Supported by grants from the National Institute of Health grants CA133085, CA182518 and CA185909 (to KL) and VA Merit Review Award BX001962 (to KL).

Todos os procedimentos seguem as diretrizes e protocolos aprovados por Georgia Regents Universidade Uso de Animais e do Comité Care. 1. Estabelecimento de Tumor cancro da mama ortotópico <ol><li> Um dia antes da experiência, a cultura de aproximadamente 2 x 10 6 células de tumor 4T1-Luc em uma placa de cultura de 10 cm em 10 ml de meio RPMI contendo 10% de FBS. Incubar a placa de cultura numa incubadora com CO 2 a 37 ° C e 5% de CO 2. </li><li> No dia da injecção de células tumorais, remover o meio de cultura da placa de cultura com vácuo, adicionam-se 5 ml de fosfato estéril salina tamponada (PBS), pH 7,4 para o prato de cultura, e agitar suavemente o prato para lavar a superfície de cultura, remover o PBS por vácuo. </li><li> Adicionar 2 ml de tripsina-EDTA (tripsina a 0,05% e EDTA 0,53 mM) para o prato de cultura, incubam-lo na incubadora de CO 2 a 37 ° C durante cerca de 5 min. Verificar as células em um microscópio invertido para certificar-se de que todas as células são destacadas do fundo do prato de cultura. &lt;/ Li&gt; <li> Extingue-se a tripsina por adição de 8 ml de meio contendo soro, as células de transferência para um tubo cónico de 15 ml e centrifugar as células a aproximadamente 450 xg durante 3 minutos à temperatura ambiente. </li><li> Remover o sobrenadante pelas células de vácuo e ressuspender em 10 ml de PBS. centrifugar células a aproximadamente 450 xg durante 3 min. Remover o sobrenadante por aspiração, e ressuspender as células em 10 ml de PBS. </li><li> Pipetar 10 ul da suspensão de células para um hemocitómetro sob a tampa de vidro. Contar as células para determinar a concentração de células. Ressuspender as células em PBS a uma densidade de 2 x 10 5 células / ml de HBSS. Transferir as células para um tubo de microcentrífuga de 1,5 ml estéril e manter as células em gelo até à sua utilização. </li><li> Use fêmea BALB / c machos de 6 - 8 semanas para a injecção de células tumorais. <ol><li> Raspar o cabelo próximo ao redor do mamilo # 3 usando um aparador de pêlos eletrônico. A injeção de 4T1 celular na glândula mamária 3 almofada de gordura gera reproducibly metástase pulmonar. Limpar a área raspada, usando o cotonete embebido em 70% etanoeu. Ressuspender as células invertendo o tubo de microcentrífuga de 2 - 3 vezes. Delicadamente aspirado de 50 ul de suspensão de células para um CC ½ 27 L 02/01 seringa de tuberculina. </li><li> Injetar as células tumorais para a almofada de gordura mamária sob o nº 3 glândula mamária de um rato BALB / c. Coloque o rato em uma gaiola limpa e voltar a gaiola para instalação de alojamento animal. Aguarde cerca de 21 - 30 dias para o desenvolvimento do tumor. O crescimento do tumor deve ser monitorado em uma base regular de acordo com as políticas da instituição para estudos tumorais. </li></ol></li></ol> 2. Ao vivo mouse Bioluminescência Imagem do crescimento do tumor <ol><li> camundongos imagem a cada 3 dias após a injecção de tumor ratos transferência para a unidade de imagem. Injectar 100 ul de luciferina (30 mg / ml em PBS) por via intraperitoneal (IP) a ratos antes da imagiologia. Após aproximadamente 10 min, anestesiar os ratos numa câmara de indução com 2% de isoflurano / O 2 de fluxo. Use a pinça para tocar a perna do mouse. Sem resposta ao toque confirma that o mouse é totalmente inconsciente. <ol><li> Colocar os ratinhos na câmara de imagem com contínuo isoflurano a 2% e O 2 a uma taxa de fluxo de 2 L / min. Colocar o rato de tal modo que a área de interesse (ou seja, a almofada mamária da injecção inicial do tumor) voltado para a câmara do sistema de imagem. Assegurar o nariz ea boca do mouse estão firmemente