Nous remercions le Dr Leonard I. Zon en laboratoire dont ces techniques ont été initialement développées; Kristen Kwan et la fin des années Chi-Bin Chien pour les dons de plasmides utilisés dans ce travail; James Lister et David Raible de l&#39;aide pour coloration des anticorps, et James Neiswender pour la micro-injection vidéo. Ce travail a été financé par le NIH subvention R00AR056899-03 à CJC

1. Dépistage du mélanome Modificateurs Onset <ol><li> Créer des clones de passerelle moyen d&#39;entrée par PCR amplifiant le cadre de pleine longueur de lecture ouvert de gènes d&#39;intérêt (GOI) et la recombinaison en utilisant les batteries BP 221 pDONR clonase II (Invitrogen). Utilisez la technologie de passerelle multi-sites (Invitrogen) pour recombiner p5E_mitfa, pME_GOI, Tol2kit N ° 302 p3E_SV40polyA 6 et miniCoopR 5 afin d&#39;ajouter des gènes d&#39;intérêt dans le cadre du promoteur mitfa dans le vecteur miniCoopR (figure 1A). </li><li> Injecter 25 picogrammes de chaque clone ainsi que 25 picogrammes Tol2 ARNm transposase dans une cellule-Tg (mitfa: BRAF V600E); p53 (lf); mitfa (LF) d&#39;embryons de poisson zèbre triplement homozygotes 7 comme décrit précédemment. Incuber les embryons injectés à 28,5 ° C. Retirez tous les embryons morts à 24 hpf. </li><li> Sélectionnez animaux transgéniques avec mélanocytes secourus à 72 hpf en plaçant la boîte de Petri contenant des embryons injectés sur une dissection microscope sous la lumière incidente sur un fond blanc (figure 1A). </li><li> Transfert à 3 réservoirs animaux L dans la pépinière de l&#39;installation poisson zèbre à 4 dpf comme décrit précédemment 8. </li><li> A 2 mois, sélectionnez les animaux avec au moins une zone de sauvetage mélanocytes supérieure à 4 mm 2 (figure 1A). Il ya une forte corrélation entre le degré de sauvetage mélanocytes à 72 hpf et le degré de sauvetage mélanocytes moins 2 mois. Lorsque les embryons ayant sauvetage mélanocytes sont cueillies à 72 hpf, la majorité d&#39;entre eux (environ 80%) ont au moins une zone de sauvetage mélanocytes supérieure à 4 mm 2 à 2 mois. </li><li> Écran animaux sélectionnés hebdomadaires pour la présence de tumeurs (figure 1B). Il ya une forte corrélation entre les changements histopathologiques et morphologiques. Transition de bénigne à maligne est comptabilisé comme un changement morphologique lorsque les lésions se soulèvent à la surface de l&#39;animal. Isoler anima porteur d&#39;une tumeurls pour étude. </li><li> Tracer des courbes de survie sans mélanome avec l&#39;âge dans les semaines en abscisse et le mélanome pour cent de la survie sans en ordonnée. Les animaux atteints de mélanocytes qui expriment un gène d&#39;intérêt sont comparés à des animaux témoins qui expriment l&#39;EGFP (enhanced green fluorescent protein) dans les mélanocytes (figure 1C). L&#39;apparition de tumeur médiane pour animaux injectés avec miniCoopR-EGFP est d&#39;environ 18 semaines. Déterminer si les deux courbes sont statistiquement différentes en utilisant un test du log-rank. </li></ol> 2. Essai de l&#39;invasion tumorale <ol><li> Sélectionnez le poisson-zèbre isolé qui développent des tumeurs dorsalement, dans une région située entre le bord postérieur du cerveau postérieur et bord antérieur de la nageoire dorsale (figure 2A). </li><li> Deux semaines après l&#39;apparition du mélanome euthanasier les poissons selon les directives spécifiées par le Groupe American Veterinary Medical Association sur l&#39;euthanasie et approuvé par l&#39;Université de Massachusetts Medical School