöglichen, wobei ein einzelner vielzelligen Sphäroid in jede Vertiefung. Einige Tumorzellen Apoptose während dieser Zeit unterziehen, so ist es wichtig, eine Zeit vor der Induktion der Apoptose zu wählen. </li></ol> 2. Mesothelzelle Monoschichtbildung <ol><li> In einer Zellkultur Haube vor die Vertiefungen von einer 6-Well-Glasboden MatTek Schale mit Fibronectin durch Zugabe von 2 ml einer 5μg Fibronectin / ml PBS-Lösung in jede Vertiefung der Schale und Inkubieren bei Raumtemperatur für 30 Minuten. Die optische Qualität der Glas-Böden in MatTek Gerichte erlauben hochauflösende mikroskopische Bildgebung. </li><li> GFP-exprimierende Mesothelzellen werden in 10% Base-Medium kultiviert. Trypsinieren eine Platte von Mesothelzellen, Spin-down in einer Tischzentrifuge (Heraeus) bei 900 UPM für 3 Minuten, den Überstand aspirieren, und in 10% Basismedium erneut zu suspendieren. DieMesothelzellen welche hier bereits die GFP exprimieren, wenn sie stammen, aber nicht markiertem Mesothelzellen können durch Transfektion mit einem Plasmid, das GFP-cDNA oder Vorinkubation der Zellen in einem grün fluoreszierenden Zellen-Tracker-Farbstoff (Invitrogen) hergestellt werden. </li><li> Nach dem 30-minütigen Inkubation Fibronektin (in Schritt 2.1), waschen Sie die Vertiefungen der MatTek Schüssel mit 2 ml PBS. </li><li> Absaugen des PBS und Platte 6 x 10 5 Mesothelzelle pro Vertiefung in jede Vertiefung der 6-Well-MatTek Schale. Inkubieren MatTek Schale in einem 37 ° C Zellkultur Inkubator über Nacht, damit die Mesothelzellen auf der Schale an und bilden eine Monoschicht. </li></ol> 3. Mesothelzelle Abstand Assay <ol><li> Verwenden Sie eine Pipette, die Eierstockkrebs Sphäroiden aus dem 96-Loch-Poly-HEMA beschichteten Platte zu sammeln. </li><li> Saugen Sie das Medium aus einem Brunnen der 6 gut MatTek Schale mit Mesothelzellen Zellmonolayer. Waschen Sie einmal mit 2 ml PBS. In all der Sphäroide aus dem 96-Loch-pspät, um eine Vertiefung der MatTek Schale (~ 3x die Anzahl von Kugeln, die er zu machen Sphäroide Landung ist für den Teil der Schale, die nicht abgebildet werden kann abgebildet werden). </li><li> Setzen Sie den MatTek Schüssel auf der Bühne eines umgekehrten Weitfeld-Fluoreszenzmikroskop ausführen kann Zeitraffer-Bildgebung für die Dauer von mindestens 8 Stunden. Verwenden Sie einen motorisierten Tisch zu Bild mehrere Positionen in der Schale, mit mehreren Sphäroid Interkalation Veranstaltungen, in einem einzigen Experiment. Wir verwenden eine Nikon Ti-E umgekehrt Motorisierte Weitfeld-Fluoreszenz Zeitraffer-Mikroskop mit integriertem Perfect Focus System und Low [20x-0.75 numerischen Apertur (NA)] Vergrößerung / NA Differential Interferenz Kontrast (DIC)-Optik, eine Nikon Halogen-Transilluminator mit 0,52 NA großem Arbeitsabstand (LWD) Kondensator, Nikon schnelle (&lt;100-Millisekunden Schaltzeit) Anregungs-und Emissions-Filter (GFP Ex 480/40, Em 525/50, RFP-mCherry Ex 575/50 Em 640/50), Sutter schnell übertragen und Epifluoreszenz Lichtweg Smart-Absperrter, eine Nikon linear-kodierte motorisiertem Tisch, ein Hamamatsu ORCA-AG gekühlt Charge-Coupled Device (CCD)-Kamera, ein custom-built Mikroskop Inkubationskammer mit Temperatur und CO 2-Steuerung, Nikon NIS-Elements AR-Software Version 3, und ein TMC Schwingungsisolation Tisch. </li><li> Die Eierstockkrebszelllinien Sphäroide wird an der Unterseite der Schale absetzen und heften sich an die Mesothelzellen Zellmonolayer. Sammle GFP, RFP und Bilder von Phase 20 + Sphäroid / Monoschicht Interaktionen, alle 10 Minuten, für 8 Stunden. </li><li> Die RFP-exprimierenden Ovarialkarzinomzellen Sphäroide wird in die GFP-exprimierenden Mesothelzelle Monoschicht ein Loch in der Monoschicht einzudringen. Nach 8 Stunden Messung der Größe der Löcher, indem es die schwarzen Löcher in den GFP-Bild mit Elements Software (oder eine andere geeignete Software wie Bild J). Normalisieren der Lochgröße auf die ursprüngliche Größe Sphäroid durch Dividieren der Lochgröße zu 8 Stunden nach der Größe des Sphäroids in der entsprechenden RFP Bild zum Zeitpunkt Null. In diesem ehemaligenreichlich, wurde das Loch Größe nur einmal gemessen, sondern es kann mehrere Male während der 8 