Wir danken Dr. Leonard I. Zon in dessen Labor diese Techniken wurden ursprünglich entwickelt, Kristen Kwan und die späten Chi-Bin Chien für Geschenke von Plasmiden in dieser Arbeit verwendet; James Lister und David Raible für die Unterstützung bei Antikörper-Färbung und James Neiswender für die Mikroinjektion Video. Diese Arbeit wurde vom NIH R00AR056899-03 bis CJC finanziert

Ein. Screening für Melanom Onset Modifiers <ol><li> Erstellen Gateways Mitteleinstieg Klone durch PCR Amplifikation des Volllängen-offenen Leserahmen von Genen von Interesse (GOI) und Rekombinieren in pDONR 221 mit BP Clonase II (Invitrogen). Verwenden Multisite Gateway-Technologie (Invitrogen) zu rekombinieren p5E_mitfa, pME_GOI, Tol2kit Nr. 302 p3E_SV40polyA 6 und miniCoopR 5 bis Genen von Interesse unter der mitfa Promotors Platz in der miniCoopR Vektor (Abbildung 1A). </li><li> Injizieren 25 Picogramm eines jeden Klons zusammen mit 25 Picogramm Tol2 Transposase mRNA in ein-Zell Tg (mitfa: BRAF V600E); p53 (lf); mitfa (lf) dreifach homozygot Zebrafischembryonen wie zuvor 7 beschrieben. Inkubieren Sie die injizierten Embryonen bei 28,5 ° C. Entfernen Sie alle toten Embryonen bei 24 hpf. </li><li> Wählen transgene Tiere mit geretteten Melanozyten bei 72 hpf, indem Sie die Petrischale mit injizierten Embryonen auf einem Seziertisch mikroskopische im Auflicht vor einem weißen Hintergrund (Abbildung 1A). </li><li> Übertragen Tiere 3 L Tanks in der Kinderstube des Zebrafisch-Anlage bei 4 dpf wie zuvor 8 beschrieben. </li><li> Nach 2 Monaten, wählen die Tiere mit mindestens einer Fläche von Melanozyten Rettungs größer als 4 mm 2 (Abb. 1A). Es besteht eine starke Korrelation zwischen dem Grad der Melanozyten Rettung bei 72 hpf und dem Grad der Melanozyten Rettung auf 2 Monate. Wenn Embryonen mit Melanocyt Rettungs bei 72 hpf gepflückt wird die Mehrzahl von ihnen (~ 80%) über mindestens eine Fläche von Melanozyten Rettungs größer als 4 mm 2 bei 2 Monaten. </li><li> Bildschirm die ausgewählten Tieren wöchentlich für das Vorhandensein von Tumoren (Abbildung 1B). Es besteht eine starke Korrelation zwischen histologischen und morphologischen Veränderungen. Übergang von gutartigen zu malignen als morphologische Änderung, wenn Läsionen werden von der Oberfläche des Tieres angehoben erfasst. Isolieren tumortragenden animals für die Studie. </li><li> Zeichnen Melanom-free Überlebenskurven mit dem Alter in Wochen auf der Abszisse und Prozent Melanom-freies Überleben auf der Ordinate. Tiere mit Melanozyten, ein Gen von Interesse exprimieren, werden im Vergleich zu Kontrolltieren, dass ausdrückliche EGFP (Enhanced Green Fluorescent Protein) in Melanozyten (1C). Die mediane Tumor Einsetzen für Tiere mit miniCoopR-EGFP injiziert wird etwa 18 Wochen. Bestimmen Sie, ob sich die beiden Kurven statistisch unterschiedlich sind mit einem Log-Rank-Test. </li></ol> 2. Tumor Invasion Assay <ol><li> Wählen getrennt Zebrafisch zu entwickeln, dass Tumore dorsal, in einem Bereich zwischen dem hinteren Rand des Rautenhirn und vorderen Rand der Rückenflosse (Abbildung 2A). </li><li> Zwei Wochen nach Melanom Beginn einschläfern die Fische nach den Richtlinien der American Veterinary Medical