Agradecemos ao Dr. Leonard I. Zon em cujo laboratório estas técnicas foram desenvolvidas inicialmente; Kristen Kwan eo falecido Chi-Bin Chien para presentes de plasmídeos utilizados neste trabalho, Tiago e David Lister Raible para assistência com coloração de anticorpos e James Neiswender para o vídeo microinjecção. Este trabalho foi financiado pelo NIH concessão R00AR056899-03 para CJC

1. Triagem para Modificadores Onset Melanoma <ol><li> Criar clones porta de entrada do meio de amplificação do PCR de comprimento completo de leitura aberta de genes de interesse (GOI) e recombinação em pDONR 221 utilizando BP clonase II (Invitrogen). Use Multisite gateway tecnologia (Invitrogen) para se recombinam p5E_mitfa, pME_GOI, Tol2kit # 302 p3E_SV40polyA 6 e miniCoopR 5 para colocar os genes de interesse sob o promotor no vector mitfa miniCoopR (Figura 1A). </li><li> Injectar 25 picogramas de cada clone, juntamente com 25 picogramas de mRNA Tol2 transposase numa única célula-Tg (mitfa: BRAF V600E), p53 (lf); mitfa (lf) de embriões zebrafish triplamente homozigóticos como descrito anteriormente 7. Incubar os embriões injetados em 28,5 ° C. Remova todos os embriões mortos em 24 hpf. </li><li> Selecione animais transgênicos com melanócitos resgatados a 72 hpf colocando a placa de Petri contendo embriões injetados em um microscop dissecare sob a luz incidente sobre um fundo branco (Figura 1A). </li><li> Transferir os animais para três tanques L no berçário da unidade zebrafish a 4 dpf como descrito anteriormente 8. </li><li> Aos 2 meses, seleccionar os animais com, pelo menos, uma área de recuperação de melanócitos maior do que 4 mm 2 (Figura 1A). Existe uma forte correlação entre o grau de recuperação dos melanócitos a 72 hpf e o grau de recuperação dos melanócitos menos 2 meses. Quando embriões com resgate de melanócitos são colhidas a 72 hpf, a maioria (~ 80%) que tem pelo menos uma área de recuperação de melanócitos maior do que 4 mm 2 em 2 meses. </li><li> Tela de os animais seleccionados semanais para a presença de tumores (Figura 1B). Há uma forte correlação entre as alterações histopatológicas e morfológica. Transição do benigno para maligno é reconhecida como uma alteração morfológica, quando lesões tornam-se levantada para fora da superfície do animal. Isolar anima rolamento tumorls para estudo. </li><li> Desenhar as curvas de sobrevida livre de melanoma com a idade em semanas na abcissa e por cento melanoma sobrevida livre na ordenada. Os animais com os melanócitos que expressam um gene de interesse são comparadas com os animais de controlo que expressam EGFP (enhanced green fluorescent protein) nos melanócitos (Figura 1C). O início médio do tumor para os animais injectados com miniCoopR-EGFP é de aproximadamente 18 semanas. Determinar se as duas curvas são estatisticamente diferentes através de um teste de log rank. </li></ol> 2. Ensaio de Invasão Tumoral <ol><li> Seleccione zebrafish isolado que desenvolvem tumores dorsalmente, numa região situada entre o limite posterior do cérebro posterior e borda anterior da barbatana dorsal (Figura 2A). </li><li> Duas semanas após o início melanoma sacrificar os peixes de acordo com as orientações especificadas pelo Painel Associação Médica Veterinária Americana sobre a