Apoiado em parte pelo NIH conceder (R43/44CA122444) e DOD BRCP concessão (W81XWH-06-1-0682) para YL.

1. Sample, Equipamento e Preparação do reagente <ol><li> Corte amostra de tecido em blocos de 3-4 mm de espessura, fixar em fresco formalina a 10% neutra tamponada por 16-32 horas à temperatura ambiente (25 º C) e inserir em parafina. Corte FFPE em seções de 5 ± 1 mM espessura dos blocos FFPE, montar seções em slides e ar seco. Nota: Os slides podem ser armazenados à temperatura ambiente sob dessecação por até 3 meses. As lâminas de tecido montadas deve ser cozido numa seco durante a 60 ° C durante 1 hora antes do ensaio RNAscope. </li><li> Traga Hibridização forno a 40 ° C. Coloque um papel umidificação no Tabuleiro do controle de umidade. Adicionar 50 ml de dH 2 O para o Livro umidificação de molhá-lo completamente. Insira a bandeja coberta para o forno para pré-aquecimento por pelo menos 30 minutos antes de usar. </li><li> Adicione 700 ml de 1x Solução Pretreat 2 (10 nl, pH 6, tampão de citrato) por diluição 10x Solução de pré-tratamento em dH2O Aqueça o 1x Pretreat 2 Solução para ferver e maintain a temperatura entre 100-104 ° C enquanto prevenindo sobre-ebulição. </li><li> Faça 3 L 1x Wash Buffer (0,1 x SSC), diluindo pré-aquecido 50x Wash Buffer em dH 2 O. </li><li> Preparar 2 x 200 ml de xileno e 2 x 200 ml de EtOH a 100% para desparafinização sob um exaustor. </li><li> Preparar a solução de coloração Hematoxilina 50% e reagente bluing (0,01% de amônia da água) para o pós-coloração sob uma coifa. </li><li> Preparar 200 ml de xileno, a 200 ml de 70%, e 2 x 200 ml de EtOH a 100% para a desidratação sob um exaustor. </li><li> Pré-aquecer as sondas alvo a 40 ° C por 10 min e trazer Detecção RTU reagente Kit à temperatura ambiente, incluindo RTU AMP1, Amp2, AMP3, AMP4, Amp 5-Brown, e Amp6-Brown, exceto DAB Chromogen Brown-A e Brown-B . </li></ol> 2. RNAscope Assay Desparafinização e desidratação Após o cozimento, Retire a parafina cortes de tecido em xileno para 2 x 5 minutos com agitação freqüente, e desidratarem EtOH a 100% durante 2 x 3 min, com agitação frequente. Secar ao ar durante 5 minutos e extrair uma barreira hidrofóbica em torno da secção de tecido com uma barreira hidrofóbica Pen. Pré-tratamentos <ol><li> Incubar as secções de tecido com Pretreat 1 durante 10 minutos a RT para extinção da actividade de peroxidase endógena, enxaguar em dH2O </li><li> Incubar as secções de tecido com Pretreat 2 mantidos à temperatura de ebulição durante 15 minutos para a recuperação de ARN, lavar duas vezes em dH2O </li><li> Incubar as secções de tecido com Pretreat 3 durante 30 min a 40 ° C durante a digestão da proteína no forno HybEZ, lavar duas vezes em dH2O </li></ol> Alvo sonda de hibridação Sondas alvo HPV-HR 7 piscina incluem: HPV16, 18, 31, 33, 35, 52 e 58. Adicionar sondas de HPV, ubiquitina C (UBC) e bacterianas sondas dapB gene separadamente em três seções do tecido adjacente. A hibridização a 40 ° C no forno durante 2hr, depois enxaguar em tampão 1x Wash por 2 x 2 min à temperatura ambiente. A amplificação de sinal <ol><li> Incubar as secções de tecido com AMP1 (pré-amplificador) durante 30 min a 40 ° C, Amp2 (fundo redutor) durante 15 min a 40 ° C, AMP3 (amplificador) durante 30 min a 40 ° C, e AMP4 (sonda de marcação) durante 15 min a 40 ° C no forno HybEZ. Depois de cada passo de hibridação, lavagem em tampão 1x de lavagem para 2 x 2 min à temperatura ambiente. </li><li> Incubar as secções de tecido com AMP5 durante 30 min e Amp6 durante 15 min a RT. Depois de cada passo de incubação, lavagem em tampão 1x de lavagem para 2 x 2 min à temperatura ambiente. </li></ol> Detecção de Sinal Incubar as secções de tecido com DAB 01:01 Mistura misturando igual volume de castanho e castanho-A-B por 10 min à temperatura ambiente, lavar duas vezes em dH2O Contracoloração <pclass = &quot;jove_content&quot;&gt; cortes de tecido mancha com solução a 50% de hematoxilina por 2 min à temperatura ambiente, lave com dH 2 O até lâminas são claras, enquanto o tecido ficar roxo. Dip desliza para 0,01% de amônia em dH 2 O para 5x e seguiu com 5 mergulhos em dH 2 O. Deslize de montagem Desidratar as secções de tecido em 70%, 100%, e 100% de EtOH, durante 2 min cada, xileno, durante 5 minutos, montagem com meios de montagem baseada em xileno.

