Diese Arbeit wurde zum Teil durch die National Institutes of Health R01 CA131990 (BFS und KRM). Live-Cell-Imaging wurde in der Mikroskopie, Imaging-und Ressourcen-Core unterstützt Zytometrie durchgeführt, zum Teil, von NIH-Center Zuschuss P30 CA22453 dem Karmanos Cancer Institute, Wayne State University und von der Perinatologie Research Branch des National Institute of Child Health and Development , Wayne State University.

1. Bereiten DQ-Substrate <ol><li> Erlauben Sie lyophilisiert DQ-Substrate auf Raumtemperatur vor dem Öffnen Fläschchen wärmen, bereiten Stammlösung von 1 mg / ml DQ-Substrat in deionisiertem Wasser, teilen sich in 50 ul Aliquots und lagern bei 4 ° C. </li></ol> Es kann notwendig sein, DQ-Substrat in einer Ultraschall-Wasserbad für ~ 5 min und Wärme auf 50 ° C, um die Dispersion zu erleichtern bewegen. <ol start="2"><li> Auftauen rBM auf Eis über Nacht bei 4 ° C; rBM sollte auf Eis zu allen Zeiten behandelt werden. </li><li> 10X Phosphat zu Kochsalzlösung (PBS), 80 g NaCl gelöst (1,37 M), 2 g KCl (0,027 M), 14,4 g Na 2 HPO 4 (1 M) und 2,4 g KH 2 PO 4 (0,02 M) in 800 ml Reinstwasser. Der pH-Wert von PBS-Puffer-Lösung auf 7,4 mit 1,0 M HCl. Bringen auf 1 Liter, durch Filtration sterilisiert. </li><li> Bereiten Kollagen I-Lösung auf Eis: 8 Teile gekühlt Kollagen I zu 1 Teil 10X PBS gekühlt. Der pH-Wertder Mischung auf 7,2 bis 7,6 unter Verwendung von sterilen 0,1 M NaOH. Überprüfen Sie pH-Wert mit pH-Streifen und auf das Endvolumen auf 10 Teile mit sterilem Wasser. </li><li> Bereiten DQ-Kollagen I: Kollagen I Matrix durch Verdünnen von DQ-Kollagen I in Kollagen I-Lösung in einem vorgekühlten Röhrchen bis zu einer Endkonzentration von 25 ug / ml und 2,4 mg / ml. </li><li> Planen DQ-Kollagen IV: rekonstituierte Basalmembran (rBM)-Matrix durch Verdünnen DQ-Kollagen IV in rBM (Cultrex oder Matrigel) in einer vorgekühlten Rohr bis zu einer Endkonzentration von 25 ug / ml und 12-15 mg / ml. Auf Eis mit sanften Pipettieren zu vermeiden Blasen zu erzeugen. </li></ol> 2. Bereiten Sie MAME Kokulturen Siehe Abbildung 1 für eine schematische Darstellung Kokulturen. <ol><li> Platz zwei rechteckige Kunststoff-Deckgläschen (sterilisiert in 70% Alkohol für 10 min und luftgetrocknet) in einem 35-mm-Kulturschale oder verwenden Sie eine ibiTreat Oberfläche 35-mm-μ-Dish. Anfahrt Hier sind für beide Deckgläser und μ-Gerichte, die wir für Studierende nutzenstirbt auf einem aufrechten Mikroskop. Für Untersuchungen an einem inversen Mikroskop, verwenden wir nur μ-Dishes. </li><li> Mischen Sie die gewünschte Anzahl von Fibroblasten mit DQ-Kollagen I: Kollagen I-Matrix. Für die Langzeit-Co-Kulturen in den Abbildungen 2 und 3 dargestellt, verwenden wir 500 Fibroblasten in 10 ul 60 ul MEDIA Plus von DQ-Kollagen I: Kollagen I-Matrix. </li><li> Unter Verwendung eines 100-ul Mikropipette sorgfältig Pipette und verteilt 70 ul von Fibroblasten: DQ-Kollagen I: Kollagen I-Matrix über die gesamte Oberfläche jedes Deckglas und lassen bei 37 ° C in einem befeuchteten Inkubator ohne CO 2 für 30 min zu verfestigen. </li><li> Transfer-35-mm-Schalen mit Deckgläsern bis 37 ° C Inkubator mit 5% CO 2 für 10 min ins Gleichgewicht kommen. </li><li> Entfernen von Inkubator und lassen Sie die 35 mm-Schalen unter der Haube, bis sie auf Raumtemperatur kommen. </li><li> In 60 ul von DQ-Kollagen IV: RBM-Matrix auf der Oberseite des erstarrten DQ-Kollagen I: Kollagen I Matrix mit eingebetteten Fibroblasten. Mit Pipettenspitze, ca.refully verbreiten DQ-Kollagen IV: RBM Matrix gleichmäßig und vermeidet Kratzer auf der Fibroblasten: DQ-Kollagen I: Kollagen I-Matrix. </li><li> Transfer-35-mm-Schalen mit Deckgläsern bis 37 ° C Inkubator mit 5% CO 2 für 10 min zu verfestigen. Während die DQ-Kollagen IV: RBM Matrix wird Erstarren trypsinieren und zählen Epithelzellen. </li><li> RBM