eutanásia e aprovado pela Universidade de Medicina de Massachusetts InsAnimal Care e do Comitê titutional Uso (IACUC). Especificamente, um peixe é colocada num prato com 0,6 mg / ml até tricaina brânquia movimento pára. </li><li> Fazer uma incisão na cavidade peritoneal de o peixe com um escalpelo e, em seguida, colocar o peixe em paraformaldeído a 4% (PFA) durante a noite a 4 ° C para a fixação. </li><li> Trata-se com 0,5 M de EDTA durante a noite a 4 ° C a descalcificação. </li><li> Fixar com formalina na cassete durante a noite, desidratar durante 1 h, claro, com xileno 3 vezes durante 1 hora e em seguida trata-se com parafina duas vezes durante 2 horas cada, a 60 ° C. </li><li> Incorporar o tecido em parafina num molde e deixar solidificar durante 30 minutos a 1 hora. </li><li> Fazer 5 mm de cada um, 50 um. As secções transversais e devem ser através de toda a lesão a fim de que o ponto mais profundo da invasão pode ser identificado. Siga pela hematoxilina e eosina. </li><li> Avaliar a invasão tumoral medindo se células tumorais permanecem fora do colágeno estrato compactum, invadir na dorsalmusculatura ou invadir ainda mais para a coluna vertebral (Figura 2B, C). Determinar diferença estatística entre os dois conjuntos de amostras. </li></ol> 3. A coloração de anticorpo de melanócitos Escala <ol><li> Perfazer com tampão PO4 80 ml ​​0,1 M de Na 2 HPO 4 e 20 ml de 0,1 M de NaH 2 PO 4. Verifique se o pH é 7,3. Tornar-se tampão de solução 2x contendo 8,0 g de sacarose, 0,15 ml de CaCl 2 0,2 M e 90 ml de 0,2 M de tampão PO4, pH 7,3. Se necessário ajustar o pH para 7,3 com NaOH ou HCl, em seguida, completar a 100 ml com tampão de PO 4. </li><li> Pesar até 300 mg de sólido PFA e adicionar a um tubo de microcentrífuga. Adicionar NaOH 5 mM para um volume igual a 4,5 x a massa, em mg, de PFA (por exemplo, 450 ul de 5 mM de NaOH e 100 mg de PFA). Aquece-se a 60-70 ° C com agitação ocasional até que o PFA é dissolvido. Girar partículas remanescentes e recuperar 20% PFA sobrenadante. Fazer o tempo todo fresco. </li><li> Prepare a solução de fixação contendo 1x fix tampão e 4% PFA. </li><li> Anestesiar os peixes em 0,17 mg / ml tricaina. </li><li> Usando a luz incidente sob um microscópio de dissecação arrancar escamas dorsais usando nítidas de ponta fina pinça, tomando cuidado para não danificar o meia melanócito contendo da escala (Figura 3A). Escalas local diretamente a pinça em uma solução de fixação ml e incubar à temperatura ambiente, ao girar, para ≥ 2 horas. Transfira o peixe em água de peixe e monitorar o peixe para confirmar a recuperação bem sucedida. </li><li> A lixívia melanócitos pigmentados lavar tabelas fixas em PBST (1x PBS pH 7,4, 0,1% de Triton X-100) 2 vezes durante 5 min à temperatura ambiente. Adicionar 1 ml de solução alcalina feita recentemente contendo 0,4 ml de KOH a 10% e 0,15 ml de 30% de H 2 O 2 em 5 ml de dH 2 O. Coloque em bloqueios parafilme ou boné para evitar que o gás de estourar os tubos abertos. Continuar até branqueamento pigmento é ido (10 min é típico) (Figura 3B, C). Lavar quatro vezes em PBST, durante 5 min à temperatura ambienteratura. </li><li> Bloco de, pelo menos, 30 min em solução de bloqueio feita de 1x PBS pH 7,4, 0,2% Triton X-100, 2 mg / ml de BSA, 1% de DMSO, 0,02% NaN 