Insprotection des animaux et le Comité titutional utilisation (IACUC). Plus précisément, un poisson est placé dans une boîte avec 0,6 mg / ml tricaïne jusqu&#39;à ce que le mouvement s&#39;arrête branchies. </li><li> Faire une incision dans la cavité péritonéale du poisson avec un scalpel, puis placer le poisson dans du paraformaldéhyde 4% (PFA) pendant une nuit à 4 ° C pour la fixation. </li><li> Traiter avec 0,5 M d&#39;EDTA nuit à 4 ° C de la détartrer. </li><li> Fixer avec du formol dans la cassette du jour au lendemain, déshydrater pendant 1 heure, clair avec du xylène 3 fois pendant 1 heure et ensuite traiter avec de la paraffine 2 fois pour 2 heures chacune à 60 ° C. </li><li> Incorporer le tissu dans de la paraffine dans un moule et le laisser se solidifier pendant 30 min à 1 h. </li><li> Assurez-5 sections um, un toutes les 50 Pm. Les sections doivent être transversal et toute la lésion sorte que le point le plus profond de l&#39;invasion peut être identifié. Suivi de l&#39;hématoxyline et éosine. </li><li> Évaluer l&#39;invasion tumorale en mesurant si les cellules tumorales restent en dehors du collagène couche compacte, envahir la dorsalemusculature ou envahir plus loin dans la colonne vertébrale (figure 2B, C). Déterminer différence statistique entre les deux séries d&#39;échantillons. </li></ol> 3. La coloration d&#39;anticorps de mélanocytes échelle <ol><li> Maquillage PO4 tampon avec 80 ml ​​0,1 M Na 2 HPO 4 et 20 ml 0,1 M NaH 2 PO 4. Assurez-vous que le pH est de 7,3. Maquillage tampon fix 2x contenant 8,0 g de saccharose, 0,15 ml 0,2 M CaCl 2 et 90 ml 0,2 M PO 4, pH 7,3. Si nécessaire, ajuster le pH à 7,3 avec NaOH ou HCl, puis compléter à 100 ml avec tampon PO 4. </li><li> Peser jusqu&#39;à 300 mg de solide PFA et ajouter dans un tube à centrifuger. Ajouter 5 mM de NaOH à un volume égal à 4,5 x la masse, en milligrammes, de PFA (par exemple 450 ul 5 mM NaOH à 100 mg PFA). Chauffer à 60-70 ° C avec agitation jusqu&#39;à occasionnelle de la PFA soit dissoute. Isoler les particules restantes et récupérer 20% de PFA surnageant. Assurez frais à chaque fois. </li><li> Préparer une solution contenant de fixation 1x fix tampon et 4% de PFA. </li><li> Anesthésier les poissons de 0,17 mg / ml tricaïne. </li><li> Utilisation de la lumière incidente sous un microscope à dissection arracher écailles dorsales pointues en utilisant une pince à pointe fine, en prenant soin de ne pas endommager la moitié des mélanocytes contenant de l&#39;échelle (figure 3A). Placer les échelles directement à partir de la pince dans 1 ml de solution de fixation et incuber à température ambiante, tout en tournant, pour ≥ 2 h. Transférer le poisson dans l&#39;eau du poisson et de surveiller le poisson pour confirmer la récupération réussie. </li><li> Pour l&#39;eau de Javel mélanocytes pigmentés laver échelles fixes dans du PBST (1x PBS, pH 7,4, 0,1% de Triton X-100) 2 fois pendant 5 min à température ambiante. Ajouter 1 ml d&#39;une solution fraîchement préparée de blanchiment contenant 0,4 ml de KOH à 10% et 0,15 ml 30% H 2 O 2 dans 5 ml de dH 2 O. Mettez serrures parafilm ou casquette pour empêcher le gaz de l&#39;éclatement des tubes ouverts. Continuer jusqu&#39;à ce que le blanchiment pigment est parti (10 min est typique) (figure 3B, C). Laver à l&#39;PBST quatre fois pendant 5 min à température ambiantetérature. </li><li> Pour bloquer au moins 30 min dans une solution composée de blocs 1x PBS pH 7,4, 0,2% de Triton X-100, 2 mg / ml de BSA, 1% de DMSO, 0,02% NaN 3 et 2% de sérum