Association Panel on Euthanasie spezifiziert und von der University of Massachusetts Medical School Ins zugelassentitutional Animal Care und Use Committee (IACUC). Insbesondere ist ein Fisch in einer Schüssel mit 0,6 mg / ml Tricain gelegt, bis gill Bewegung stoppt. </li><li> Einen Einschnitt in die Bauchhöhle des Fisches mit einem Skalpell und dann den Fisch in 4% Paraformaldehyd (PFA) über Nacht bei 4 ° C zur Fixierung. </li><li> Gönnen mit 0,5 M EDTA über Nacht bei 4 ° C bis zu entkalken. </li><li> Fix mit Formalin in Kassette Nacht für 1 Stunde zu entwässern, klar mit Xylol 3 mal 1 Stunde und dann behandeln mit Paraffin 2 mal für 2 Stunden jeweils bei 60 ° C. </li><li> Einbetten des Gewebes in Paraffin in einer Form und lassen es für 30 min bis 1 h zu verfestigen. </li><li> Machen Sie 5 &amp;mgr; m Schnitte, einer alle 50 um. Die Abschnitte sollten quer und durch die gesamte Läsion so daß der tiefste Punkt der Invasion identifiziert werden kann. Folgen von Hämatoxylin und Eosin-Färbung. </li><li> Bewerten Tumorinvasion durch Messung, ob Tumorzellen außerhalb der kollagenen Schicht compactum bleiben, in den dorsalen eindringenMuskulatur oder weiteren dringen in die Wirbelsäule (Abbildung 2B, C). Bestimmen statistische Differenz zwischen den beiden Sätzen von Abtastwerten. </li></ol> 3. Antikörperfärbung of Scale Melanozyten <ol><li> Make-up PO4-Puffer mit 80 ml ​​0,1 M Na 2 HPO 4 und 20 ml 0,1 M NaH 2 PO 4. Stellen Sie sicher, dass der pH-Wert beträgt 7,3. Schmink 2x fix Puffer enthaltend 8,0 g Saccharose, 0,15 ml 0,2 M CaCl 2 und 90 ml 0,2 M PO 4-Puffer, pH 7,3. Falls notwendig den pH-Wert auf 7,3 mit NaOH oder HCl dann bis zu 100 ml mit PO 4-Puffer. </li><li> Wiegen bis zu 300 mg feste PFA und fügen Sie ein Mikrozentrifugenröhrchen. Hinzufügen 5 mM NaOH bis zu einem Volumen gleich 4,5 x Masse in mg, PFA (z. B. 450 ul 5 mM NaOH bis 100 mg PFA). Wärme bei 60-70 ° C unter gelegentlichem Schütteln, bis das PFA gelöst ist. Spin down verbleibenden Partikeln und erholen 20% PFA Überstand. Machen frischen jeder Zeit. </li><li> Bereiten Fixierung Lösung mit 1x fix Puffer und 4% PFA. </li><li> Anästhesieren den Fisch in 0,17 mg / ml Tricain. </li><li> Mit einfallende Licht unter einem Binokular zupfen Rückenschuppen mit scharfen spitzen Pinzette, kümmert sich nicht um die Melanozyten-haltige Hälfte der Skala (3A) zu beschädigen. Skalen erfolgt direkt von der Zange in 1 ml Fixierungslösung und inkubieren bei Raumtemperatur, Drehen, für ≥ 2 hr. Übertragen Sie die Fische in Fisch Wasser und überwachen die Fische erfolgreiche Wiederherstellung zu bestätigen. </li><li> Zum Bleichen pigmentierten Melanozyten waschen Pauschaltabellen in PBST (1x PBS pH 7,4, 0,1% Triton X-100) 2 mal für 5 min bei Raumtemperatur. 