Ringraziamo il Dott. Leonard I. Zon nel cui laboratorio queste tecniche sono state inizialmente sviluppate, Kristen Kwan e alla fine Chi-Bin Chien per i regali di plasmidi utilizzati in questo lavoro, James e David Lister Raible per l&#39;assistenza con la colorazione degli anticorpi, e James Neiswender per la microiniezione video. Questo lavoro è stato finanziato dalla sovvenzione del NIH R00AR056899-03 al CJC

1. Screening per Modificatori Onset Melanoma <ol><li> Crea Gateway cloni entrata di mezzo con PCR amplificando il full-length open reading frame di geni di interesse (GOI) e ricombinando in pDONR 221 con BP clonase II (Invitrogen). Utilizzare Multisite Gateway tecnologia (Invitrogen) per ricombinare p5E_mitfa, pME_GOI, Tol2kit # 302 p3E_SV40polyA 6 e miniCoopR 5 per spostare geni di interesse sotto il promotore mitfa nel vettore miniCoopR (Figura 1A). </li><li> Iniettare 25 picogrammi di ogni clone insieme con 25 picogrammi Tol2 mRNA trasposasi in una cella-Tg (mitfa: BRAF V600E), p53 (lf); mitfa (lf) embrioni di zebrafish triplamente omozigoti come descritto in precedenza 7. Incubare gli embrioni iniettati a 28,5 ° C. Rimuovere eventuali embrione è morto a 24 HPF. </li><li> Selezionare animali transgenici con melanociti salvati a 72 HPF ponendo la capsula Petri contenente gli embrioni iniettati su una microscop dissezionee sotto la luce incidente su uno sfondo bianco (Figura 1A). </li><li> Di trasferire gli animali a 3 serbatoi L nel vivaio della struttura zebrafish a 4 dpf come descritto in precedenza 8. </li><li> A 2 mesi, selezionare gli animali con almeno una zona di salvataggio melanociti superiore a 4 mm 2 (Figura 1A). Vi è una forte correlazione tra il grado di soccorso melanociti a 72 HPF e il grado di soccorso melanociti a 2 mesi. Quando embrioni con salvataggio melanociti vengono raccolte a 72 HPF, la maggior parte di essi (~ 80%) avranno almeno uno spazio di salvataggio melanociti maggiore di 4 mm 2 a 2 mesi. </li><li> Schermo gli animali selezionati settimanali per la presenza di tumori (Figura 1B). Vi è una forte correlazione tra i cambiamenti istopatologici e morfologiche. Transizione da benigna a maligna è riconosciuto come un cambiamento morfologico quando le lesioni diventano sollevato dalla superficie dell&#39;animale. Isolare anima tumore cuscinettols di studio. </li><li> Disegna melanoma senza curve di sopravvivenza con l&#39;età in settimane in ascissa e la percentuale di sopravvivenza libera melanoma in ordinata. Animali con melanociti che esprimono un gene di interesse sono confrontati agli animali di controllo che esprimono EGFP (enhanced green fluorescent protein) nei melanociti (Figura 1C). L&#39;insorgenza tumorale mediano per animali iniettati con miniCoopR-EGFP è circa 18 settimane. Determinare se le due curve sono statisticamente differenti utilizzando un log rank test. </li></ol> 2. Invasion test Tumore <ol><li> Selezionare zebrafish isolato che sviluppano tumori dorsalmente, in una regione compresa tra il confine posteriore del romboencefalo e margine anteriore della pinna dorsale (Figura 2A). </li><li> Due settimane dopo l&#39;insorgenza del melanoma eutanasia il pesce secondo le linee guida indicate dalla American Veterinary Medical Association Panel sull&#39;eutanasia e approvato dalla University