3, e 2% de soro de cordeiro. Deixar de fora NaN 3 se desenvolver com a reação HRP. </li><li> Incubar com anticorpo primário em solução bloqueadora durante a noite à temperatura ambiente. Você precisa de ter, pelo menos, 400 ul de solução sobre as amostras para mantê-los submergidos. </li><li> Lave as escalas 3 vezes durante 30 min em PBST, à temperatura ambiente. </li><li> Incubar com o anticorpo secundário de 2 horas a durante a noite em solução de bloqueio à temperatura ambiente. </li><li> Mancha por 10 min com PBST DAPI uM + 0.1. </li><li> Lavar 3 vezes durante 5 min com PBST. </li><li> Montar as escalas em uma lâmina de vidro em uma gota de Vectashield de modo que o lado côncavo da balança está voltado para baixo no sentido da lâmina e colocar uma lamela de vidro sobre eles. Vedar as extremidades da lâmina com unha polonês clara e observar as lâminas num microscópio de fluorescência (Figura 3D, E, F, G </strong&gt;). </li></ol> 4. Transplante de Ensaio <ol><li> Prepare destinatário 2-3 meses de idade casper 9 peixes através da irradiação com 25 Gy de radiação gama, um dia antes do transplante 10. Permitir que os peixes se recuperar em peixes de água. </li><li> Euthanize um peixe portadores de tumor tal como previamente descrito (secção 2.2). </li><li> Cortar o tumor e colocá-lo em uma placa de Petri com aproximadamente 5 ml de filtro esterilizado 0.9x PBS com 5% de FBS. Dado o tumor com uma lâmina de barbear, enquanto em solução. </li><li> Trabalhar à temperatura ambiente, tritura-se com uma pipeta P1000 para obter células únicas. Perfazer o volume para 25 ml com filtro esterilizado 0,9 x PBS com 5% de FBS. </li><li> Filtrar através de filtro de 40 mesh uM. </li><li> Girar em uma centrífuga de bancada Eppendorf 5810R a 1.500 rpm (453 rcf) durante 10 min. </li><li> Remover o sobrenadante e fazer a suspensão das células a uma concentração aproximadamente desejado (assume-se 10 7 células de tumor de 100 mm 3). </li><li> Calcular tele número exato de células utilizando um hemocitómetro e dilui-se a suspensão de células de modo a que o volume final de injecção é de cerca de 5 ul. Por exemplo, se a 50.000 células são para ser injectada, a suspensão de células deve ser diluído para uma concentração final de 10000 células / uL. Flick o tubo que contém a suspensão de células a cada poucos minutos para evitar aglomeração das células. </li><li> Lavar um calibre 26 (ponta cónica) 701 N 10 seringa Hamilton ul 2-3 vezes com etanol a 100% e 0,9 x PBS. Carregar a seringa com 5 uL da suspensão de células. </li><li> Anestesiar os peixes destinatário irradiado em 0,17 mg / ml tricaina. Coloque o peixe de lado sobre um Kimwipe húmido (Figura 4A). </li><li> Estabilizar os peixes com uma mão e inserir a agulha com o bisel voltado para cima em um ângulo de 45 ° no flanco do peixe acima da cavidade peritoneal a meio caminho entre o limite posterior do cérebro posterior e borda anterior da barbatana dorsal. Suavemente o êmbolo (Figura 4B). &lt;/ Li&gt; <li> Permitir que o peixe se recuperar a água de peixe fresco e observar o peixe por dia, durante o enxerto do tumor (Figura 4C). Se o enxerto tenha ocorrido, o crescimento continuado e o desenvolvimento da doença pode também 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>