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S., 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+intestinal+stem+cell+markers+Bmi1+and+Lgr5+identify+two+functionally+distinct+populations.">The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (2), 466-471 (2012).</li><li>Payne, R. 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=Viable+circulating+tumour+cell+detection+using+multiplex+RNA+in+situ+hybridisation+predicts+progression-free+survival+in+metastatic+breast+cancer+patients.">Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients.</a> Br. J. Cancer. 106 (11), 1790-1797 (2012).</li><li>Bordeaux, J. 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Todos os autores são empregados pelo próprio estoque e em diagnósticos avançados celulares, Inc.

O ensaio RNAscope HPV permitiu a visualização direta de E6/E7 mRNA in situ na cabeça associada ao HPV e carcinoma de células escamosas pescoço. O ensaio RNAscope é totalmente compatível com o tecido do tumor rotineiramente fixo e preserva a morfologia do tecido para as correlações histopatológicas (Figura 2). Uma vantagem chave do ensaio em relação aos métodos RNAscope CISH convencionais é que especificamente amplifica os sinais de hibridação (Figuras 2B e 2E) sem amplificação do ruído de fundo (Figuras 2C e 2F). Na prática, o procedimento de ensaio RNAscope HPV pode ser concluída dentro de 8 horas ou convenientemente dividido em dois dias. O ensaio RNAscope HPV tem sido usado para determinar a infecção por HPV na cabeça e pescoço, carcinoma de células escamosas 12-16, que demonstra a sensibilidade 97% e especificidade de 93% utilizando qRT-PCR, como o método de referência 16. Chr convencionalISH omogenic para HR-HPV DNA é altamente específico, mas tem uma sensibilidade de 80% ~ 12. Imuno-histoquímica (IHQ) coloração para o celular substituto marcador p16 demonstra excelente sensibilidade, mas podem gerar resultados falsos positivos 15,18, especialmente na cabeça e pescoço nonoropharyngeal 15. O atual método &quot;padrão ouro&quot; de qRT-PCR para HPV E6/E7 detecção do RNAm requer tecido fresco congelado para melhores resultados e é tecnicamente complexo, o que limita seu uso apenas para o laboratório de pesquisa. Além disso, requer a extração de RNA que torna impossível correlacionar expressão HR-HPV E6/E7 mRNA com a histopatologia. Há vários fatores críticos para o sucesso do ensaio RNAscope. Primeiro, para obter melhores resultados, os tecidos devem ser fixados em formalina a 10% fresco neutro tamponado à temperatura ambiente durante 16-32 horas de acordo com as diretrizes da ASCO / CAP 19. Em segundo lugar, o forno HybEZ é altamente recomendado, uma vez que permitemé um óptimo controlo de temperatura e umidade para a hibridização da sonda e as etapas de amplificação de sinal. Em terceiro lugar, é importante para remover o excesso de buffers residuais antes de cada passo, mas ainda manter a secção de tecido de secagem durante qualquer uma dessas etapas. O procedimento RNAscope manual descrita aqui tem sido completamente automatizada em um sistema de corrediça-coloração automóvel comercial 10. Isso deve facilitar muito a normalização das condições de ensaio e salvar o trabalho manual precioso em laboratórios de patologia clínica. Além disso, o software de análise de imagem dedicado foi desenvolvido 10 para identificar automaticamente as células e sinais de coloração em um slide digitalizado, o que deve ajudar a eliminar a subjetividade e melhorar a reprodutibilidade na pontuação. Em resumo, o ensaio RNAscope HPV detecta a presença de transcritos de E6/E7 de HPV-RH in situ de mRNA em tecidos FFPE. Tem um fluxo de trabalho que é familiar para labora patologia clínicatories por permitir a visualização direta destes em cortes de tecido. Ele oferece uma plataforma ideal para o exame (FFPE) amostras de tecido, e pode ser facilmente adotado por laboratórios de patologia de diagnóstico.

