O trabalho dos autores FCO, DF, JN, DJH e GDS mencionado acima é financiado pelo Escritório Cientista Chefe, número de concessão: ETM37 e apoiado pelo Reino Unido Centro de Pesquisa do Câncer Medicina Experimental do Câncer. O trabalho de AL é financiado pelo Royal College of Surgeons de Edinburgh Robertson Trust, o Confiança Melville para o cuidado ea cura do câncer e do Conselho de Pesquisa Médica. IO é apoiado por uma Sociedade Real de Edimburgo Fellowship Governo escocês co-financiado pelas Acções Marie Curie e do UK Medical Research Council. Os autores gostariam de agradecer SCOTRRCC co-candidatos e colaboradores para suas discussões úteis sobre alguns dos temas abordados neste trabalho.

1. Identificação de morfológica e molecular Heterogeneidade Tumor <ol><li> Os tumores removidos do congelador a -80 ° C e mantido em gelo seco. </li><li> Dividir os tumores em secções de cerca de 1 cm 3. Mapear a posição original de cada secção do tumor em relação ao outro e etiqueta com um nome único. Armazenar as amostras em criotubos individuais a -80 ° C até que esteja pronto para uso. </li><li> Amostras do revestimento em outubro e cortar em um criostato a -22 ° C. </li><li> Amostras corados com Hematoxilina e Eosina método contracoloração. </li><li> Análise de microscopia de cortes congelados para garantir que eles são de natureza ccRCC, tumor viável e de classificação (de baixo grau, de alto grau ou mista de grau baixo / alto, desafiando a diferenciar os graus Fuhrman 1 a 4 na seção de congelados). Figura 1 a &amp; b (coloração H &amp; E de grau baixo e elevado misto). </li><li> Selecione até 4 amostras de cada região morfologicamente diferentes dentro de cada tumor para extração de proteínas. </li></ol> 2. Extracção de proteínas a partir de amostras de tumores <ol><li> Corte amostra de tumor de outubro </li><li> Lugar 50-75 mg de tecido em tubos de 2 ml com 990 ul de tampão de lise [Tris 50 mM-HCl (pH 7,5), 5 mM de EGTA (pH 8,5), 150 mM de NaCl] suplementado com aprotinina (Sigma A6279) (10 mg / ml), a fosfatase cocktail inibidor 2 (Sigma, P5716), cocktail de inibidores de fosfatase 3 (Sigma, P0044) e um cocktail de inibidores de protease (Roche, 11836153001). </li><li> Adicionar uma única esfera de aço de 5 mm de cada tubo e homogeneíza-se a 50 Hz, durante 5 min duas vezes utilizando um TissueLyser, verificando o nível de homogeneização, após cada período de 5 min. </li><li> Transfira a amostra homogeneizada novo tubo de 2 ml usando uma pipeta de sair da esfera de aço para trás. </li><li> Adicionar 10 ul de Triton X-100 a cada uma das amostras antes da centrifugação a 13.000 xg durante 30 min a 4 ° C. </li><li> Transfira os sobrenadantes para tubos de microcentrífuga fresco. </li><li> Determinar a concentração de proteína utilizando o BCA ensaio (Figura 2). </li><li>Normalizar as concentrações de proteína a 1 mg / ml. </li></ol> 3. Validação de anticorpo <ol><li> Preparar as amostras de proteína (extraído de linhas de células ou tecidos adequados) para transferência de Western e executado em um gel de 10% SDS-PAGE. </li><li> Transferir as amostras para uma membrana de nitrocelulose durante a noite a 4 ° C. </li><li> Bloquear a membrana no tampão de Li-Cor Odyssey bloqueio (50:50 diluída em PBS) durante 1 hora à temperatura ambiente. </li><li> Diluir os anticorpos primários em Li-Cor Tampão de Bloqueio Odyssey (50:50 diluída em PBS) na diluição recomendada os fabricantes tipicamente 1 em 1000. </li><li> Incubar membrana em anticorpos primários durante a noite a 4 ° C. </li><li> Tornar-se 0,1% de PBS-Tween 20 (PBS-T, 1 ml Tween 20/1 L de PBS). </li><li> Lavar membrana em PBS-T a temperatura ambiente durante 5 min (x3). </li><li> Diluir fluorescente marcado com anticorpos secundários em Tampão de Bloqueio Odyssey (diluído em PBS, 50:50) 0,01% de SDS a diluição 1:10000 (1,5 μl/15 ml). </li><li> Incubar em membrana secundáriaanticorpos à temperatura ambiente durante 45 min com agitação suave - é importante proteger a membrana a partir da luz até ao momento em que tenha sido finalmente digitalizado. </li><li> Lavar membrana em PBS-T a temperatura ambiente durante 5 min (x3), mantendo-se a membrana no escuro. </li><li> Lavar membrana em PBS à temperatura ambiente durante 5 min (x3) para remover Tween20 residual, mais uma vez mantendo a membrana no escuro. </li><li> Deite membrana plana sobre um pedaço de papel de filtro no escuro e deixar secar ao ar - permitindo que a membrana de secar pode melhorar o sinal e reduzir o fundo, mas tornar-se inútil para remoção e re-sondagem. </li><li> Digitalização da membrana no scanner Odyssey LiCor. Manter a membrana no escuro até ao momento em que tenha sido digitalizada. </li><li> Apenas seleccionar anticorpos que geram uma única banda predominante no peso molecular correcto. Figura 3 mostra exemplos de anticorpos aceitáveis ​​e inaceitáveis ​​para uso com RPPA. </li></ol> 4. RPPA PriNTING <ol><li> Lisados ​​de proteínas foram depositados em lâminas de nitrocelulose revestidos de vidro (Fastslides-Whatman) usando um MicroGrid II robótica observador. As lâminas utilizadas continham duas almofadas sobre o qual as amostras foram manchados. Cada bloco foi manchado com amostras idênticas, neste caso, 100 amostras. Outros formatos disponíveis incluem 1, 8 e 16 lâminas almofada. Quanto maior o número de almofadas de quanto menor for o número de amostras que podem ser depositados em cada um. </li><li> Uma série de cinco diluições de 2 vezes foram feitas a partir de cada amostra e cada uma delas foi então visto em triplicado (resultando num total de 15 pontos por amostra). </li></ol> 5. Detecção de Proteína RPPA <ol><li> Lâminas molhados em tampão LiCor excesso de bloqueio (50:50 diluída em PBS). Incubar à temperatura ambiente durante 1 hora numa plataforma de agitação. </li><li> Preparar 