of Massachusetts Medical Ins ScuolaCura degli animali e del Comitato titutional uso (IACUC). In particolare, un pesce è posto in un piatto con 0,6 mg / ml fino a quando il movimento tricaine branchia si ferma. </li><li> Praticare un&#39;incisione nella cavità peritoneale del pesce con un bisturi e poi mettere il pesce in 4% paraformaldeide (PFA) per una notte a 4 ° C per il fissaggio. </li><li> Trattare con EDTA 0,5 M per una notte a 4 ° C per decalcificare. </li><li> Fissare con formalina nel cassetto durante la notte, disidratare per 1 ora, chiaro con xilene 3 volte per 1 ora e poi trattare con paraffina 2 volte per 2 ore ciascuna a 60 ° C. </li><li> Incorporare il tessuto in paraffina in uno stampo e farlo solidificare per 30 minuti a 1 ora. </li><li> Fai 5 sezioni micron, una ogni 50 micron. Le sezioni devono essere trasversale e attraverso l&#39;intera lesione in modo che il punto più profondo della invasione può essere identificato. Segui con ematossilina eosina e colorazione. </li><li> Valutare l&#39;invasione del tumore misurando se le cellule tumorali rimangono al di fuori del collagene strato compactum, invadere la dorsalemuscolatura o invadere ulteriormente nella colonna vertebrale (Figura 2B, C). Determinare differenza statistica tra le due serie di campioni. </li></ol> 3. Colorazione anticorpale dei melanociti Scala <ol><li> Costituiscono PO4 tampone con 80 ml ​​0,1 M Na 2 HPO 4 e 20 ml di 0,1 M NaH 2 PO 4. Verificare che il pH è 7.3. Make up tampone 2x soluzione contenente 8,0 g di saccarosio, 0,15 ml di 0,2 M CaCl 2 e 90 ml di 0,2 M PO 4, pH 7.3. Se necessario regolare il pH a 7,3 con NaOH o HCl poi portare a 100 ml con tampone PO 4. </li><li> Pesare fino a 300 mg di solido PFA e aggiungere in una provetta. Aggiungere 5 mM NaOH ad un volume pari a 4,5 x massa, in mg, di PFA (ad esempio 450 ul 5 mM NaOH a 100 mg PFA). Termico a 60-70 ° C con occasionale agitazione fino a quando il PFA è sciolto. Spin down particelle rimanenti e recuperare il 20% PFA surnatante. Fai fresco ogni volta. </li><li> Preparare la soluzione di fissaggio contenente 1x fix tampone e 4% PFA. </li><li> Anestetizzare il pesce in 0,17 mg / ml tricaine. </li><li> Utilizzo di luce incidente sotto un microscopio da dissezione cogliere squame dorsali con taglienti a punta fine pinze, facendo attenzione a non danneggiare il melanocita contenente la metà della scala (Figura 3A). Posizionare le scale direttamente dalle pinze in 1 ml di soluzione di fissaggio e incubare a temperatura ambiente, mentre si gira, per ≥ 2 ore. Trasferire il pesce in acqua pesci e monitorare il pesce per confermare il recupero di successo. </li><li> Per candeggina melanociti pigmentati lavare scale fisse in PBST (1x PBS pH 7,4, 0,1% Triton X-100) due volte per 5 minuti a temperatura ambiente. Aggiungere 1 ml di soluzione di candeggina espressi contenente 0,4 ml di KOH al 10% e 0,15 ml 30% H 2 O 2 in 5 ml di dH 2 O. Mettere su serrature parafilm o tappo per impedire al gas di scoppio dei tubi aperti. Continuare sbiancamento fino pigmento è andata (10 min tipico) (Figura 3B, C). Lavare in PBST quattro volte per 5 min a temperatura ambienteteratura. </li><li> Blocco per almeno 30 min in soluzione di blocco costituito da 1x PBS pH 7,4, 0,2% Triton X-100, 2 mg / ml BSA, 1% DMSO, 0,02% NaN 3 e 