Contas de infecção pelo papilomavírus humano de alto risco (HR-HPV) para cerca de 5% de todos os cânceres em todo o mundo 1. A incidência de câncer de orofaringe associadas ao HPV tem aumentado nas últimas décadas, especialmente entre os homens. Carcinoma HPV-positivos orofaríngeo de células escamosas (OPSCC) provavelmente constituem a maioria de todos os cânceres de cabeça e pescoço, nos Estados Unidos nos próximos 20 anos 2. Carcinomas de células escamosas da orofaringe causados ​​pelo HPV estão associados com a sobrevivência favorável e status HPV tumor é um fator prognóstico forte e independente para a sobrevivência 3. Evidência para a ativação da transcrição dos oncogenes virais E6 e E7 é considerada o padrão ouro para a presença de HPV clinicamente relevante 4. No entanto, a detecção de ARNm de E6/E7 de tecido de tumor fresco pela técnica de RT-PCR não é prático na prática clínica de rotina e tem sido limitada aos laboratórios de investigação. Recentemente, nós have desenvolveu uma tecnologia de RNA ISH romance chamado RNAscope, que permite a detecção multiplex em células individuais com uma única molécula de RNA sensibilidade em formalina parafina fixo amostras de tecido incorporados (FFPE) 5-10. Nós fornecemos três linhas de evidência para uma única molécula de detecção 5. Primeiro, o design e sistema de amplificação de sinal de sonda RNAscope permitiu a detecção de uma única cópia HER2 alvos de DNA genômico em HeLa e SK-BR-3 linhas celulares. Em segundo lugar, quando comparado com os sinais de ADN genómico de HER2, a distribuição das intensidades de fluorescência do sinal HER2 mRNA pontos em células HeLa era consistente com uma molécula por ponto. Em terceiro lugar, o número de sinais de HER2 de mRNA por célula pontos combinados intimamente o número de cópias de mRNA HER2 estimado por um ensaio de quantificação baseado em solução, apoiando ainda mais a detecção de uma única molécula. Além disso, a coloração de contraste de núcleos com DAPI em microscopia fluorescente ou com hematoxilina em microscopia de campo claro permite a visualização de núcleos individuais, which por sua vez, permite a detecção e quantificação de alvos de ARN sobre uma base de uma única célula 10. A capacidade de analisar a expressão do gene in situ em amostras clínicas de rotina faz RNAscope uma plataforma promissora para o diagnóstico da patologia, especialmente os ensaios baseados em secção de tecido FFPE 10,11. Nós desenvolvemos um ensaio de HPV baseada em RNAscope para detectar ARNm de E6/E7 de genótipos de HPV de alto risco (HPV16, 18, 31, 33, 35, 52, e 58), utilizando um conjunto de sondas específicas de genótipo. Nossos estudos recentes em OPSCC têm mostrado que o ensaio RNAscope HPV é altamente sensível e específico para determinar o status do HPV em tecidos FFPE 12-17, e também informa o prognóstico em OPSCC 12,16. O princípio da tecnologia RNAscope foi anteriormente descrito 5. Aqui, nós descrevemos o protocolo de ensaio RNAscope completa e demonstrar a sua utilização na detecção de HR-HPV em secções de tecidos tumorais FFPE.