Stunden Experiment gemessen werden zum besseren Verständnis der Dynamik der Einlagerung. </li></ol> 4. Repräsentative Ergebnisse In diesem Beispiel, verglichen wir die Fähigkeit von Mesothelzellen Abstand OVCA433 Ovarialkarzinomzellen Sphäroide, die gedämpft Expression von Talin-1 bis OVCA433 Sphäroide zu kontrollieren. OVCA433 Sphäroide aus jeder Gruppe wurden in einen MatTek Schale mit ZT Mesothelzelle Monoschichten zugegeben. Sechs Sphäroide aus jeder Gruppe wurden alle 10 Minuten für 8 Stunden (Abbildung 4, Movie 3, Movie 4) abgebildet. Die Löcher in der Monoschicht durch den sich ausbreitenden kugeln wurden gemessen und sechs Positionen aus jeder Gruppe wurden gemittelt. 4 zeigt, dass die durchschnittliche Fläche der lichten Weite von Talin 1 Zuschlag Sphäroide erzeugt wesentlich kleiner als die durchschnittliche Fläche durch Steuerung Sphäroide geschaffen wurde, was darauf hindeutet, dass Talin für Mesothelzellen Clearance durch O erforderlichVCA433 Eierstockkrebs Sphäroide. <img alt="1" src="/files/ftp_upload/3888/3888fig1.jpg" /> Abbildung 1. Eierstockkrebs Metastasen. Primäre Tumoren der Eierstöcke entwickeln sich entweder aus dem Eierstock Oberflächenepithel oder Eileiter. Tumorzellen / Cluster brechen aus dem Primärtumor und sammeln sich in der Bauchhöhle. Tumorzellen können dann aggregieren, um mehrzellige Sphäroide zu bilden. Sphäroide dann auf die Mesothelzellen Zellmonolayern Auskleidung der Bauchhöhle zu befestigen. Die Mesothelzellen von unterhalb des beigefügten Eierstockkrebs Sphäroids ausgeschlossen, so dass die Kügelchen den Zugriff auf die zugrunde liegende Basalmembran zu gewinnen. Movie 1. Eierstockkrebs Metastasen. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">Hier klicken, um Film anzusehen</a> . <img alt="2" src="/files/ftp_upload/3888/3888fig2.jpg" /> Abbildung 2. </strong&gt; Mesothelzellen Linie die Oberfläche der menschlichen Peritoneum und von unterhalb Ovarialkarzinomzellen Implantate ausgeschlossen. <img alt="Abbildung 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> Abbildung 3. Mesothelzellen Abstand Assay. Eierstockkrebs Sphäroide durch Inkubation von 100 ausgebildet RFP-exprimierenden Eierstockkrebs Zellen pro Vertiefung in einem Poly-HEMA beschichteten 96-Well-Rundboden Kulturschale bei 37 ° C für 16 Stunden. Poly-HEMA verhindert, dass die Zellen aus Befestigung an der Kulturschale, so dass die Zellen in Suspension verbleiben und aneinander haften, um einen Cluster pro Vertiefung zu bilden. Mesothelzellen Zellmonoschichten werden durch Beschichten 6x10 5 Mesothelzellen pro Well in einer 6-Well-Fibronectin beschichteten MatTek Schale und Inkubation der Platte bei 37 ° C für 16 Stunden hergestellt. Die Sphäroide werden dann an den MatTek Schale mit Mesothelzellen Monoschicht übertragen und die beiden Zellpopulationen werden alle 10 Minuten für 8 Stunden unter Verwendung eines Nikon Ti-E I abgebildetnverted Motorisierte Weitfeld-Fluoreszenz Zeitraffer-Mikroskop und Software-Elemente. Movie 2. Mesothelzellen Abstand Assay. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">Hier klicken, um Film anzusehen</a> . <img alt="Abbildung 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> Abbildung 4. Dämpfung von Talin-1-Expression in OVCA433 Sphäroide sinkt Mesothelzellen Clearance Fähigkeit. OVCA433 Sphäroide (rot) mit und ohne abgeschwächte Expression von Talin-1 durften zu befestigen und dringen in eine ZT Mesothelzellen Monolayer (grün). Die beiden Zellpopulationen wurden alle 10 Minuten für 8 Stunden mit einer Nikon Ti-E umgekehrt Motorisierte Weitfeld-Fluoreszenz Zeitraffer-Mikroskop und Software-Elemente abgebildet. Die Grafik zeigt, dass Talin 1 Dämpfungsignifikant abnimmt Mesothelzelle Clearance (Quantil-Plot mit grünen Balken an der Mittel). Movie 3. Kontrolle OVCA433 Sphäroide (rot) in eine Invasion Mesothelzellen Monolayer (grün). <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">Hier klicken, um Film anzusehen</a> . Movie 4. Dämpfung von Talin-1-Expression in OVCA433 Sphäroide (rot) nimmt Mesothelzellen (grün)-Clearance Fähigkeit. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">Klicken Sie hier, um Film anzusehen</a> .