Matrix: Platz 50 ul einer epithelialen / Tumor Zellsuspension auf Deckgläsern mit DQ-Kollagen IV beschichtet. Platz 35-mm-Schalen mit den Deckgläschen in eine 37 ° C Inkubator. Erlauben Sie 40 bis 60 min für die Zellen, um zu DQ-Kollagen IV anhängen: RBM-Matrix. Für die langfristige Kokulturen in den 2 und 3 dargestellt, verwenden wir 2500 Epithel oder Tumorzellen. </li><li> 2 ml Kulturmedium mit 2% rBM zu jedem 35-mm-Schale. Wenn Sie jegliche medikamentöse Behandlungen, sollten die Medikamente in dieser Zeit hinzugefügt werden. </li><li> Inkubieren gewünschten Zeit vor der Bildgebung. Für die langfristige Kulturen, Medien sollten alle 3-4 Tage gewechselt werden. Die Zellen aufrechterhalten werden kannin ihrer normalen Kulturmedium. Für MAME Kokulturen von Brust-Zelllinien verwenden wir Epithelial Basal Medium (MEBM-PRF) mit MEGM SingleQuots ergänzt. </li></ol> 3. Führen 4D (3D + Zeit) Imaging an lebenden MAME Kokulturen durch konfokale Mikroskopie <ol><li> Für den Proof-of-Prinzip Studien dargestellt, wurden MAME Kokulturen mit einem fluoreszierenden Farbstoff Zelle Tracking gemäß unserer öffentlichen Verfahren 1 bezeichnet. Nach dem Waschen mit PBS wurden die Co-Kulturen mit 5 uM CellTracker Orange MEGM Weg für 45 min bei 37 ° C-Inkubator inkubiert, gewaschen mit PBS gewaschen und mit vorgewärmtem MEGMmedium für 30 min bei 37 ° C mit 5% CO 2-Inkubator vor dem Belichten . </li><li> Bild MAME Kokulturen leben bei niedriger Vergrößerung (10X, Wasser Eintauchen Linse) auf einem Zeiss LSM 510 META-konfokalen Mikroskop. </li><li> Optische Schnitte in Intervallen in der gesamten Tiefe der Struktur erfasst. Für unsere Studien, erfassen wir optische Schnitte bei 10um Intervalle. </li><li> Verwenden Sie optische Schnitte, um Bilder in 3D zu rekonstruieren. Wir verwenden Endgeschwindigkeit, Software, um 3D-Rekonstruktionen zu erzeugen. </li><li> Machen Sie Filme von den 3D-Rekonstruktionen, so dass Strukturen, Zell: Zell-Interaktionen und Wechselwirkungen mit der umgebenden Matrix visualisiert werden können. Wir haben Filme mit Quiktime Player 7. </li></ol> 4. Repräsentative Ergebnisse Das MAME Kokulturen, die wir entwickelt sind eine gefügig experimentelles Modell für Live-Cell-Imaging in Echtzeit von Wechselwirkungen zwischen den verschiedenen zellulären Bestandteilen, die eine Brust-Tumor und dessen Mikroumgebung umfassen. Hier zeigen wir die Fähigkeit des MAME Modelle zu Zelle rekapitulieren: Zell-Interaktionen während der Brustkrebs-Progression und von lebenden Zellen, um diese Interaktionen über die Zeit zu folgen. Wir zeigen die Fähigkeit, Bild-Wechselwirkungen zwischen einem präinvasive MCF10.DCIS menschlichen Brust-Zelllinie und eine menschliche Brustkrebs-assoziierten Fibroblasten-Linie, WS-12TI, überein Zeitraum von mehr als drei Wochen in Kultur. Die Co-Kulturen wurden bei 3, 16 und 23 Tage abgebildet. Optische Schnitte durch das gesamte Volumen der Co-Kulturen wurden in 3D und repräsentative Beispiele rekonstruiert werden für jede der drei Zeitpunkten in 2 gezeigt. Die rote Fluoreszenz in 2 stellt den MCF10.DCIS und WS-12TI Zellen. Um zwischen den beiden Zelltypen und für die langfristige Kokulturen zu diskriminieren, kann man transfizieren oder transduzieren einzelnen Zelltypen mit fluoreszierenden Proteinen vor der Gründung der Kulturen. Ein MAME Kokultur MCF10.DCIS und WS-12TI Zellen, die unterschiedlich mit fluoreszierenden Proteinen für die Identifikation markiert ist in 3 dargestellt. Durch die Integration von Substraten, in diesem Fall DQ-Kollagen, in die MAME Kokulturen, können wir bewerten und zu quantifizieren Veränderungen über die Zeit bei der Proteolyse mit Übergang in einem invasiven Phänotyp assoziiert. Wir haben Methoden zur Quantifizierung der fluoreszierenden Spaltprodukte nachgewiesen, dass Ergebnisvon DQ-Kollagenabbau 