Agradecemos al Dr. Leonard I. Zon en cuyo laboratorio estas técnicas fueron desarrolladas inicialmente; Kwan Kristen y el difunto Chi-Bin Chien para los regalos de los plásmidos utilizados en este trabajo, James Lister y Raible David para obtener ayuda con la tinción de anticuerpos, y por James Neiswender el vídeo de microinyección. Este trabajo fue financiado por el NIH subvención R00AR056899-03 a CJC

1. La detección de Onset Modificadores Melanoma <ol><li> Cree clones de entrada de puerta de enlace media por PCR amplificando el marco de cuerpo entero de lectura abierta de los genes de interés (GOI) y recombinación en pDONR 221 con BP Clonase II (Invitrogen). Use Multisite tecnología de Gateway (Invitrogen) para recombinarse p5E_mitfa, pME_GOI, Tol2kit # 302 p3E_SV40polyA 6 y miniCoopR 5 para colocar los genes de interés bajo el promotor en el vector mitfa miniCoopR (Figura 1A). </li><li> Inyectar 25 picogramos de cada clon junto con 25 picogramos Tol2 transposasa mRNA en una célula Tg (mitfa: BRAF V600E); p53 (lf); mitfa (lf) embriones de pez cebra triplemente homocigotos como se ha descrito previamente 7. Se incuban los embriones inyectados a 28,5 ° C. Retire los embriones muertos a las 24 HPF. </li><li> Seleccione los animales transgénicos con melanocitos rescatados a 72 HPF, colocando la placa de Petri que contiene los embriones inyectados en una disección microscópicase con luz incidente sobre un fondo blanco (fig. 1A). </li><li> Transfiera los animales a 3 tanques de L en el vivero de la instalación de pez cebra a 4 dpf a lo descrito previamente 8. </li><li> A los 2 meses, seleccionar los animales con al menos un área de rescate melanocitos mayor que 4 mm 2 (Figura 1A). Hay una fuerte correlación entre el grado de rescate de melanocitos en 72 HPF y el grado de rescate de melanocitos a los 2 meses. Cuando los embriones con rescate de los melanocitos se recogió en 72 hpf, la mayoría de ellos (~ 80%) tendrá al menos un área de rescate melanocitos mayor que 4 mm 2 a los 2 meses. </li><li> Pantalla de los animales seleccionados semanales para detectar la presencia de tumores (Figura 1B). Hay una fuerte correlación entre los cambios histopatológicos y morfológicas. Transición de benigno a maligno se reconoce como un cambio morfológico cuando las lesiones se elevó fuera de la superficie del animal. Aislar anima con el tumorls de estudio. </li><li> Dibujar libres melanoma curvas de supervivencia con la edad en semanas en las abscisas y el porcentaje de supervivencia libre de melanoma en la ordenada. Los animales con los melanocitos que expresan un gen de interés se comparan con los animales control que expresa EGFP (proteína verde fluorescente) en los melanocitos (Figura 1C). La aparición de tumores mediana para los animales inyectados con miniCoopR-EGFP es de aproximadamente 18 semanas. Determinar si las dos curvas son diferentes estadísticamente mediante la prueba de log-rank. </li></ol> 2. Ensayo de invasión tumoral <ol><li> Seleccione pez cebra aislado que desarrollan tumores dorsalmente, en una región entre el límite posterior del cerebro posterior y el borde anterior de la aleta dorsal (Figura 2A). </li><li> Dos semanas después de la aparición del melanoma eutanasia a los peces según las directrices especificadas por la American Veterinary Medical Association Panel sobre la eutanasia y aprobado por