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Der &quot;Ausverkauf Mesothelzellen Assay&quot; präsentiert hier verwendet Zeitraffer-Mikroskopie, um die Wechselwirkungen von Eierstockkrebs mehrzelligen Sphäroiden und Mesothelzellen Zellmonolayern überwachen, in der großen räumlichen und zeitlichen Detail. Zuvor hatten mehrere Gruppen 8-14 Endpunkt-Assays verwendet werden, um zu zeigen, dass Eierstock-Krebszellen zu befestigen und dringen in Mesothelzelle Monoschichten. Dieser Assay ist einzigartig, da sie verwendet fluoreszenzmarkierten Zellen Tumorz...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog-Zahl </td><td> Kommentare </td></tr><tr><td> OVCA433 Eierstockkrebszellen </td><td></td><td></td><td> Geschenk von Dr. Dennis Slamon </td></tr><tr><td> ZT Mesothelzellen </td><td></td><td></td><td> Geschenk von Dr. Tan Ince </td></tr><tr><td> Medium 199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> Cell-Anwendungen Inc. </td><td> 117-500 </td><td></td></tr><tr><td> FBS-hitzeinaktiviertem </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> Pen-Strep </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Platten mit 96 Vertiefungen </td><td> Corning Costar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polyhydroxyethylmethacrylat (Poly-HEMA) </td><td> Sigma Aldrich </td><td> 192066-25G </td><td> Für Poly-HEMA-Lösung aufzulösen 6 mg Poly-HEMA-Pulver in 1 ml 95% EtOH </td></tr><tr><td> EtOH </td><td> Pharmco-Aaper </td><td> 111ACS200 </td><td> Verdünnen Sie bis 95% in dH 2 0 </td></tr><tr><td> Zellkultur Haube </td><td> NuAire </td><td> NU-425-300 </td><td></td></tr><tr><td> Zellkultur-Inkubator </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> Inkubator für Poly-HEMA-Platten </td><td> Labline Instruments </td><td> Kaiserliche III 305 </td><td></td></tr><tr><td> Tischzentrifuge </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 gut Glasboden-Schüssel </td><td> MatTek corp. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> Fibronektin </td><td> Sigma </td><td> F1141-1MG </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21 bis 040-CV </td><td></td></tr><tr><td> Zeitraffer-Mikroskop: </td><td></td><td></td><td></td></tr><tr><td> Mikroskop </td><td> Nikon </td><td></td><td> Ti-E Inverted Motorisierte Fluoreszenz-Zeitraffer-Mikroskop mit integriertem Perfect Focus System </td></tr><tr><td> Objektiv </td><td> Nikon </td><td></td><td> 20X-0.75 numerische apeture </td></tr><tr><td> Halogen-Transilluminator </td><td> Nikon </td><td></td><td> 0,52 NA Kondensor für langen Arbeitsabstand </td></tr><tr><td> Anregungs-und Emissionsfilter </td><td> Chroma Single-Pass-Filter in Nikon-Gehäuse </td><td></td><td> GFP-Ex 480/40, Em 525/50 RFP-mCherry Ex 575/50 Em 640/50 </td></tr><tr><td> Übertragen und Epifluoresce Lichtweg </td><td> Sutter </td&gt; <td></td><td> Smart-Fensterläden </td></tr><tr><td> Linear-kodierte Motortisch </td><td> Nikon </td><td></td><td></td></tr><tr><td> Gekühlte Charged-Coupled Device-Kamera </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Mikroskop Inkubationskammer mit Temperatur und CO 2-Steuerung </td><td> custom-built </td><td></td><td></td></tr><tr><td> Schwingungsisolierung Tisch </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elements-Software </td><td> Nikon </td><td></td><td> Version 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