2% di siero di agnello. Lasciare fuori NaN 3 se in via di sviluppo con la reazione HRP. </li><li> Incubare con l&#39;anticorpo primario in soluzione di blocco notte a temperatura ambiente. È necessario disporre di almeno 400 ml di soluzione sui campioni per tenerli sommersi. </li><li> Lavare le scale 3 volte per 30 minuti in PBST a temperatura ambiente. </li><li> Incubare con anticorpo secondario da 2 ore a tutta la notte in soluzione di blocco a temperatura ambiente. </li><li> Colorare per 10 minuti con PBST + 0,1 DAPI pM. </li><li> Lavare 3 volte per 5 minuti con PBST. </li><li> Montare le scale su un vetrino in una goccia di Vectashield modo che il lato concavo della bilancia rivolto verso il vetrino e posizionare un coprioggetto di vetro su di loro. Sigillare i bordi del vetrino con smalto trasparente e osservare i vetrini al microscopio a fluorescenza (figura 3D, E, F, G </strong&gt;). </li></ol> 4. Trapianto Assay <ol><li> Preparare destinatario 2-3 mesi vecchio casper 9 pesci irradiando con 25 Gy di raggi gamma un giorno prima del trapianto 10. Lasciare che il pesce per il recupero in acqua pesci. </li><li> Eutanasia di un portatore di tumore pesci come descritto in precedenza (Sezione 2.2). </li><li> Tagliare il tumore e metterlo in una capsula di Petri con circa 5 ml di filtro sterilizzato 0.9x PBS con il 5% di FBS. Tagliare il tumore con un rasoio mentre in soluzione. </li><li> Lavorare a temperatura ambiente, con una pipetta triturare P1000 per ottenere cellule singole. Portare al volume di 25 ml con filtro sterilizzato 0.9x PBS con il 5% di FBS. </li><li> Filtrare su filtro a 40 pM a rete. </li><li> Spin in una centrifuga Eppendorf 5810R tavolo a 1.500 giri al minuto (453 rcf) per 10 min. </li><li> Rimuovere il surnatante e fare sospensione cellulare alla concentrazione di circa desiderato (assumere 10 7 celle a mm 100 3 tumore). </li><li> Calcola il tegli esatto numero di cellule utilizzando un emocitometro e diluire la sospensione cellulare in modo che il volume di iniezione finale è di circa 5 microlitri. Ad esempio, se 50.000 cellule deve essere iniettato, la sospensione cellulare deve essere diluito ad una concentrazione finale di 10.000 cellule / microlitro. Lievemente la provetta contenente la sospensione cellulare ogni pochi minuti per evitare grumi delle cellule. </li><li> Lavare un calibro 26s (punta conica) 701 N siringa da 10 microlitri Hamilton 2-3 volte con il 100% di etanolo e 0.9x PBS. Caricare la siringa con 5 ml di sospensione cellulare. </li><li> Anestetizzare il pesce irradiato destinatario in 0,17 mg / ml tricaine. Pesce posto su un lato su un Kimwipe umido (Figura 4A). </li><li> Stabilizzare il pesce con una mano e inserire l&#39;ago con lo smusso rivolto verso l&#39;alto ad un angolo di 45 ° nel fianco del pesce al di sopra della cavità peritoneale circa a metà strada tra il confine posteriore del cervello posteriore e il bordo anteriore della pinna dorsale. Delicatamente lo stantuffo (Figura 4B). &lt;/ Li&gt; <li> Permettono di recuperare il pesce in acqua pesce fresco e osservare i pesci giornaliera per l&#39;attecchimento del tumore (Figura 4C). Se si è verificato l&#39;attecchimento, crescita e lo sviluppo della malattia possono essere rispettate. </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>