d&#39;agneau. Laissez de côté NaN 3 si le développement de la réaction HRP. </li><li> Incuber avec l&#39;anticorps primaire en solution bloquer pendant une nuit à température ambiante. Vous devez avoir au moins 400 pi de solution sur les échantillons pour les maintenir immergés. </li><li> Laver les échelles 3 fois pendant 30 min dans du PBST à la température ambiante. </li><li> Incuber avec l&#39;anticorps secondaire de 2 heures ou toute la nuit dans une solution de bloc à température ambiante. </li><li> Colorer pendant 10 min avec du PBST + DAPI uM 0,1. </li><li> Laver 3 fois pendant 5 min avec du PBST. </li><li> Monter les échelles sur une lame de verre dans une goutte d&#39;Vectashield sorte que le côté concave de la balance tournée vers le bas en direction de la lame et de placer une lamelle couvre-objet en verre sur eux. Scellez les bords de la lame avec vernis à ongles transparent et observez les lames sous un microscope à fluorescence (figure 3D, E, F, G </strong&gt;). </li></ol> 4. Transplantation Assay <ol><li> Préparer destinataire 2-3 mois vieux casper 9 poissons par irradiation avec 25 Gy d&#39;irradiation gamma un jour avant la transplantation 10. Permettre aux poissons de se remettre dans l&#39;eau du poisson. </li><li> Euthanasier un poisson porteur d&#39;une tumeur comme décrit précédemment (section 2.2). </li><li> Coupez la tumeur et le mettre dans une boîte de Pétri avec environ 5 ml de filtre stérilisé 0.9x PBS avec 5% de FBS. Dés la tumeur avec un rasoir tandis que dans la solution. </li><li> Travailler à la température ambiante, on triture avec une pipette P1000 pour obtenir des cellules individuelles. Compléter le volume à 25 ml avec du PBS 0.9x stérilisée par filtration avec 5% de FBS. </li><li> Filtrer à travers filtre 40 mesh uM. </li><li> Faites tourner dans une centrifugeuse Eppendorf 5810R table à 1.500 tours par minute (453 rcf) pendant 10 min. </li><li> Enlever le surnageant et faire suspension cellulaire à une concentration d&#39;environ désiré (en supposant 10 7 cellules pour une tumeur 100 mm 3). </li><li> Calculer til précise le nombre de cellules à l&#39;aide d&#39;un hémocytomètre et diluer la suspension cellulaire de sorte que le volume d&#39;injection final est d&#39;environ 5 pl. Par exemple, si 50.000 cellules doivent être injectés, la suspension cellulaire doit être dilué à une concentration finale de 10.000 cellules / ul. Basculez le tube contenant la suspension cellulaire à chaque quelques min pour empêcher l&#39;agglutination des cellules. </li><li> Laver une jauge 26s (pointe conique) 701 N seringue de 10 ul Hamilton 2-3 fois avec de l&#39;éthanol à 100% et 0.9x PBS. Chargez la seringue avec 5 pi de la suspension cellulaire. </li><li> Anesthésier le poisson receveur irradié dans 0,17 mg / ml tricaïne. Placer le poisson sur le côté sur une Kimwipe humide (figure 4A). </li><li> Stabiliser le poisson avec une main et insérez l&#39;aiguille avec le biseau vers le haut à un angle de 45 ° dans le flanc du poisson au-dessus de la cavité péritonéale à mi-chemin entre le bord postérieur du cerveau postérieur et bord antérieur de la nageoire dorsale. Appuyer doucement sur ​​le piston (figure 4B). &lt;/ Li&gt; <li> Permettre aux poissons de se remettre à l&#39;eau les poissons frais et observer les poissons par jour pendant la prise de greffe tumorale (figure 4C). Si la prise de greffe a eu lieu, la croissance continue et le développement des maladies peuvent également être observés. </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>