Não há conflitos de interesse declarados.

O método miniCoopR permite a expressão de genes de interesse em melanócitos zebrafish. Esta abordagem toma vantagem do facto de que o gene do peixe-zebra mitfa age células autonomamente. Por este motivo, os melanócitos resgatados pelo vector miniCoopR são determinados para conter o minigene e qualquer gene de interesse ao qual ele está fisicamente acoplados. Melanócitos resgatados são claramente visíveis e podem ser obtidos de animais que foram injectados como embriões de uma única célula. Um grau ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Recombinação reagentes de gateway </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td> Referência 5 </td></tr><tr><td> Anticorpo Mitfa </td><td></td><td></td><td> Referência 5 </td></tr><tr><td> FITC de cabra anti-coelho 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> Referência 9 </td></tr><tr><td> 701N Seringa 10 ul </td><td> Hamilton / Fisher </td><td> 14-824 </td><td> &amp;nbsp; </td></tr><tr><td> 40 uM 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

Estudos genômicos de cânceres humanos produziram uma riqueza de informações sobre os genes que são alterados em tumores 1,2,3. Um desafio proveniente destes estudos é que muitos genes é alterada, e isso pode ser difícil distinguir as alterações genéticas que levaram tumorigénese de que aqueles surgiu incidentalmente durante a transformação. Para fazer esta distinção é benéfico ter um ensaio que pode medir quantitativamente o efeito de um gene alterado em iniciação do tumor e de outros processos que permitem que os tumores de persistir e disseminar. Apresentamos aqui um meio rápido para pesquisar um grande número de modificadores candidatos de melanoma no peixe-zebra utilizando um modelo de 4 autóctone tumor que inclui os passos necessários para a iniciação e manutenção de melanoma. Um reagente chave no presente ensaio é o vector miniCoopR, que acopla a cópia de tipo selvagem do factor especificação mitfa melanócito para uma cassete na qual a recombinação gateway candidato melAnoma genes podem ser recombinados 5. O vector tem um minigene miniCoopR resgate mitfa que contém o promotor, quadros de leitura aberta e região 3 &#39;não traduzida do gene de tipo selvagem mitfa. Ele permite fazer construções de comprimento total, utilizando quadros de leitura abertos de modificadores de melanoma candidatos. Estes clones individuais podem então ser injetado em uma única célula de Tg (mitfa: BRAF V600E), p53 (lf); embriões mitfa (LF) zebrafish. O vector miniCoopR fica integrado por Tol2 mediada por transgénese 6 e resgates melanócitos. Porque eles são fisicamente acoplados ao resgate mitfa minigene, genes candidatos são expressos em melanócitos resgatados, alguns dos quais irão transformar e desenvolver-se em tumores. O efeito de um gene candidato para a iniciação melanoma e as propriedades das células de melanoma pode ser medida utilizando as curvas de sobrevida livre de melanoma, ensaios de invasão, coloração de anticorpos e ensaios de transplante.

Uma célula-Tg (mitfa: BRAF V600E), p53 (lf); mitfa embriões (lf) zebrafish foram injectados com o vector contendo o oncogene miniCoopR melanoma SETDB1 5 ou EGFP, cada um sob o controlo do promotor de mitfa. Embriões com resgate de melanócitos foram selecionados e permitiu a amadurecer. Aos 2 meses de idade, com os animais de resgate melanócito maior do que 4 mm 2 foram selecionados. Os animais foram examinados semanalmente para melanomas. Curvas de ...

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

Uma forma rápida de tela para modificadores de melanoma, utilizando um modelo de tumor zebrafish autóctone é apresentado. Leva vantagem de o vector miniCoopR que permite a expressão de genes candidatos de melanoma nos melanócitos. Um método para obtenção das curvas de sobrevida livre de melanoma, um ensaio de invasão, um protocolo para a coloração de anticorpos de melanócitos de escala e um ensaio de transplante de melanoma são descritos.

Triagem para Modificadores Melanoma usando um modelo de tumor Zebrafish autóctone

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 visualizações.  University of Massachusetts Medical School. Uma forma rápida de tela para modificadores de melanoma, utilizando um modelo de tumor zebrafish autóctone é apresentado. Leva vantagem de o vector miniCoopR que permite a expressão de genes candidatos de melanoma nos melanócitos. Um método para obtenção das curvas de sobrevida livre de melanoma, um ensaio de invasão, um protocolo para a coloração de anticorpos de melanócitos de escala e um ensaio de transplante de melanoma são descritos.

Vídeo: 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-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

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

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.

Avaliar as alterações teratogênicos em um modelo de Zebrafish de exposição fetal ao álcool

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 Linhas tumor neuroendócrino de células como um modelo tridimensional para o estudo da terapia de tumores humanos Neuroendócrino

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).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos 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.

Um modelo Zebrafish de Diabetes Mellitus e Memória 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.

A Novel de alta resolução<em&gt; In vivo</em&gt; Técnica de Imagem para estudar a resposta dinâmica de estruturas intracranianas ao crescimento do tumor e Terapêutica

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.

Um Modelo Possível Zebrafish da doença renal policística: Knockdown de<em&gt; Wnt5a</em&gt; Faz com cistos no peixe-zebra Rins

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 de co-cultura para Interaction 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.

Isolado hepática Perfusão como um tratamento para o fígado Metástases de Uveal Melanoma

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.

Avaliação do tumor infiltrantes Leucócitos Subconjuntos em um modelo de tumor subcutâneo

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