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		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="39">
			<td height="60" rowspan="2" style="height:60px;width:145px;">SuperFrost Plus slides</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">12-550-15</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:145px;">HybEZ Oven, Tray, and Rack</td>
			<td style="width:145px;">Advanced Cell Diagnostics</td>
			<td style="width:145px;">310011, 310012, 310014</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:145px;">RNAscope 2.0 FFPE Reagent Kit - Brown</td>
			<td style="width:145px;">Advanced Cell Diagnostics</td>
			<td style="width:145px;">310035</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:145px;">RNAscope HPV-HR7 Probe for HPV 16,18,31,33,35,52 and 58, E6/E7 mRNA</td>
			<td style="width:145px;">Advanced Cell Diagnostics</td>
			<td style="width:145px;">312351</td>
			<td style="width:145px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:145px;">ImmEdge Hydrophobic Barrier Pen</td>
			<td style="width:145px;">Vector Laboratory</td>
			<td style="width:145px;">H-4000</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">100% EtOH</td>
			<td rowspan="2" style="width:145px;">American Master Tech Scientific</td>
			<td rowspan="2" style="width:145px;">ALREAGAL</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:145px;">Xylene&#160;</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">X3P-1GAL</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
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		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">Gill&#8217;s Hematoxylin I</td>
			<td rowspan="2" style="width:145px;">American Master Tech Scientific</td>
			<td rowspan="2" style="width:145px;">HXGHE1LT</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
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		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">Ammonia hydroxide</td>
			<td rowspan="2" style="width:145px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:145px;">320145-500mL</td>
			<td rowspan="2" style="width:145px;"></td>
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		<tr height="21">
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		<tr height="59">
			<td height="80" rowspan="2" style="height:80px;width:145px;">Cover Glass 24x50 mm</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">12-545-F</td>
			<td rowspan="2" style="width:145px;"></td>
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		<tr height="21">
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		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:145px;">Hot Plate</td>
			<td rowspan="2" style="width:145px;">Fisher Scientific</td>
			<td rowspan="2" style="width:145px;">11-300-49SHP</td>
			<td rowspan="2" style="width:145px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="99">
			<td height="120" rowspan="2" style="height:120px;width:145px;">Drying Oven</td>
			<td rowspan="2" style="width:145px;"></td>
			<td rowspan="2" style="width:145px;"></td>
			<td rowspan="2" style="width:145px;">Capable of holding temperature at 60 &#177; 1&#176;C</td>
		</tr>
		<tr height="21">
		</tr>
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			<td height="120" rowspan="2" style="height:120px;width:145px;">Water Bath</td>
			<td rowspan="2" style="width:145px;"></td>
			<td rowspan="2" style="width:145px;"></td>
			<td rowspan="2" style="width:145px;">Capable of holding temperature at 40 &#177; 1&#176;C</td>
		</tr>
		<tr height="21">
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rnascope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma

O &quot;padrão ouro&quot; para detecção de HPV oncogênico é a demonstração de HPV de alto risco transcricionalmente ativo no tecido tumoral. No entanto, a detecção de E6/E7 mRNA por transcrição Polymerase Chain Reaction reversa quantitativa (qRT-PCR) requer a extração de RNA que destrói o tecido tumoral contexto crítico para a correlação morfológica e tem sido difícil de ser adotada na prática clínica rotineira. Nosso RNA recentemente desenvolvido em tecnologia de hibridização in situ, RNAscope, permite a visualização direta de RNA em, (FFPE) tecido embebido em parafina fixado em formalina com sensibilidade única molécula e resolução de uma única célula, o que permite altamente sensível e específico na análise in situ de qualquer biomarcador RNA em amostras clínicas de rotina. O ensaio RNAscope HPV foi concebido para detectar o ARNm de E6/E7 de genótipos de HPV de alto risco (HPV 16, 18, 31, 33, 35, 52, e 58), utilizando um conjunto de sondas específicas de genótipo. Ela demonstrou excelente sensibilidadee especificidade em relação ao método atual &quot;padrão ouro&quot; de detectar E6/E7 mRNA por qRT-PCR. Estado HPV determinado pela RNAscope é fortemente prognóstico do resultado clínico em pacientes com câncer de orofaringe.