2,3. Wir normalisieren Abbauprodukte in der gesamten 3D-Volumen des MAME Kokulturen auf einer Basis pro Zelle durch Markierung und Zählung Zellkerne. Darüber hinaus können wir lokalisieren den Abbau der extrazellulären und intrazellulären Kompartimenten und Quantifizierung der gesamten, intra-und extrazellulären Abbauprodukten wie wir zuvor 2 beschrieben. Die grüne Fluoreszenz in den 2 und 3 für Spaltprodukte der beiden DQ-Kollagen Substrate. Zu diesem Zeitpunkt sind differentiell-markierten Kollagene nicht zur Verfügung, würde uns erlauben, zwischen Abbau von Kollagen Typ IV und dem Abbau von Kollagen Typ I zu unterscheiden. Die Interaktionen in der MAME Kokulturen sind dynamisch und können im Laufe der Zeit ändern. Um zeitliche und dynamische Informationen zu erhalten, kann die gleiche Sichtfeld bebilderten und optische Schnitte durch die gesamte Probe des Volumens wiederholt über einen Zeitraum von Minuten bis zu Wochen verabreicht werden,somit das Sammeln von Daten in 4-D. In Abbildung 2 zeigen wir ziemlich dramatischen Veränderungen, die mehr als 23 Tagen auftreten: Veränderungen in der Zellzahl, Zellmigration und Zell-Interaktionen, Struktur Bände, Struktur Formen, Abweichungen der Strukturen von der Kugelgestalt, invasive Auswüchse und Proteolyse. Darüber hinaus demonstrieren wir auch eine allgemeine Veränderung des Volumens aufgrund der MAME Kokulturen Beeinträchtigung der umgebenden extrazellulären Matrix in diesem Zeitraum. An drei Tagen in 2A dargestellt, ist das Volumen um 110.000 3. Im Gegensatz dazu ist das Volumen des MAME Kokulturen bei 16 und 23 Tage nur 46.250 um 3, wie in den 2B und C dargestellt <img alt="1" src="/files/ftp_upload/3661/3661fig1.jpg" /> Abbildung 1. Schematische Darstellung der MAME Kokulturen. Kunststoff-Deckglas wird mit Kollagen I überzogen, mit einem DQ-Kollagen I und Fibroblasten (Magenta). Eine zweite Schicht aus rekonstituierten Basalmembran (rBM) mit DQ-collagen IV wird angefügt. Tumorzellen (rot) sind auf der Oberseite ausplattiert und 2% RBM in Kulturmedien ist überlagert (weiße Punkte) mit jedem Wechsel der Medien. Grün steht für die fluoreszierenden Spaltprodukte des DQ-Kollagen I und IV. <img alt="2" src="/files/ftp_upload/3661/3661fig2.jpg" /> Abbildung 2. MAME Kokulturen von MCF10.DCIS menschlichen Brust Zellen und WS-12TI menschlichen Brust Fibroblasten. Die Fibroblasten wurden in Kollagen I / DQ-Kollagen I und überlagert mit RBM mit DQ-Kollagen IV eingebettet. Die DCIS Zellen wurden dann auf der rBM überlagert und mit Medien, die 2% rBM ausgesät. In den 23 Tage in Kokultur hier dargestellten es Zellproliferation, die Degradation der DQ-Kollagen IV und I (grün) und Kollagen-Matrices wie durch die Reduzierung des Volumens des Co-Kulturen angegeben. Die Zellen wurden durch Markierung mit CellTracker Orange in situ vor der Bildgebung (rot) lokalisiert. Die gleichen Live-Co-Kulturen wurden im Laufe der 23 Tage mit Bildern von C eingefangen beobachtetonfocal Mikroskopie bei 3, 16 und 23 Tagen der Kokultur. Die sich daraus ergebenden optischen Scheiben wurden in 3D mit Endgeschwindigkeit, Software rekonstruiert. Vergrößerung, 10X. <img alt="Abbildung 3" src="/files/ftp_upload/3661/3661fig3.jpg" /> Abbildung 3. MAME 16 Tage Kokulturen von MCF10.DCIS menschlichen Brust Zellen und WS-12TI menschlichen Brust, dass Fibroblasten Express RFP (rot) und YFP (weiß) sind. Kokulturen wurden hergestellt und analysiert wie in 2. Die beiden Zelltypen hier gezeigt hatte, wurden jedoch unterschiedlich, so dass sie voneinander unterschieden werden könnten beschriftet. Die Bilder mit höherer Vergrößerung in dieser Figur, um den in 2 verglichen, zeigen Proteolyse von DQ-Kollagen IV an der Oberfläche der Zellen und DCIS diffuse Proteolyse von DQ-Kollagen I in Bereichen nahe den Fibroblasten. Vergrößerung, 20X.