la Universidad de Massachusetts Medical School InsCuidado de Animales y el empleo Comisión titucional (IACUC). Específicamente, un pez se coloca en un plato con 0,6 mg / ml hasta tricaína movimiento branquial se detiene. </li><li> Hacer una incisión en la cavidad peritoneal de los peces con un escalpelo y luego coloque el pescado en 4% de paraformaldehído (PFA) durante la noche a 4 ° C para su fijación. </li><li> Tratar con 0,5 M EDTA durante la noche a 4 ° C para descalcificar. </li><li> Fijar con formalina en casete durante la noche, se deshidratan durante 1 hora, claro con xileno 3 veces durante 1 hora y luego se trata con parafina 2 veces durante 2 horas cada una a 60 ° C. </li><li> Insertar el tejido en parafina en un molde y dejar que se solidifique durante 30 min a 1 hr. </li><li> Hacer 5 micras secciones, una cada 50 micras. Las secciones deben ser transversal y a través de toda la lesión para que el punto más profundo de invasión puede ser identificado. Siga con hematoxilina y eosina. </li><li> Evaluar la invasión tumoral midiendo si las células tumorales permanecen fuera de la colágena compactum estrato, invadir la dorsalmusculatura o invadir adicional en la columna vertebral (Figura 2B, C). Determinar la diferencia estadística entre los dos grupos de muestras. </li></ol> 3. Antibody tinción de melanocitos Escala <ol><li> Maquillaje PO4 tampón con 80 ml ​​de 0,1 M Na 2 HPO 4 y 20 ml de 0,1 M NaH 2 PO 4. Asegúrese de que el pH es de 7,3. Maquillaje solución tampón 2x que contiene 8,0 g de sacarosa, 0,15 ml 0,2 M CaCl 2 y 90 ml de 0,2 M tampón de PO 4, pH 7,3. Si es necesario ajustar el pH a 7,3 con NaOH o HCl completar hasta 100 ml con tampón de PO 4. </li><li> A pesar hasta 300 mg PFA sólido y añade a un tubo de microcentrífuga. Añadir 5 mM NaOH a un volumen igual a la masa x 4,5, en mg, de PFA (por ejemplo, 450 ul 5 mM NaOH a 100 mg PFA). Se calienta a 60-70 ° C con agitación ocasional hasta que el PFA se disuelve. Decantar las partículas restantes y recuperar el 20% PFA sobrenadante. Haga fresca cada vez. </li><li> Preparar la solución de fijación que contiene 1x fix tampón y 4% PFA. </li><li> Se anestesia el pescado en 0,17 mg / ml tricaína. </li><li> Usando la luz incidente bajo un microscopio de disección arrancar escamas dorsales agudas utilizando pinzas de punta fina, teniendo cuidado de no dañar el medio que contiene los melanocitos de la escala (Figura 3A). Escalas lugar directamente desde las pinzas en 1 ml de solución de fijación y se incuba a temperatura ambiente, mientras se gira, por ≥ 2 hr. Transferir el pez en el agua los peces y controlar a los peces a confirmar una recuperación exitosa. </li><li> Para blanquear melanocitos pigmentados lavar escalas fijas en PBST (1x PBS pH 7,4, 0,1% Triton X-100) 2 veces durante 5 min a temperatura ambiente. Añadir 1 ml de lejía recién preparada que contiene 0,4 ml de solución de KOH al 10% y 0,15 ml de 30% de H 2 O 2 en 5 ml de dH 2 O. Ponga cerraduras Parafilm o gorra para evitar que el gas se rompa los tubos abiertos. Continuar hasta decoloración de pigmento se ha ido (10 min es típico) (Figura 3B, C). Lavar cuatro veces en PBST durante 5 min a temperatura ambientetura. </li><li> Bloquear por lo menos durante 30 minutos en solución de bloqueo compuesta por 1x PBS pH 7,4, 0,2% Triton X-100, 2 mg / ml de BSA, 1% de DMSO, 0,02% NaN 3 y 2% suero