800 ul de anticorpos primários em LiCor tampão de bloqueio (diluído em PBS, 50:50) para as concentrações desejadas e manter em gelo. </li><li> Montar as lâminas em (i) Clip Chip o único frameou (ii) a quatro &#39;FastFrame&#39; porta-lâminas compartimento de modo que uma vedação apertada é formado entre a lâmina e a câmara de incubação. </li><li> Remover o tampão residual a partir de poços e adicionar 600 uL de anticorpo primário às cavidades respectivas. </li><li> Colocar a lâmina e câmara em uma caixa selada húmida e incubar no balanço da plataforma durante a noite a 4 ° C. </li><li> Tornar-se 0,1% de PBS-Tween 20 (PBS-T, 100 ul de Tween 20/100 ml de PBS). </li><li> Retirar as lâminas da câmara e remova cuidadosamente os anticorpos primários de cada poço. </li><li> Adicionar 600 ul de PBS-T e lavagem das lâminas em balançar plataforma à temperatura ambiente durante 5 min (X3). </li><li> Prepare fluorescentemente marcadas com anticorpos secundários através de diluição em tampão de bloqueio Odyssey (diluído em PBS, 50:50) 0,01% de SDS a 1:2.000 de diluição (1 ul / 2 ml), em primeiro lugar. </li><li> Remover o tampão dos poços e adicionar 600 ul de anticorpos marcados com fluorescência, secundárias às cavidades respectivas. Incubar anticorpos secundários à temperatura ambiente durante 45 min com agitação suave - loÉ importante proteger a membrana a partir da luz até ao momento em que tenha sido finalmente digitalizado. </li><li> Remover anticorpos secundários de lavagem bem e rapidamente (3x) em 600 ul de PBS-T, à temperatura ambiente. Retirar a lâmina de suporte, a transferência para um recipiente adequado e lavar em excesso de PBS-T, durante 15 minutos, mantendo-se a membrana no escuro. </li><li> Remover o PBS-T e da membrana de lavagem adicional com PBS à temperatura ambiente durante 15 min para remover o residual de Tween-20, mais uma vez mantendo a membrana no escuro. </li><li> Seca-se o forno Fastslide em 50 ° C durante 10 min e, em seguida, digitalizar no digitalizador Odyssey Li-Cor. Mantenha o slide no escuro até que tenha sido digitalizado. </li><li> Digitalizar o slide a 680 nm e / ou 800 nm, dependendo do anticorpo secundário / anticorpos utilizados. Por duas cores de detecção de usar sempre altamente cross-adsorvidas anticorpos secundários para minimizar reatividade cruzada. A selecção cuidadosa de anticorpos primários e secundários é necessário para a detecção de duas cores. De importância primária é a selecção dediferentes espécies hospedeiras (coelho e rato, por exemplo) para os dois anticorpos primários. Isso permite que a discriminação por anti-coelho e rato anti-anticorpos secundários que são marcadas com corante com espectros de emissão facilmente distinguíveis. </li><li> Os arquivos de imagem são salvos como arquivos. Tiff. Figura 4 (imagem de arquivos digitalizados). </li></ol> 6. Análise de Dados <ol><li> Lançamento do Software MicroVigene (VigeneTech, Carlisle, MA, EUA). </li><li> Abra. Tiff arquivo de imagem contendo a digitalização do slide RPPA. </li><li> Seleccionar um ficheiro de modelo predefinido que terá uma grelha para sobrepor a imagem do slide RPPA. </li><li> Clique no botão definir regiões de interesse (ROI), para abrir a grade. </li><li> Posicione a grade sobre os pontos RPPA. Figura 6a (imagem da grade sobre a imagem). </li><li> Clique no botão Selecionar ALL destacar tudo o ROI. </li><li> Clique em Localizar tudo. MicroVigene vai automaticam encontrar o ROI, encontrar os pontos, subtrair o fundo, remova a poeira e quantificar pontos. </li><li> Clique no botão Exibir curva de diluição para trazer os resultados para todas as amostras no slide RPPA. </li><li> Clique em Salvar Dados diluição. </li><li> À medida que cada amostra é impresso em 5 pontos a cada diluição em triplicado, são 15 pontos a analisar, o que reduz o risco de erros e melhora a qualidade do ajustamento de curva. MicroVigene produz uma logística de 4 parâmetros de registo de modelo de algoritmo &quot;Supercurve&quot; (Figura 6b), que incorpora todos os pontos para produzir uma curva sigmoidal de antigénio-anticorpo cinética de ligação. A suposição é que os mesmos anticorpos antígeno-cinética de ligação está ocorrendo em cada ponto da amostra, mesmo em diferentes amostras, assim, tomando todos os pontos em uma matriz para atender a uma curva de resposta comum pode aumentar a confiança do ajuste de curva 10,11 Y = a + ((ba) / (1 + e-(c * d-ln (x))) em que x é o dilution factor e Y é a intensidade do sinal. As amostras podem ser analisadas comparativamente, usando o valor de y0, que, em nossa análise correspondia ao valor de y no ponto médio dos valores de x, após o mapeamento aqueles para o supercurve. </li><li> Exportar os dados em Microsoft Excel e enredo y0 como na Figura 7. </li><li> Variância proteína intratumoral foi calculado separadamente para tratados e não tratados pacientes tratados em um quadro ANOVA. Distribuições de variância combinando dados a partir de todas as proteínas analisadas foram comparados pelo teste de Mann-Whitney (TMV). Variações intratumorais para proteínas individuais foram comparadas pelo teste F, onde os pressupostos de normalidade e homocedasticidade realizadas, respectivamente avaliada utilizando o Lillefours e testes Fligner; taxa de descoberta de falsas (FDR) correção foi aplicada 12. A expressão diferencial de proteínas entre as amostras dos pacientes tratados e não tratados foi testada para cada proteína utilizando teste t de Student, onde normalidade e homocedasticidade suposiçãos foram atendidas, caso contrário, MWT foi realizada; FDR foi aplicado sobre os valores combinados t-teste e MWT 12. Significado da expressão de proteínas e correlação de Pearson variância para as proteínas foram estimados usando uma abordagem padrão [R referência] e FDR aplicada 12. </li></ol>