estabelecidos dentro do tubo anestésico. Os animais devem ser anestesiados por normas veterinárias na instituição. </li></ol></li><li> Adquirir imagem de bioluminescência com uma variedade de diferentes tempo de exposição. Por exemplo, definir a exposição a 30, campo de visão (FOV) de 25, altura do objeto de 1,5 cm, e obter imagens fotográficas luminescentes com baixo consumo de energia de raios-X. Defina as unidades de medida a &quot;luminosidade&quot;. <ol><li> Utilize uma gama de cores linear automática com um máximo de cerca de unidades de 2,4 x 10 7 de radiância. Depois de adquirir imagens iniciais, ajustar as configurações e obter uma imagem otimizada-(FiguraS 3 &amp; 4). </li></ol></li><li> Voltar ratos para limpar gaiolas e garantir ratos recuperar a consciência com a deambulação antes de retornar os animais para a instalação de habitação. </li><li> Analisar os dados com o software de imagem estar associada ao instrumento de imagem. Usando um programa de imagem, desenhe uma linha fechada em torno da região de interesse (ROI). Use o software de imagem para determinar a intensidade esplendor do ROI. Grave as intensidades relativas de cada ROI para cada ratinho. NOTA: intensidades de radiância mais altos indicam maiores níveis de células luminescentes, e, portanto, maior o crescimento do tumor. </li><li> Visualmente examine o mouse a cada 3 dias para efeitos adversos. NOTA: Os ratos serão pesados ​​uma vez por semana. Uma perda de peso de 15% de peso corporal serão considerados efeito adverso e os ratinhos portadores de tumor serão sacrificados tal como descrito no passo 5. Os ratinhos com tumores ulcerados também serão sacrificados. </li></ol> 3. A remoção cirúrgica dos tumores primários NÃOTE: tesouras autoclave, pinça e os grampos de sutura e aplicador de clipes de feridas (Figura 1). <ol><li> Realizar a cirurgia num hote estéril para manter uma condição esterilizada para reduzir o risco de infecção. Um dia antes da cirurgia, raspar a área em torno dos tumores dos ratos portadores de tumor com um aparador de cabelo elétrico. </li><li> Aproximadamente 2-4 horas antes da cirurgia, os ratos alimentam comprimidos mastigáveis ​​carprofeno formulados para ratos a uma dose de 5 mg / kg de peso corporal. </li><li> Colocar os ratos numa câmara contendo 2% de isoflurano em oxigénio durante cerca de 30 seg. Continuamente anestesiar o rato usando um aparelho de anestesia durante todo o período cirúrgica. </li><li> Use uma vara de algodão estéril para aplicar vet pomada para os olhos do mouse para evitar a secura e sob anestesia. Use a pinça para tocar a perna do mouse. Sem resposta ao toque confirma que o mouse é totalmente inconsciente. </li><li> Desinfectar a área da pele ao redor do local do tumor com três alternadatoalhetes de um matagal sabão desinfetante. Limpe a área cirúrgica com álcool a 70% wipe. </li><li> Usar um bisturi esterilizado (Figura 1) para cortar uma pequena incisão próximo do tumor. </li><li> Insira a ponta de um par de tesouras esterilizados para a incisão para cortar a pele em torno do tumor para expor o nódulo de tumor (Figura 2A). Segurar o tumor com fórceps estéreis e usar a tesoura para separar o tumor a partir da pele (Figura 2B). Guardar a amostra de tumor por congelação de -80 ° C ou congelador através da fixação em solução de formalina a 10% para uso futuro. </li><li> Fechar o local cirúrgico com clipes para feridas de 9 mm, usando o aplicador auto-grampo (Figura 1). Devolver o mouse para a gaiola limpa com roupa de cama autoclavado. Os animais devem ser monitorados por diretrizes institucionais. NOTA: O rato geralmente recupera a consciência suficiente e começa a mover-se na gaiola de aproximadamente 10 - 30 min após a cirurgia. </li><li> Administrar carprofen pararatinhos novamente 24 horas após a