Nessun conflitto di interessi dichiarati.

Il metodo miniCoopR permette l&#39;espressione di geni di interesse in melanociti zebrafish. Questo approccio sfrutta il fatto che il gene zebrafish mitfa agisce autonomamente cellulare. Per questo motivo, melanociti recuperati dal vettore miniCoopR sono certi di contenere il minigene e qualsiasi gene di interesse a cui è fisicamente accoppiati. Melanociti salvati sono chiaramente visibili e possono essere ottenuti in animali che sono stati iniettati come monocellulari embrioni. Una misura definita di chimeris...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Gateway ricombinazione reagenti </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> Riferimento 5 </td></tr><tr><td> Mitfa anticorpo </td><td></td><td></td><td> Riferimento 5 </td></tr><tr><td> FITC goat anti-rabbit IgG </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 Zebrafish </td><td></td><td></td><td> Riferimento 9 </td></tr><tr><td> 701N siringa da 10 microlitri </td><td> Hamilton / Fisher </td><td> 14-824 </td><td> &amp;nbsp; </td></tr><tr><td> 40 pM filtro </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

Studi di genomica dei tumori umani hanno prodotto una grande quantità di informazioni sui geni che sono alterati nei tumori 1,2,3. Una sfida derivante da questi studi è che molti geni sono alterati, e può essere difficile distinguere alterazioni genetiche che hanno spinto tumorigenesi da coloro che nasce accidentalmente durante la trasformazione. Per questa distinzione è utile avere un saggio che può quantitativamente misurare l&#39;effetto di un gene alterato sull&#39;iniziazione tumore e altri processi che consentono tumori di persistere e diffondere. Qui vi presentiamo un mezzo rapido per lo screening un gran numero di candidati modificatori di melanoma in zebrafish con un autoctono modello 4 tumore che comprende passaggi necessari per l&#39;iniziazione melanoma e la manutenzione. Un reagente chiave in questo saggio è il vettore miniCoopR, che accoppia una wild-type copia del melanociti fattore specifica mitfa ad una cassetta di ricombinazione Gateway in cui candidato melAnoma geni possono essere ricombinati 5. Il vettore ha una miniCoopR minigene salvataggio mitfa che contiene il promotore, di lettura aperta e 3&#39;-non tradotta regione del gene di tipo selvaggio mitfa. Ci permette di fare costrutti con full-length fasi di lettura aperta di modificatori melanoma candidati. Questi cloni singoli possono poi essere iniettato nella cella singola Tg (mitfa: BRAF V600E), p53 (lf); mitfa (lf) embrioni di zebrafish. Il vettore miniCoopR viene integrata da Tol2-mediata transgenesi 6 e salva melanociti. Perché sono fisicamente accoppiati al salvataggio mitfa minigene, geni sono espressi in melanociti recuperati, alcuni dei quali si trasformano e svilupparsi in tumori. L&#39;effetto di un gene candidato per l&#39;inizio del melanoma e delle cellule di melanoma può essere misurato con melanoma senza curve di sopravvivenza, saggi di invasione, colorazione degli anticorpi e saggi di trapianto.

Unicellulare Tg (mitfa: BRAFV600E), p53 (lf); mitfa (lf) embrioni di zebrafish sono stati iniettati con il vettore contenente il miniCoopR oncogene melanoma SETDB1 5 o EGFP, ciascuna sotto il controllo del promotore mitfa. Gli embrioni con salvataggio melanociti sono stati selezionati e lasciati maturare. A 2 mesi di età, con animali di salvataggio melanociti superiore a 4 mm 2 sono stati selezionati. Gli animali sono stati sottoposti a screening settimanale per...

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 modo rapido per lo screening del melanoma modificatori che utilizzano un modello di zebrafish tumore autoctona è presentato. Si avvale del vettore miniCoopR che permette l&#39;espressione di geni candidati melanoma nei melanociti. Un metodo per ottenere melanoma senza curve di sopravvivenza, un saggio di invasione, un protocollo per la colorazione degli anticorpi dei melanociti di scala e di un saggio di trapianto melanoma sono descritti.

Screening per Modificatori melanoma utilizzando un modello Zebrafish Tumore autoctone

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 Views.  University of Massachusetts Medical School. Un modo rapido per lo screening del melanoma modificatori che utilizzano un modello di zebrafish tumore autoctona è presentato. Si avvale del vettore miniCoopR che permette l'espressione di geni candidati melanoma nei melanociti. Un metodo per ottenere melanoma senza curve di sopravvivenza, un saggio di invasione, un protocollo per la colorazione degli anticorpi dei melanociti di scala e di un saggio di trapianto melanoma sono descritti.

Video: 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-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

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

Ricerca

Medicina

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.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

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).

Paziente coltura cellulare derivato e isolamento di CD133<sup&gt; +</sup&gt; Putativi staminali alle cellule tumorali di 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.

Un modello Zebrafish di diabete mellito e di memoria metabolica

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.

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

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 possibile modello Zebrafish della malattia del rene policistico: Knockdown di<em&gt; Wnt5a</em&gt; Provoca cisti in Zebrafish Reni

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.

Tridimensionale Co-cultura Modello di tumore 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.

Isolata epatica perfusione come Trattamento di metastasi epatiche di melanoma uveale

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.

Valutazione del tumore infiltrante leucociti sottoinsiemi in un tumore sottocutaneo modello

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

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Iniezione di cellule singenici del topo melanoma per determinare il loro potenziale metastatico nei polmoni

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...