<ol>
	<li>Stewart, G. D., O'Mahony, F. C., Powles, T., Riddick, A. C., Harrison, D. 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=What+can+molecular+pathology+contribute+to+the+management+of+renal+cell+carcinoma.">What can molecular pathology contribute to the management of renal cell carcinoma.</a> Nat. Rev. Urol. 8, 255-265 (2011).</li><li>Rini, B. I., Michaelson, M. D., Rosenberg, J. E., Bukowski, R. M., Sosman, 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=Antitumor+activity+and+biomarker+analysis+of+sunitinib+in+patients+with+bevacizumab-refractory+metastatic+renal+cell+carcinoma.">Antitumor activity and biomarker analysis of sunitinib in patients with bevacizumab-refractory metastatic renal cell carcinoma.</a> J. Clin. Oncol. 26, 3743-3748 (2008).</li><li>Swanton, C., Larkin, J. M., Gerlinger, M., Eklund, A. C., Howell, 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=Predictive+biomarker+discovery+through+the+parallel+integration+of+clinical+trial+and+functional+genomics+datasets.">Predictive biomarker discovery through the parallel integration of clinical trial and functional genomics datasets.</a> Genome Med. 2, 52 (2010).</li><li>Cortes, J., Jabbour, E., Kantarjian, H., Yin, C. C., Shan, 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=Dynamics+of+BCR-ABL+kinase+domain+mutations+in+chronic+myeloid+leukemia+after+sequential+treatment+with+multiple+tyrosine+kinase+inhibitors.">Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors.</a> Blood. 110, 4005-4011 (2007).</li><li>Gerlinger, M., Rowan, A. J., Horswell, S., Larkin, J., Endesfelder, 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=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N. Engl. J. Med. 366, 883-892 (2012).</li><li>Fisher, R., Larkin, J., Swanton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter+and+intratumour+heterogeneity:+a+barrier+to+individualized+medical+therapy+in+renal+cell+carcinoma.">Inter and intratumour heterogeneity: a barrier to individualized medical therapy in renal cell carcinoma.</a> Front. Oncol. 2, 49 (2012).</li><li>Yachida, S., Jones, S., Bozic, I., Antal, T., Leary, 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=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467, 1114-1117 (2010).</li><li>O'Mahony, F. C., Faratian, D., Varley, J., Nanda, J., Theodoulou, 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+use+of+automated+quantitative+analysis+to+evaluate+epithelial-to-mesenchymal+transition+associated+proteins+in+clear+cell+renal+cell+carcinoma.">The use of automated quantitative analysis to evaluate epithelial-to-mesenchymal transition associated proteins in clear cell renal cell carcinoma.</a> PLoS One. 7, e31557 (2012).</li><li>Spurrier, B., Ramalingam, S., Nishizuka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse-phase+protein+lysate+microarrays+for+cell+signaling+analysis.">Reverse-phase protein lysate microarrays for cell signaling analysis.</a> Nat. Protoc. 3, 1796-1808 (2008).</li><li>Hu, J., He, X., Baggerly, K. A., Coombes, K. R., Hennessy, B. T., 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=Non-parametric+quantification+of+protein+lysate+arrays.">Non-parametric quantification of protein lysate arrays.</a> Bioinformatics. 23, 1986-1994 (2007).</li><li>Wang, X., Dong, Y., Jiwani, A. J., Zou, Y., Pastor, 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=Improved+protein+arrays+for+quantitative+systems+analysis+of+the+dynamics+of+signaling+pathway+interactions.">Improved protein arrays for quantitative systems analysis of the dynamics of signaling pathway interactions.</a> Proteome Sci. 9, 53 (2011).</li><li>Benjamini, Y., Hochberg, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+False+Discovery+Rate:+A+Practical+and+Powerful+Approach+to+Multiple+Testing.">Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing.</a> Journal of the Royal Statistical Society Series B (Methodological). 57, 289-300 (1995).</li></ol>