1 ml frisch zubereiteten Bleichlösung mit 0,4 ml 10% KOH und 0,15 ml 30% H 2 O 2 in 5 ml mit dH 2 O. Setzen Sie auf Parafilm oder Kappe Sperren Gas aus Platzen die Röhren offen zu verhindern. Weiter Bleichen bis Pigment ist weg (10 min ist typisch) (3B, C). Waschen in PBST viermal 5 min bei Raumtemptur. </li><li> Block für mindestens 30 min in Block Lösung aus 1x PBS pH 7,4, 0,2% Triton X-100, 2 mg / ml BSA, 1% DMSO, 0,02% NaN 3 und 2% Lammserum hergestellt. Weglassen NaN 3, wenn die Entwicklung mit HRP-Reaktion. </li><li> Inkubieren mit primärem Antikörper in Block Lösung über Nacht bei Raumtemperatur. Sie müssen mindestens 400 ul Lösung über die Proben, um sie untergetaucht. </li><li> Waschen Sie die Waage 3 mal für 30 min in PBST bei Raumtemperatur. </li><li> Inkubieren mit sekundärem Antikörper von 2 h bis über Nacht in Block-Lösung bei Raumtemperatur. </li><li> Stain für 10 min mit PBST + 0,1 uM DAPI. </li><li> Waschen 3 mal für 5 min mit PBST. </li><li> Montieren an der Waage auf einem Glasträger in einem Tropfen Vectashield so dass die konkave Seite der Waage nach unten zugewandt Schiebers und legen ein Deckglas darüber. Seal die Ränder der Folie mit klarem Nagellack und beachten Sie die Objektträger unter einem Fluoreszenzmikroskop (Abbildung 3D, E, F, G </strong&gt;). </li></ol> 4. Transplantation Assay <ol><li> Bereiten Empfänger 2-3 Monate alten casper 9 Fische durch Bestrahlung mit 25 Gy Gamma-Bestrahlung einen Tag vor der Transplantation 10. Lassen Sie die Fische in Fisch Wasser erholen. </li><li> Euthanize eine tumortragenden Fische wie zuvor beschrieben (Abschnitt 2.2). </li><li> Schneiden Sie den Tumor und steckte es in eine Petrischale mit ca. 5 ml filtersterilisiert 0.9x PBS mit 5% FBS. Würfeln, den Tumor mit einer Rasierklinge, während in Lösung. </li><li> Arbeiten bei Raumtemperatur mit einem P1000 Pipette, um einzelne Zellen zu verreiben. Stellen Sie die Lautstärke auf 25 ml mit Filter sterilisiert 0.9x PBS mit 5% FBS. </li><li> Anschließend wird durch 40 uM Sieb-Filter. </li><li> Spin in einer Eppendorf 5810R Tischzentrifuge bei 1.500 Umdrehungen pro Minute (453 rcf) für 10 min. </li><li> Entfernen des Überstandes und machen Zellsuspension auf etwa gewünschten Konzentration (10 7 Zellen annehmen, für einen 100 mm 3 Tumor). </li><li> Berechnen ter exakte Zellzahl unter Verwendung eines Hämozytometers und verdünnt das Zellsuspension so dass die endgültige Einspritzmenge ungefähr 5 ul ist. Wenn zum Beispiel 50.000 Zellen injiziert werden können, sollte die Zellsuspension auf eine Endkonzentration von 10.000 Zellen / ul verdünnt werden. Blättern Sie durch die Röhrchen mit der Zellsuspension alle paar Minuten bis Verklumpen der Zellen zu verhindern. </li><li> Waschen Sie ein 26s Gauge (Abschrägung tip) 701 N 10 ul Hamilton-Spritze 2-3 mal mit 100% Ethanol und 0,9 x PBS. Legen Sie die Spritze mit 5 ul der Zellsuspension. </li><li> Anästhesieren des bestrahlten Empfängers Fische in 0,17 mg / ml Tricain. Legen Sie den Fisch auf seiner Seite auf einem feuchten Kimwipe (Abbildung 4A). </li><li> Stabilisieren Sie den Fisch mit einer Hand und führen Sie die Nadel mit der Fase nach oben in einem Winkel von 45 ° in die Flanke der Fisch über die Bauchhöhle etwa auf halbem Weg zwischen der hinteren Begrenzung des Hinterhirn und vorderen Rand der Rückenflosse. Vorsichtig drücken Sie den Kolben (4B). &lt;/ Li&gt; <li> Lassen Sie die Fische in frischen Fisch Wasser erholen und beobachten die Fische täglich für Tumor-Transplantation (4C). Wenn Transplantation stattgefunden hat, können weitere Wachstum und die Entwicklung der Krankheit beobachtet werden. </li></ol>