<ol>
	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

Wir haben nichts zu offenbaren.

Wie gezeigt, kann die MAME Kokulturen für Live-Cell-Imaging in Echtzeit von Wechselwirkungen zwischen den verschiedenen zellulären Bestandteilen, die eine Brust-Tumor und dessen Mikroumgebung umfassen verwendet werden. In laufenden Studien in unserem Labor haben wir MAME Kokulturen verwendet werden, um proteolytische Stoffwechselwege mit Übergang von DCIS zu einem invasiven duktalen Karzinoms verbunden sind, sowie die Wechselwirkungen zwischen proteolytische Stoffwechselwege und andere Wege in diesem Übergang, wie C...

<table border="1"><tbody><tr><td> Reagenz / Ausrüstung </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Rekonstituierte Basalmembran (Cultrex) </td><td> Trevigen </td><td> 3445-005-01 </td><td> Vergleichbar mit Matrigel (BD Biosciences) </td></tr><tr><td> Kollagen I </td><td> Zusammenhalt Laboratories </td><td> 5005-B </td><td></td></tr><tr><td> DQ-Substrate (Kollagen I, IV) </td><td> Invitrogen </td><td> DQ-col I-D12060 DQ-col IV D12052 </td><td></td></tr><tr><td> 22-mm Kunststoff-Deckgläschen </td><td> Fisher Scientific Co. </td><td> 12-547 </td><td> Halbiert, lassen Sie in 70% Ethanol für 10 min an der Luft trocknen und vor dem Gebrauch. </td></tr><tr><td> Zellkulturmedium </td><td> Lonza </td><td> MEBM-PRF CC-3153 MEGM CC-4136 </td><td> Phenolrot-freiem </td></tr><tr><td> ibiTreat Oberfläche μ-Dish </td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Endgeschwindigkeit, Software </td><td> Perkin-Elmer </td><td> Version 5.5 </td><td> Andere Marken der Imaging-Software lassen sich 3D-Rekonstruktionen und Filme zu erzeugen. </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