Fluxo de trabalho de ensaio RNAscope HPV O ensaio RNAscope tem um fluxo de trabalho altamente simplificada, que é semelhante à de IHC (Figura 1). Ele consiste em quatro etapas principais: pré-tratamentos, hibridação, amplificações de sinal e de detecção. Ele emprega uma estratégia de design da sonda única que garante sinal de alta fidelidade amplificação 5. No ensaio RNAscope HPV, os sete conjuntos de sonda HR-HPV são reunidas, todos reconhecidos pelo mesmo sistema de amplificação de sinal ligado a peroxidase de rábano (HRP). Sinais de hibridação específicos são detectados como precipitados castanhos formados por reacção catalisada por HRP cromogénico utilizando DAB como substrato, o qual pode ser facilmente visualizadas por microscopia de campo brilhante standard. Coloração Representante para detecção do HPV A Figura 2 mostra exemplos de imagens de cortes corados FFPE de cabeça e carcinoma de células escamosas pescoço. No caso de HPV-positivos (Fig.ure 2A), as sondas de HR-HPV detectado sinais puntiformes fortes especificamente nas células tumorais. A sonda detectada UBC numerosos sinais citoplasmáticos puntiformes em ambas as células tumorais e as células do estroma (figura 2B). A sonda dapB gene bacteriano demonstrou um fundo limpo (Figura 2C). No caso de HPV-negativo, tanto a sonda HR-HPV e a sonda dapB detectado nenhum sinal (Figuras 2D e 2F), enquanto que os sinais fortes foram detectados utilizando a sonda de UBC (Figura 2E). Neste ensaio, UBC serve como controlo positivo para avaliar a qualidade do RNA de tecido e dapB como controlo negativo para os sinais espontâneos. A pontuação para determinação do estatuto de HPV envolve examinar todas as três lâminas para cada caso. O nível de coloração no controlo deslizante negativo (dapB) corrediça é utilizada como ponto de corte: positividade HPV é definida pela presença de citoplasmática punctata e / ou coloração nuclear que era anteriormente o sinal no slide dabpB. <pclass = &quot;jove_content&quot; fo: manter-together.within-page = &quot;always&quot;&gt; <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51426/51426fig1highres.jpg" src="/files/ftp_upload/51426/51426fig1.jpg" /> Figura 1. Fluxograma de ensaio RNAscope. Ilustração de fluxo de trabalho de ensaio RNAscope. O ensaio RNAscope contém quatro etapas principais, pré-tratamentos, a hibridação sonda alvo, amplificação de sinal e detecção do sinal. O procedimento de ensaio completo pode ser concluído em 8 horas. <a href="https://www.jove.com/files/ftp_upload/51426/51426fig1highres.jpg" target="_blank">Clique aqui para ver imagem ampliada.</a> <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/51426/51426fig2highres.jpg" src="/files/ftp_upload/51426/51426fig2.jpg" /> Figura 2. Imagens exemplo de lâminas RNAscope coradas seções FFPE corados com sondas para HR-HPV, UBC (controle positivo) e dapB (controle negativo).a partir de dois casos HNSCC. AC. Caso de HPV positivo. A, expressão de E6/E7 do HPV HR-mRNA nas células tumorais. Inset, aumento de 40 vezes para mostrar sinais puntiformes. B e C) cortes de tecidos adjacentes que apresentam coloração positiva UBC (B) e negativo para dapB (C), respectivamente DF) HPV-negativos caso. D). Nenhuma coloração para HPV E6/E7 mRNA , similar ao controle negativo dapB (F). E) UBC coloração positiva. <a href="https://www.jove.com/files/ftp_upload/51426/51426fig2highres.jpg" target="_blank">Clique aqui para ver imagem ampliada.</a>

Watch this Scientific Journal Video about RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma at JoVE.com

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

A presença de HPV de alto risco no tecido do tumor de cabeça e pescoço está associada a resultados favoráveis. O RNA recentemente desenvolvido na técnica de hibridização in situ chamado RNAscope permite a visualização direta de HPV E6/E7 mRNA em cortes de tecido FFPE.