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O método aqui apresentado RPPA representa uma alternativa para a produção elevada amplamente utilizada mas comparativamente baixa taxa de transferência western blot técnica de análise de proteínas. O método permite que centenas de amostras para ser semi-quantitativamente analisadas e comparadas simultaneamente permitindo a comparação directa de proteínas-chave através de uma grande variedade de linhas celulares e amostras de tecido. Multiplexagem com espécies diferentes de anticorpos aumenta ainda mais o po...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> aprotinina </td><td> Sigma </td><td> A6279 </td></tr><tr><td> cocktail inibidor de fosfatase duas </td><td> Sigma </td><td> P5726 </td></tr><tr><td> cocktail de inibidores de fosfatase 3 </td><td> Sigma </td><td> P0044 </td></tr><tr><td> cocktail inibidor de protease </td><td> Roche </td><td> 11836153001 </td></tr><tr><td> Triton X-100 </td><td> Triton-X </td><td> T8787 </td></tr><tr><td> Li-Cor Odyssey tampão de bloqueio </td><td> Li-Cor </td><td> 927-40000 </td></tr><tr><td> TissueLyser </td><td> Qiagen </td><td> 85600 </td></tr><tr><td> MicroGrid II robótica spotter </td><td> Biorobotics </td><td></td></tr><tr><td> Quatro FastFrame &#39;suporte de slides baía </td><td> Whatman </td><td> 10486001 </td></tr><tr><td> Deslize FAST - 2-Pad </td><td> Whatman </td><td> 10485317 </td></tr><tr><td> IRDye Cabra 680LT anti-IgG </td><td> Licor </td><td> 926-68020 </td></tr><tr><td> IRDye Cabra 800CW anti-IgG de coelho </td><td> Licor </td><td> 926-32211 </td></tr></tbody></table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