de cordero. Dejar de lado NaN 3, si el desarrollo de la reacción con HRP. </li><li> Incubar con anticuerpo primario en solución de bloqueo durante la noche a temperatura ambiente. Debe tener al menos 400 l de solución sobre las muestras para mantenerlas sumergidas. </li><li> Lavar las escalas 3 veces durante 30 minutos en PBST a temperatura ambiente. </li><li> Incubar con anticuerpo secundario de 2 horas a toda la noche en solución de bloqueo a temperatura ambiente. </li><li> Teñir durante 10 min con PBST DAPI mu M + 0,1. </li><li> Lavar 3 veces durante 5 min con PBST. </li><li> Montar la balanza sobre un portaobjetos de vidrio en una gota de Vectashield de modo que el lado cóncavo de la balanza está orientado hacia abajo hacia el portaobjetos y colocar un cubreobjetos de cristal sobre ellos. Selle los bordes de la diapositiva con esmalte de uñas transparente y observar las diapositivas con un microscopio de fluorescencia (Figura 3 D, E, F, G </strong&gt;). </li></ol> 4. Trasplante de Ensayo <ol><li> Preparar destinatario 2-3 meses de edad casper 9 peces por irradiación con 25 Gy de irradiación gamma, un día antes del trasplante 10. Permitir a los peces a recuperarse en el agua los peces. </li><li> Eutanasia a un pez portador del tumor como se ha descrito anteriormente (Sección 2,2). </li><li> Corte el tumor y lo puso en una caja de Petri con filtro de aproximadamente 5 ml de PBS esterilizado 0.9x con FBS al 5%. Cortar el tumor con una maquinilla de afeitar mientras está en solución. </li><li> Trabajando a temperatura ambiente, se tritura con una pipeta P1000 para obtener células individuales. Completar el volumen a 25 ml con PBS esterilizado por filtración 0.9x con FBS al 5%. </li><li> Filtrar a través de filtro de 40 de malla. </li><li> Haga girar en una centrífuga de mesa Eppendorf 5810R a 1.500 rpm (453 fcr) durante 10 min. </li><li> Eliminar el sobrenadante y crea suspensión de células a la concentración de aproximadamente deseado (suponga 10 7 células para un 100 mm 3 tumor). </li><li> Calcular tque el número de células exacta usando un hemocitómetro y se diluye la suspensión de células de modo que el volumen de inyección final es de aproximadamente 5 l. Por ejemplo, si las células son 50.000 a inyectar, la suspensión celular se diluyó a una concentración final de 10.000 células / mL. Flick el tubo que contiene la suspensión de células cada pocos minutos para evitar la aglutinación de las células. </li><li> Lave un calibre 26s (punta biselada) jeringa l 701 N 10 Hamilton 2-3 veces con etanol al 100% y PBS 0.9x. Cargar la jeringa con 5 l de la suspensión celular. </li><li> Se anestesia el pez receptor irradiado en 0,17 mg / ml tricaína. Coloque el pescado en su lado en una Kimwipe húmedo (Figura 4A). </li><li> Estabilizar el pescado con una mano e inserte la aguja con el bisel hacia arriba en un ángulo de 45 ° en el flanco del pez encima de la cavidad peritoneal a medio camino entre el límite posterior del cerebro posterior y el borde anterior de la aleta dorsal. Suavemente el émbolo (Figura 4B). &lt;/ Li&gt; <li> Permiten a los peces recuperar en peces de agua dulce y observar los peces diaria para el injerto del tumor (Figura 4C). Si el injerto se ha producido, el continuo crecimiento y desarrollo de la enfermedad también puede ser observado. </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>