Wir haben 3D-Kokultur-Modelle entwickelt, die wir als MAME (m ammary ein rchitecture und m icroenvironment ngineering e), und ihnen werden für Live-Cell-Imaging in Echtzeit von Zell: Zell-Interaktionen. Unser oberstes Ziel war es, Modelle, die die Architektur des präinvasive Brustläsionen ihre Progression zu einem invasiven Phänotyp zu studieren rekapitulieren zu entwickeln. Insbesondere haben wir Modelle der Interaktion zwischen den prä-maligne Zellen Brustepithelzellen Varianten und anderen Zelltypen der Tumor-Mikroumgebung, die in verstärkt oder verringert wird das Fortschreiten der präinvasive Brustepithelzellen invasive duktale Karzinome verwickelt sind zu analysieren. Andere Zelltypen, die bisher untersucht sind Myoepithelzellen, Fibroblasten, Makrophagen und Blut und Lymphe mikrovaskuläre Endothelzellen. Zusätzlich zu den MAME Modelle, die zur zellulären Wechselwirkungen innerhalb der Brust während rekapitulieren werdenTumorprogression, haben wir vergleichbare Modelle für die Progression von Prostatakrebs entwickelt. Hier veranschaulichen wir die Verfahren für die Festlegung der 3D-Kokulturen zusammen mit der Verwendung von lebenden Zellen und einem funktionellen Proteolyse-Assay, um den Übergang von Co-Kulturen von Brust-duktales Carcinoma in situ (DCIS) und Fibroblasten zu einer invasiven Phänotyp im Laufe der Zeit folgen, in in diesem Fall mehr als 23 Tage in Kultur. Die Co-Kulturen MAME bestehen aus mehreren Schichten. Fibroblasten sind in der unteren Schicht von Kollagen Typ I eingebettet. Auf daß eine Schicht aus rekonstituierte Basalmembran (rBM), auf dem DCIS Zellen geimpft worden. Eine abschließende Deckschicht aus 2% rBM enthalten ist und aufgefüllt mit jedem Wechsel der Medien. Um das Bild Proteolyse mit dem Fortschreiten zu einem invasiven Phänotyp assoziiert, verwenden wir Farbstoff-abgeschreckten (DQ) fluoreszierenden Matrix Proteine ​​(DQ-Kollagen I mit der Schicht aus Kollagen I und Kollagen IV-DQ mit der mittleren Schicht der rB vermischteM) und beobachten lebende Kulturen mit Hilfe der konfokalen Mikroskopie. Optische Schnitte werden erfasst, verarbeitet und rekonstruiert in 3D mit Endgeschwindigkeit, Visualisierungs-Software. Im Laufe der 23 Tage in MAME Kokulturen, vermehren sich die Zellen DCIS und verbinden sich zu großen invasive Strukturen. Fibroblasten wandern und in diese Strukturen eingebunden invasive geworden. Fluoreszierende proteolytische Fragmente der Kollagene in Verbindung mit der Oberfläche des DCIS Strukturen gefunden, intrazellulär, und auch in der umgebenden Matrix dispergiert. Wirkstoffe, die proteolytische, Chemokin / Cytokin und Kinasen oder Veränderungen in der zellulären Zusammensetzung des Co-Kulturen der Invasivität reduzieren kann, was darauf hindeutet, dass MAME Modelle vorklinischen Screens für neue therapeutische Ansätze können verwendet werden.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

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

Wir haben 3D-Kokultur-Modelle für Live-Cell-Imaging entwickelt in Echtzeit von Interaktionen zwischen Brust-Tumor-Zellen und andere Zellen in ihrer Mikroumgebung, die Einfluss auf Progression zu einem invasiven Phänotyp. Diese Modelle können als Bildschirme für präklinische Arzneimittel auf parakrine-induzierte proteolytische, Chemokin / Zytokin-und Kinase-Signalwege in Invasivität-Ziels dienen.

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 ...

mame-models-for-4d-live-cell-imaging-tumor-microenvironment

Research

JoVE Journal

Medicine

14.3K Aufrufe.  Wayne State University. Wir haben 3D-Kokultur-Modelle für Live-Cell-Imaging entwickelt in Echtzeit von Interaktionen zwischen Brust-Tumor-Zellen und andere Zellen in ihrer Mikroumgebung, die Einfluss auf Progression zu einem invasiven Phänotyp. Diese Modelle können als Bildschirme für präklinische Arzneimittel auf parakrine-induzierte proteolytische, Chemokin / Zytokin-und Kinase-Signalwege in Invasivität-Ziels dienen.

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

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Orthotopen Xenografting of Human Luciferase-Tagged malignen peripheren Nervenscheide Tumorzellen für In vivo Prüfung von Kandidaten Therapeutika

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Forschung

Medizin

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Evaluation of Cancer Stem Cell Migration Mit Abschottung Mikrofluidiksysteme und Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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 ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Live-Imaging of Drug Responses in der Tumor-Mikroumgebung in Mausmodellen von Brustkrebs

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel Hochauflösende<em&gt; In vivo</em&gt; Bildtechnik, um die Dynamik der intrakranielle Strukturen zu Tumorwachstum und Therapeutics Studieren

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

Hochfrequenz-Ultraschall-Bildgebung der Maus Halslymphknoten

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Ein Hirntumor / Organotypischen Co-Kultursystem für die Untersuchung Tumor-Microenvironment und gezielte Arzneimitteltherapie

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Biolumineszenz-basierte Tumor Quantifizierung Verfahren zur Überwachung Tumorprogression und Therapieeffekte bei den Maus-Lymphom-Modelle

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Ex-vivo-Live-Imaging von Lungenmetastasen und deren Mikromilieu

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...