Actualmente não existe tratamento curativo para cancro metastático da célula clara de células renais, o mais comum dos variante da doença. Um factor chave neste resistência ao tratamento é considerado a complexidade molecular da doença 1. Terapia-alvo, tais como o inibidor da tirosina cinase (TKI)-sunitinib têm sido utilizados, mas apenas 40% dos pacientes respondem, com a grande maioria destes pacientes recidivantes dentro de 1 ano 2. Como tal a questão da resistência intrínseca e adquirida em pacientes com câncer de células renais é altamente relevante 3. Para estudar a resistência a inibidores da tirosina quinase, com o objetivo final de desenvolver tratamentos eficazes e personalizados, tecido seqüencial após um período específico de terapia-alvo é necessária, uma abordagem que tinha sido bem sucedida em mielóide crônica 4 leucemia. No entanto, a aplicação de uma tal estratégia em carcinoma de células renais é complicada pelo elevado nível de both heterogeneidade inter-e intra-tumoral, o que é uma característica do carcinoma de células renais, 5,6, assim como outros tumores sólidos 7. Heterogeneidade Intertumoral devido a diferenças transcriptomic e genética está bem estabelecido, mesmo em pacientes com apresentação semelhante, estágio e grau do tumor. Além disso, é evidente que existe uma grande heterogeneidade (intratumoral) morfológica no RCC, a qual é susceptível de representar ainda maior heterogeneidade molecular. Mapeamento detalhado e classificação de tumores CCR por análise morfológica e classificação combinada Fuhrman permite a seleção de áreas representativas para análise proteômica. Análise de proteínas baseada em 8 RCC é atraente devido a sua ampla disponibilidade em laboratórios de patologia, no entanto, a sua aplicação pode ser problemática devido à disponibilidade limitada de anticorpos específicos 9. Devido à natureza dot blot dos arrays de proteínas, em fase reversa (RPPA), a especificidade do anticorpo deve be pré-validado, como o controlo de qualidade rigoroso de tais anticorpos utilizados é de importância primordial. Apesar desta limitação, o formato dot blot não permite a miniaturização do ensaio, permitindo a impressão de centenas de amostras para uma lâmina de nitrocelulose único. Slides impressos podem então ser analisadas de uma forma semelhante à análise de Western com o uso de alvos específicos anticorpos primários e anticorpos secundários marcados com fluorescência, permitindo a multiplexagem. A expressão diferencial de proteínas em todas as amostras numa lâmina pode então ser analisados ​​simultaneamente através da comparação do nível relativo de fluorescência de uma forma mais rentável e de alto rendimento.