No hay conflictos de interés declarado.

El método miniCoopR permite la expresión de genes de interés en los melanocitos de pez cebra. Este enfoque tiene la ventaja del hecho de que el gen de pez cebra mitfa actúa células autónoma. Por esta razón, los melanocitos rescatadas por el vector miniCoopR son determinados para contener el minigen y cualquier gen de interés al que está acoplado físicamente. Melanocitos rescatados son claramente visibles y pueden ser obtenidos en los animales que fueron inyectados como una sola célula de los embrione...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Reactivos de puerta de enlace de recombinación </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> Referencia 5 </td></tr><tr><td> Mitfa anticuerpo </td><td></td><td></td><td> Referencia 5 </td></tr><tr><td> FITC cabra anti-conejo de anticuerpos 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 pez cebra </td><td></td><td></td><td> Referencia 9 </td></tr><tr><td> 701N 10 l Jeringa </td><td> Hamilton / Fisher </td><td> 14-824 </td><td> Ynbsp; </td></tr><tr><td> 40 mM de 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

Los estudios genómicos de cáncer en humanos han arrojado una gran cantidad de información acerca de los genes que están alterados en los tumores 1,2,3. Un desafío que surge de estos estudios es que muchos genes están alterados, y puede ser difícil distinguir las alteraciones genéticas que impulsaron la tumorigénesis de aquellos que surgió incidentalmente durante la transformación. Para hacer esta distinción es beneficioso tener un ensayo que puede medir cuantitativamente el efecto de un gen alterado en la iniciación del tumor y otros procesos que permiten a los tumores a persistir y difundir. Aquí presentamos un medio rápido para detectar un gran número de candidatos modificadores de melanoma en el pez cebra utilizando un modelo de tumor 4 autóctona que abarca los pasos necesarios para la iniciación y el mantenimiento de melanoma. Un reactivo clave en este ensayo es el vector miniCoopR, que acopla una copia de tipo salvaje del factor de especificación mitfa melanocitos a un casete de recombinación Gateway en el que candidato melAnoma genes pueden ser recombinados 5. El vector miniCoopR tiene un minigen rescate mitfa que contiene el promotor, marco de lectura abierta y la región 3 &#39;no traducida del gen de tipo salvaje mitfa. Nos permite hacer construcciones con larga duración marcos de lectura abierta de modificadores de melanoma candidatos. Estos clones individuales pueden ser inyectados en célula única Tg (mitfa: BRAF V600E); p53 (lf); mitfa (lf) embriones de pez cebra. El vector miniCoopR se integra por Tol2 transgénesis mediada 6 y rescata a los melanocitos. Debido a que están físicamente acoplado a la minigen mitfa rescate, genes candidatos se expresa en los melanocitos rescatadas, algunas de las cuales se transforman y se desarrollan en tumores. El efecto de un gen candidato en la iniciación melanoma y propiedades celulares de melanoma se puede medir usando libres melanoma curvas de supervivencia, los ensayos de invasión, tinción de anticuerpos y ensayos de trasplante.

Uno de células Tg (mitfa: BRAF V600E); p53 (lf); mitfa (lf) embriones de pez cebra fueron inyectados con el vector que contiene el oncogén miniCoopR melanoma SETDB1 5 o EGFP, cada uno bajo el control del promotor mitfa. Los embriones con rescate de melanocitos se seleccionaron y se dejó madurar. A los 2 meses de edad de los animales con rescate de melanocitos mayor que 4 mm 2 fueron seleccionados. Los animales fueron examinados semanalmente para melanomas. Cur...

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

Una forma rápida de pantalla para los modificadores de melanoma utilizando un modelo de pez cebra tumor autóctona se presenta. Se aprovecha de la miniCoopR vector que permite la expresión de los genes candidatos de melanoma en los melanocitos. Un método para obtener gratis melanoma curvas de supervivencia, un ensayo de invasión, un protocolo para la tinción de anticuerpos de los melanocitos de escala y un ensayo de trasplante de melanoma se describen.

La detección de Modificadores melanoma utilizando un modelo de pez cebra Tumor autóctona

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 vistas.  University of Massachusetts Medical School. Una forma rápida de pantalla para los modificadores de melanoma utilizando un modelo de pez cebra tumor autóctona se presenta. Se aprovecha de la miniCoopR vector que permite la expresión de los genes candidatos de melanoma en los melanocitos. Un método para obtener gratis melanoma curvas de supervivencia, un ensayo de invasión, un protocolo para la tinción de anticuerpos de los melanocitos de escala y un ensayo de trasplante de melanoma se describen.

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-fotón imágenes de invasión de células tumorales en un modelo de ratón ortotópico de carcinoma oral de células escamosas

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

Investigación

Medicina

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.

Evaluación de los cambios teratogénicos en un modelo de pez cebra de la exposición fetal al alcohol

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.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

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).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de 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 modelo de pez cebra de la diabetes mellitus y la memoria metabólica

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.

Una novela de alta resolución<em&gt; En vivo</em&gt; Técnica de escaneo para el Estudio de la respuesta dinámica de estructuras intracraneales al crecimiento del tumor y Terapéutica

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 posible modelo de pez cebra de la enfermedad renal poliquística: Derribo de<em&gt; Wnt5a</em&gt; Causas Los quistes en el pez cebra Riñones

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.

Tridimensional Modelo Co-cultura para la interacción tumor estromal

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.

Aislado hepática de perfusión como un tratamiento para las metástasis hepáticas de melanoma uveal

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.

Evaluación de tumor infiltrante de leucocitos subconjuntos en un modelo de tumor subcutáneo

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