Eierstockkrebs ist die fünfte führende Ursache von Krebs verursachten Todesfälle in den Vereinigten Staaten ein. Trotz eines positiven ersten Reaktion auf Therapien, entwickeln 70 bis 90 Prozent der Frauen mit Eierstockkrebs neue Metastasen, und die Rezidivrate ist oft tödlich 2. Es ist daher notwendig, zu verstehen, wie sekundäre Metastasen entstehen, um bessere Behandlungen für Zwischen-und Spätstadium Eierstockkrebs entwickeln. Ovarialkarzinom Metastasen tritt auf, wenn bösartige Zellen aus dem Primärtumor Ort lösen und zu verbreiten, in der gesamten Bauchhöhle. Die disseminierten Zellen können vielzelligen Cluster oder Sphäroide, die entweder ungebunden bleiben wird, oder das Implantat auf Organe in der Bauchhöhle 3 (Abbildung 1, Movie 1) zu bilden. Alle Organe in der Bauchhöhle mit einem einzigen, kontinuierlichen, Schicht von Mesothelzellen 4-6 (2) ausgekleidet ist. Allerdings sind Mesothelzellen abwesend von untenPeritoneal Tumormassen, wie durch eine elektronenmikroskopische Aufnahme von Studien ausgeschnitten humanem Tumorgewebe Abschnitten 3,5-7 (2) offenbart. Dies deutet darauf hin, dass Mesothelzellen von der Unterseite der Tumormasse sind von einem unbekannten Prozess ausgeschlossen. Zurück in vitro Experimente zeigten, dass primäre Ovarialkarzinom Zellen effizienter zu befestigen extrazellulären Matrix, als Mesothelzellen 8, und neuere Studien zeigten, dass die primäre peritoneale Mesothelzellen tatsächlich eine Barriere für Eierstockkrebs Zelladhäsion und Invasion (im Gegensatz zu Adhäsion und Invasion im Vergleich auf Substraten, die nicht mit Mesothelzellen bedeckt waren) 9,10. Dies würde darauf hindeuten, dass Mesothelzellen als Barriere gegen Eierstock-Krebs Metastasen handeln. Die zellulären und molekularen Mechanismen, durch die Eierstockkrebszellen diese Barriere zu durchbrechen, und schließt die Mesothel haben bis vor kurzem unbekannt geblieben. Hier beschreiben wir the-Methodik für ein in-vitro-Test, dass die Modelle der Wechselwirkung zwischen Eierstockkrebszelllinien Sphäroide und Mesothelzellen in vivo (Abbildung 3, Movie 2). Unser Protokoll wurde von bisher beschriebenen Methoden zur Analyse Eierstocktumorzelllinien Interaktionen mit Mesothelzellen Monoschichten 16.8 angepasst, und wurde zum ersten Mal in einem Bericht zeigt, dass Eierstocktumorzellen verwenden einen Integrin-abhängige Aktivierung von Myosin und Zugkraft auf den Ausschluss von der Förderung beschrieben Mesothelzellen von unter einem Tumor Sphäroid 17. Dieses Modell nutzt die Vorteile der Zeitraffer-Fluoreszenz-Mikroskopie, die beiden Zellpopulationen in Echtzeit zu überwachen und bietet räumliche und zeitliche Informationen über die Wechselwirkung. Die Eierstockkrebs exprimieren rot fluoreszierendes Protein (RFP), während die Mesothelzellen grün fluoreszierendes Protein (GFP). RFP-exprimierenden Eierstockkrebszelllinien Sphäroiden auf die GFP-exprimierenden Mesothelzellen Monoschicht zu befestigen. Die Sphäroide Verbreitung, einzudringen, undzwingen die Mesothelzellen beiseite schaffen ein Loch in der Monoschicht. Dieses Loch wird visualisiert als der negative Raum (schwarz) in der GFP-Bild. Die Fläche des Loches kann dann gemessen werden, um quantitativ die Unterschiede hinsichtlich des Clearance-Aktivität zwischen der Kontrolle und Populationen von Eierstockkrebs und / oder Mesothelzellen werden. Dieser Assay erfordert nur eine kleine Anzahl von Eierstockkrebszellen (100 Zellen pro Sphäroid x 20-30 Sphäroide pro Bedingung), so ist es möglich, diesen Assay unter Verwendung von wertvollen primären Tumorzelle Proben. Ferner kann dieser Assay einfach für Hochdurchsatz-Screening angepasst werden.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