<ol>
	<li>Beroukhim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

Keine Interessenskonflikte erklärt.

Die miniCoopR Methode ermöglicht die Expression von Genen von Interesse im Zebrafisch Melanozyten. Dieser Ansatz nutzt die Tatsache, dass der Zebrafisch mitfa Gen-Zelle autonom wirkt. Aus diesem Grund sind Melanozyten durch die geretteten miniCoopR Vektor um das bestimmte Minigen und jedes Gen von Interesse, auf die sie physisch gekoppelt ist, enthalten. Gerettet Melanozyten sind deutlich sichtbar und können bei Tieren, die als Einzel-Zell-Embryonen injiziert wurden erzielt werden. Eine angegebene Grad der Ch...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Gateway-Rekombination Reagenzien </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> Referenz 5 </td></tr><tr><td> Mitfa Antikörper </td><td></td><td></td><td> Referenz 5 </td></tr><tr><td> FITC Ziegen-Anti-Kaninchen-IgG-Antikörper </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> Vectashield </td><td> Vector Labs </td><td> H-1000 </td><td></td></tr><tr><td> casper Zebrafisch </td><td></td><td></td><td> Referenz 9 </td></tr><tr><td> 701N 10 ul Spritze </td><td> Hamilton / Fisher </td><td> 14-824 </td><td> &amp;nbsp; </td></tr><tr><td> 40 uM Filter </td><td> BD Falcon / Fisher </td><td> 352340 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr></tbody></table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

Genomic Studien der menschlichen Krebsarten haben eine Fülle von Informationen über die Gene, die in Tumoren 1,2,3 verändert werden erbracht. Eine Herausforderung, die aus diesen Studien ist, dass viele Gene verändert werden, und es kann schwierig sein, genetische Veränderungen, Tumorgenese aus, dass diejenigen entstanden zeitweilig während der Transformation fuhren zu unterscheiden. Um diesen Unterschied zu zeichnen ist es vorteilhaft, einen Assay, die quantitativ messen kann die Wirkung eines veränderten Gens auf Tumorinitiation und andere Prozesse, die Tumoren zu persistieren und verbreiten ermöglichen müssen. Hier präsentieren wir Ihnen eine schnelle Möglichkeit, eine große Anzahl von Kandidaten Melanom Modifikatoren im Zebrafisch mit einem autochthonen Tumormodell 4, Schritte zur Melanom Initiierung und Wartung umfasst screenen. Ein Schlüssel Reagenz in diesem Assay ist die miniCoopR Vektor, welche Paare eine Wildtyp-Kopie des mitfa Melanocyt Spezifikation Faktor an einen Gateway Rekombinationskassette in welche Kandidaten melAnomalien Gene 5 rekombiniert werden. Der Vektor weist eine miniCoopR mitfa rettende Minigen, der den Promoter, offenen Leserahmen und 3&#39;-untranslatierten Region des Wildtyp-mitfa Gen enthält. Es ermöglicht uns, Konstrukte mit voller Länge open reading frames von Kandidaten Melanom Modifikatoren. P53 (lf);; mitfa (lf) Zebrafischembryonen: Diese einzelnen Klone können dann in einzelne Zelle Tg (BRAF V600E mitfa) injiziert werden. Die miniCoopR Vektor wird von Tol2-vermittelte Transgenese 6 integriert und rettet Melanozyten. Weil sie physisch mit dem mitfa rettende Minigen gekoppelt sind, werden Kandidatengene gerettet Melanozyten, von denen einige zu transformieren und entwickeln sich zu Tumoren exprimiert. Die Wirkung eines Kandidatengens auf Melanom Initiierung und Melanomzelle Eigenschaften können unter Verwendung Melanom Überleben Kurven Invasionsassays, Antikörperfärbung und Transplantation Assays werden.