Um exemplo de uma lâmina de RPPA digitalizada pode ser visto na Figura 4 (i), com ambos os canais 680 e 800 nm indicadas. Separando as imagens por comprimento de onda, a Figura 4 (ii) permite que cada bloco na corrediça RPPA a ser analisado e a expressão da proteína individual determinada Figura 4 (iii). Como pode ser visto na Figura 4 (iii) a expressão de proteínas individuais através das amostras é o único com gelsolina tendo um elevado níve...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA permite a expressão da proteína de centenas de amostras, impressos em lâminas de nitrocelulose para serem interrogados ao mesmo tempo, utilizando anticorpos marcados com fluorescência. Esta técnica tem sido utilizada para estudar o efeito do tratamento com medicamentos na heterogeneidade claro carcinoma de células renais.

O uso de matrizes de fase reversa Proteínas (RPPA) para explorar variação Protein Expression individuais dentro de câncer de células renais

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

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The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

33.5K visualizações.  University of Edinburgh. RPPA permite a expressão da proteína de centenas de amostras, impressos em lâminas de nitrocelulose para serem interrogados ao mesmo tempo, utilizando anticorpos marcados com fluorescência. Esta técnica tem sido utilizada para estudar o efeito do tratamento com medicamentos na heterogeneidade claro carcinoma de células renais.

Vídeo: The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

Uso de um sistema de suspensão de peso para oclusão da artéria renal isolada

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Medicina

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Mapeamento de heterogeneidade de expressão da proteína em tumores usando imunofluorescência Quantitative

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Produção e Detecção de Espécies Reativas de Oxigênio (ROS) em Câncer

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Purificação e Agregação do Domínio proteína precursora de amilóide intracelular

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Identificação baseada em Microarray de Individual HERV Loci Expressão: Aplicação a descoberta de biomarcadores em câncer de próstata

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Tridimensional Cultura Ensaio para Explorar Cancer Invasividade celular e formação de tumor Satélite

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Um método eficiente de crivagem para isolar intactos glomérulos do rim de rato adulto

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

Implementação de precisão do teste de dose mínima de eritema (MED) para avaliar a variação individual na resposta inflamatória humana

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

Entrega direta de medicamentos ao rim através da artéria renal

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...