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

Die Mesothelzellen-Clearance-Test hier beschriebenen nutzt die Vorteile der fluoreszenzmarkierten Zellen und Zeitraffer-Video-Mikroskopie zu visualisieren und quantitativ zu messen die Wechselwirkungen von Eierstockkrebs mehrzelligen Sphäroiden und Mesothelzelle Monoschichten. Dieser Test modelliert die frühen Schritte der Metastasierung Eierstockkrebs.

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 ...

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

14.4K Aufrufe.  Harvard Medical School. Die Mesothelzellen-Clearance-Test hier beschriebenen nutzt die Vorteile der fluoreszenzmarkierten Zellen und Zeitraffer-Video-Mikroskopie zu visualisieren und quantitativ zu messen die Wechselwirkungen von Eierstockkrebs mehrzelligen Sphäroiden und Mesothelzelle Monoschichten. Dieser Test modelliert die frühen Schritte der Metastasierung Eierstockkrebs.

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Experimentelle Metastase Assay

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

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Medizin

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME Modelle für die 4D-Bildgebung lebender Zellen von Tumor: Mikroumgebung Wechselwirkungen, die Einfluss auf maligne Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodale Bildgebung von Angiogenese in einem Nude Rattenmodell der Brustkrebs Knochenmetastasen mittels Magnetresonanztomographie, volumetrische Computertomographie und Ultraschall

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotypische Dreidimensionale<em&gt; In Vitro</em&gt; Modellierung von Stromal-Epithelial Interaktionen beim Eierstockkrebs Initiation und Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Models von Knochenmetastasen

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Ein orthotopen Mausmodell der menschlichen Prostatakrebs-Metastasen

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

In vitro Anreicherung des Eierstockkrebs Tumor-initiierenden Zellen

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Die Quantifizierung von Intraperitonealtest Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modellierung der frühen Schritte der Eierstockkrebs Verbreitung in einer organotypischen Kultur der Menschen Peritonealhöhle

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...