cirurgia. Carprofeno é para analgesia pós-cirurgia e prescrição da analgesia pós-operatória deve ser verificada por meio de consulta directa com o pessoal veterinário da instituição. Remova os clipes ferida após a ferida cicatriza (geralmente dentro de 5 - 7 dias após a cirurgia). Remova os clipes de feridas usando o removedor de auto-clip. </li></ol> 4. Ao vivo mouse bioluminescência imagem de metástase de tumor <ol><li> ratinhos imagem a cada 3 dias após a cirurgia tal como descrito no passo 2. </li></ol> 5. Validação de metástases pulmonares Usando India Ink inflação dos Pulmões portadores de tumor NOTA: Para validar os resultados de imagem tumor vivo luciferase, execute a inflação nanquim dos pulmões do rato portador de tumor de quantificar nódulos tumorais. Células 4T1 também metástase para outros órgãos e tecidos (Figura 5) e, por conseguinte, o exame histológico destes órgãos para validar a metástase do tumor deve ser realizada, se necessário. Este lun uso de protocolog metástase como um exemplo. <ol><li> Manter os ratos em sua gaiola casa existente para a eutanásia. Quando alojados com outros animais, mudar esses ratos não sendo sacrificados a uma gaiola diferente. </li><li> Remover a parte superior do filtro gaiola e substitua com a parte superior do filtro CO 2. Ligue o cilindro comprimido CO2 gasoso (100%), que está ligado à parte superior do filtro de CO 2. Definir a taxa de fluxo de 2 L / min e mantê-CO 2 por pelo menos 10 min. Se os ratos são ainda respiração no final de 10 min, deixar o CO2 em até pelo menos 1 min após ratinhos parar de respirar. Desligue CO 2 controle do cilindro e do medidor de vazão. Espere por mais 3 minutos e retire a parte superior do filtro. </li><li> Coloque o rato sacrificado em sua parte traseira em uma placa de poliestireno. Pin as pernas para garantir acesso livre à traqueia. Pulverize os ratos com 70% de etanol. </li><li> Usando uma tesoura, corte ao longo da linha média do mid-abdómen do rato através da caixa torácica e para cima em direção ao salivariar glândulas. Observe a traqueia. Use fórceps para remover tecidos que circundam a traqueia para expor a traqueia. </li><li> Passe uma ponta de pipeta debaixo da traqueia. Segurando a ponta com uma mão, levante com cuidado a traqueia para cima e longe do corpo. </li><li> Rodar a plataforma segurando o mouse 180 °. Use um CC ½ 27 G 1/2 seringa de tuberculina a injectar India Ink (10% nanquim e 0,1% de hidróxido de amónio) para os pulmões através da traqueia. Completamente inflar os pulmões com tinta até que uma forte resistência é sentida. </li><li> Use uma tesoura para cortar a traqueia. Use uma pinça para segurar o mouse e inserir um outro conjunto de fórceps sob os pulmões e puxe os lobos pulmonares para fora do mouse. Lavar brevemente pulmões em um copo contendo água. </li><li> Em uma coifa química, transferir os pulmões para um frasco de cintilação de vidro contendo 3 ml de solução de Fekete (etanol a 50%, 6% de formaldeído, e 3% de ácido acético glacial). Tapar o frasco para evitar a evaporação da solução. NOTA: Os tecidos podem ser armazenados em solução de Fekete indefinidamente. </li><li> Após alguns minutos, observa nódulos tumorais como pontos brancos sobre os pulmões pretas (Figura 5). O nódulo tumoral branco é visível por olhos. Transferir os pulmões para um prato numa hotte e contar o número de manchas brancas. Cada mancha branca representa um único nódulo tumoral metastático. </li></ol>

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Mice were purchased from the Jackson Laboratory and housed in the Georgia Regents University animal facility. Experiments and care/welfare were in agreement with federal regulations and an approved protocol by the Georgia Regents University IACUC committee. The authors have nothing to disclose.