RNAscope para<em&gt; Em situ</em&gt; Detecção de Papilomavírus Humano transcricionalmente ativo em Cabeça e Pescoço Carcinoma de células escamosas

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

rnascope-for-situ-detection-transcriptionally-active-human

Research

JoVE Journal

Medicine

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

27.1K visualizações.  Advanced Cell Diagnostics, Inc..  A presença de HPV de alto risco no tecido do tumor de cabeça e pescoço está associada a resultados favoráveis. O RNA recentemente desenvolvido na técnica de hibridização in situ chamado RNAscope permite a visualização direta de HPV E6/E7 mRNA em cortes de tecido FFPE. 

Vídeo: RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

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

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Cultura primária de Vestibular Humano Schwanomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

o<em&gt; In Ovo</em&gt; Chick Corioalantóide membrana (CAM) como um Ensaio Xenoenxerto modelo eficiente de carcinoma hepatocelular

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a &#34;side population&#34; (SP). As the SP cells represent a low percentage (&#60;5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of &#946;-catenin.

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Isolamento e Caracterização de uma cabeça e pescoço carcinoma espinocelular subpopulação Tendo-tronco características celulares

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

Abordagem do Canal Semicircular posterior para a orelha interna Gene entrega no rato Neonatal

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Na Vivo Análise morfométrica de humano nervos cranianos, utilizando imagens de ressonância magnética da doença doença ouvidos e orelhas de audição Normal

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Porcine As a Training Module for Head and Neck Microvascular Reconstruction

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes have been available for years in many surgical fields. We used the female (because they are easy to handle for the procedure) Yorkshire pigs for the head and neck reconstruction by harvesting the deep inferior epigastric artery perforator or the superior epigastric artery perforator flap. The anastomosis site (neck skin defect or tracheal wall defect) was prepared via the dissection of the common carotid artery and the internal jugular vein, in which 3.5&#215; loupe magnification was used for anastomosis as we use on human cases in real life. This procedure demonstrates a new training method using a reliable learning model and provides a detailed anatomy in a live scenario. We focused on the ischemia time, harvesting, vessel anastomosis, and designing the flap to fit the defect site. This model improves tissue handling and with the use of proper instruments can be repeated many times so that the surgeon is fully confident before starting the surgery on humans.

Here we present a protocol for the use of the pig superior epigastric artery perforator flap as a learning module for head and neck microvascular reconstruction.

Suínos como um módulo de treinamento para reconstrução Microvascular, cabeça e pescoço

Live models that resemble surgical conditions of humans are needed for training free-flap harvesting and anastomosis. Animal models for training purposes ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

A membrana Chorioalantoica Chick in vivo modelo para avaliar invasão perineural em câncer de cabeça e pescoço

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Learning Modern Laryngeal Surgery in a Dissection Laboratory

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is almost completely reserved to a few referral centers that deal with a large proportion of this pathology. Practice on human specimens is not always possible for ethical, economic, or availability reasons. The aim of this study is to provide a reproducible method for the organization of a laryngeal laboratory on ex vivo animal models where it is possible to approach, learn, and refine laryngeal techniques. Porcine and ovine larynges are ideal, affordable, models to simulate laryngeal surgery given their similarity to the human larynx in their anatomical layout and tissue composition. Herein, the surgical steps of transoral laser surgery, open partial horizontal laryngectomy, and total laryngectomy are reported. The merging of endoscopic and exoscopic views guarantees an inside-out perspective, which is vital for the comprehension of the complex laryngeal anatomy. The method was successfully adopted during three sessions of a dissection course &#34;Lary-Gym&#34;. Further perspectives on robotic surgical training are described.

The purpose of this paper is to illustrate how to organize a reproducible laboratory for laryngeal surgery on affordable and closely similar animal laryngeal models in order to improve anatomical and surgical knowledge and skills.

Aprendendo cirurgia laríngea moderna em um laboratório de dissecção

Surgery for laryngeal malignancies requires millimetric accuracy from the different endoscopic and open techniques available. Practice of this surgery is ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Preparação de andaimes renais descelularizados em ratos

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...