Ein-Zell-Tg (mitfa: BRAF V600E); p53 (lf); mitfa (lf) Zebrafischembryonen wurden mit dem Vektor, der die miniCoopR Melanom Onkogen SETDB1 5 oder EGFP, jeder unter der Kontrolle des Promotors mitfa injiziert. Embryonen mit Melanozyten Rettung wurden ausgewählt und reifen lässt. Bei 2 Monate alten Tieren mit Melanocyt Rettungs größer als 4 mm 2, wurden ausgewählt. Die Tiere wurden untersucht wöchentlich für Melanome. Tumorinzidenz Kurven für den Erwachsenen...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Eine schnelle Weg zum Bildschirm für die Melanom-Modifikatoren mit einem Zebrafisch autochthonen Tumormodell vorgestellt. Es nutzt die miniCoopR Vektor, der zur Expression von Melanom-Kandidat Gene in Melanozyten ermöglicht. Verfahren zur Melanom Überleben Kurven, eine Invasion Assay, ein Protokoll für Antikörperfärbung Maßstab von Melanozyten und Melanomzellen eine Transplantation Assay erhalten werden beschrieben.

Screening für Melanom Modifiers mit einem Zebrafisch Autochthone Tumormodell

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

screening-for-melanoma-modifiers-using-zebrafish-autochthonous-tumor

Research

JoVE Journal

Medicine

13.6K Aufrufe.  University of Massachusetts Medical School. Eine schnelle Weg zum Bildschirm für die Melanom-Modifikatoren mit einem Zebrafisch autochthonen Tumormodell vorgestellt. Es nutzt die miniCoopR Vektor, der zur Expression von Melanom-Kandidat Gene in Melanozyten ermöglicht. Verfahren zur Melanom Überleben Kurven, eine Invasion Assay, ein Protokoll für Antikörperfärbung Maßstab von Melanozyten und Melanomzellen eine Transplantation Assay erhalten werden beschrieben.

Serie: Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

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

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

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

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

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

Forschung

Medizin

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Die Beurteilung Teratogene Änderungen in einem Zebrafisch-Modell der Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Menschliche neuroendokrinen Tumor-Zelllinien als dreidimensionales Modell für das Studium der menschlichen neuroendokrinen Tumor-Therapie

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Patienten abgeleitet Zellkultur und Isolierung von CD133<sup&gt; +</sup&gt; Mutmaßliche Cancer Stem Cells von Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Ein Zebrafisch Modell des Diabetes mellitus und Stoffwechselkrankheiten Speicher

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

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 ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Eine mögliche Zebrafisch-Modell der Kidney Disease: Knockdown von<em&gt; Wnt5a</em&gt; Ursachen Zysten in Zebrafisch-Nieren

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Dreidimensionale Co-Kultur-Modell für Tumor-Stroma-Interaktion

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolierte Leberperfusion als Behandlung für Lebermetastasen von Aderhautmelanom

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Bewertung der Tumor-infiltrierenden Leukozyten-Untergruppen in einer subkutanen Tumormodell

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...