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La méthode miniCoopR permet l&#39;expression de gènes d&#39;intérêt dans les mélanocytes de poisson zèbre. Cette approche tire parti du fait que le gène poisson zèbre mitfa agit cellulaire-autonome. Pour cette raison, les mélanocytes sauvés par le vecteur miniCoopR êtes certain de contenir le minigène et tout gène d&#39;intérêt auquel il est couplé physiquement. Rescued mélanocytes sont clairement visibles et peuvent être obtenus chez les animaux qui ont été injectés dans des embryons unice...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Réactifs de recombinaison passerelle </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> Référence 5 </td></tr><tr><td> Anticorps Mitfa </td><td></td><td></td><td> Référence 5 </td></tr><tr><td> FITC de chèvre anti-IgG de lapin </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 le poisson-zèbre </td><td></td><td></td><td> Référence 9 </td></tr><tr><td> 701N seringue de 10 ul </td><td> Hamilton / Fisher </td><td> 14-824 </td><td> &amp;nbsp; </td></tr><tr><td> 40 uM de filtre </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

Les études génomiques de cancers humains ont révélé une mine de renseignements sur les gènes qui sont altérés dans les tumeurs 1,2,3. Un défi découlant de ces études est que de nombreux gènes sont altérés, et il peut être difficile de distinguer les altérations génétiques qui ont poussé la tumorigenèse de ceux qui se leva d&#39;ailleurs lors de la transformation. Pour faire cette distinction, il est utile d&#39;avoir un test qui peut mesurer quantitativement l&#39;effet d&#39;un gène modifié sur l&#39;initiation de la tumeur et d&#39;autres procédés qui permettent tumeurs à persister et à diffuser. Nous présentons ici un moyen rapide pour cribler un grand nombre de modificateurs de mélanome chez le poisson zèbre candidats à l&#39;aide d&#39;un 4 tumeurs autochtones modèle qui englobe les étapes nécessaires à l&#39;initiation et le maintien du mélanome. Un réactif clé dans ce test est le vecteur miniCoopR, qui couple une copie de type sauvage de la spécification facteur mitfa mélanocytes à une cassette de recombinaison dans lequel la passerelle candidate melAnoma gènes peuvent être recombinées 5. Le vecteur a un minigène miniCoopR sauvetage mitfa qui contient le promoteur, le cadre de lecture ouvert et la région 3 &#39;non traduite du gène de type sauvage mitfa. Il nous permet de faire des constructions à l&#39;aide de longue durée cadres ouverts de lecture de modificateurs de mélanome candidats. Ces clones individuels peuvent ensuite être injecté dans la cellule unique Tg (mitfa: BRAF V600E); p53 (lf); embryons de poisson zèbre mitfa (lf). Le vecteur miniCoopR obtient intégré par Tol2 médiée par transgénèse 6 et sauve mélanocytes. Parce qu&#39;ils sont physiquement couplé à la minigène mitfa sauvetage, les gènes candidats sont exprimés dans les mélanocytes rescapés, dont certains vont transformer et se développer en tumeurs. L&#39;effet d&#39;un gène candidat à l&#39;initiation du mélanome et des cellules de mélanome peut être mesurée à l&#39;aide des courbes de survie sans mélanome, des essais d&#39;invasion, la coloration d&#39;anticorps et les essais de transplantation.

Une cellule-Tg (mitfa: BRAF V600E); p53 (lf); embryons de poisson zèbre mitfa (LF) ont été injectés avec le vecteur contenant l&#39;oncogène miniCoopR mélanome SETDB1 5 ou EGFP, chacun sous le contrôle du promoteur mitfa. Embryons sauvetage mélanocytes ont été sélectionnés et ont permis de mûrir. À 2 mois d&#39;âge avec des animaux sauvetage mélanocytes supérieure à 4 mm 2 ont été sélectionnés. Les animaux ont été examinés par se...

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

Un moyen rapide pour le dépistage de modificateurs de mélanome en utilisant un modèle de tumeur poisson zèbre autochtone est présenté. Il tire profit du vecteur miniCoopR qui permet l&#39;expression de gènes candidats de mélanome dans les mélanocytes. Une méthode pour obtenir des courbes de survie sans mélanome, un dosage invasion, un protocole pour la coloration d&#39;anticorps de mélanocytes échelle et un essai de transplantation mélanome sont décrits.

Dépistage du mélanome Modificateurs l&#39;aide d&#39;un modèle de tumeur Zebrafish Autochtone

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 vues.  University of Massachusetts Medical School. Un moyen rapide pour le dépistage de modificateurs de mélanome en utilisant un modèle de tumeur poisson zèbre autochtone est présenté. Il tire profit du vecteur miniCoopR qui permet l'expression de gènes candidats de mélanome dans les mélanocytes. Une méthode pour obtenir des courbes de survie sans mélanome, un dosage invasion, un protocole pour la coloration d'anticorps de mélanocytes échelle et un essai de transplantation mélanome sont décrits.

Vidéo: 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-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

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

Recherche

Médecine

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.

Évaluer les changements tératogènes dans un modèle de poisson zèbre de l&#39;exposition d&#39;alcoolisation fœtale

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.

L&#39;homme neuroendocriniennes lignées cellulaires tumorales comme un modèle tridimensionnel pour l&#39;étude de la thérapie des tumeurs neuro-endocrines humaines

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).

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

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.

Un modèle de poisson zèbre du diabète sucré et de la mémoire métabolique

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 haute résolution<em&gt; In vivo</em&gt; Technique d&#39;imagerie pour étudier la réponse dynamique des structures intracrâniennes à la croissance tumorale et de thérapeutique

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.

Un modèle de poisson zèbre possible de la maladie polykystique des reins: Knockdown de<em&gt; Wnt5a</em&gt; Causes kystes chez le poisson zèbre reins

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.

En trois dimensions modèle de co-culture tumeur stromale-Interaction

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.

Perfusion hépatique isolée comme un traitement pour les métastases hépatiques de mélanome uvéal

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.

Évaluation des leucocytes infiltrant les tumeurs sous-ensembles dans un Modèle de tumeur sous-cutanée

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