Muitos tipos de modelos de ratinhos transgénicos de metástases de cancro da mama têm sido desenvolvidos. 1 Estes ratinhos transgénicos têm uma incidência elevada de tumor variando entre 60 a 100%. No entanto, a incidência de metástases destes ratinhos transgénicos é muito menor do que a incidência de tumores (14 - 100%). As células de tumor metastizar para LN e pulmões na maioria destes modelos de ratinho transgénicos de metástases de cancro da mama. A latência metástase varia de modelo para ...

De acordo com a American Cancer Society, o cancro da mama é a forma mais frequentemente diagnosticado de câncer em mulheres nos Estados Unidos. A detecção precoce em combinação com terapias específicas recentemente desenvolvidos reduziu significativamente a mortalidade do cancro da mama nas últimas duas décadas. No entanto, o câncer de mama ainda é a segunda principal causa de morte relacionada ao câncer em mulheres nos Estados Unidos 1. A maioria das mortes de pacientes com câncer de mama são devido a metástase das células tumorais. Infelizmente, a maioria do cancro da mama invasivo e é frequentemente metastiza para o nódulo linfático e, subsequentemente, para órgãos distantes, incluindo osso, pulmão, fígado e cérebro. 1,2 Não existe actualmente nenhuma terapia eficaz para o cancro da mama metastático. Portanto, o desenvolvimento de agentes quimioterapêuticos e imunoterapêuticos para suprimir o cancro da mama metastático é de grande importância. Vários cancro da mama mouse modelos de metástase espontânea têm been desenvolvidas para estudar os mecanismos moleculares subjacentes a progressão de células de tumor da mama e de metástases e para ser usados ​​como modelos para o desenvolvimento de agentes terapêuticos. 1,3-5 No entanto, a maioria destes modelos de murganho são modelos de tumor genéticas que, ao mesmo tempo excelentes modelos para mecanicista estudos, não são adequados para testar agentes terapêuticos uma vez que a metástase leva meses a desenvolver-se estes modelos genéticos, exigindo, assim, a administração dispendiosos de longo prazo dos agentes anti-cancro. progressão tumoral 6,7 Monitorização em ratinhos vivo também é tecnicamente exigente. Em contraste, um modelo de metástases de cancro da mama transplantado tem as vantagens de progressão tumoral termo curto e fácil de seguimento da progressão do tumor em murganhos vivos. O modelo de ratinho 4T1 metástase espontânea ortotópico de cancro da mama é um modelo de tumor transplantado tal. 8 Neste modelo, as células de tumor da mama são transplantadas para a almofada de gordura mamaria de estabelecer nódulos de tumor primário. O tumor primáriopode então ser removido cirurgicamente como em pacientes com cancro da mama humano. Células de tumor 4T1 são altamente invasivo. 8,9, 3,10 Quase todos os ratinhos desenvolvem metástases em 30 dias após o transplante do tumor para dentro a almofada de gordura mamária portador de tumor. No entanto, os tumores 4T1 crescer agressivamente nos sítios primários e muitas vezes os tamanhos dos tumores exceder os limites que são permitidos na maioria dos protocolos de origem animal. Nesta fase, as metástases são muitas vezes micrometástases. Portanto, é essencial para remover tumores primários para permitir o progresso metástase para testar agentes terapêuticos. Aqui, nós relatamos o estabelecimento de um procedimento simples e ainda sensível do transplante do tumor primário, a remoção cirúrgica do tumor primário e bioluminescência quantificação baseadas em imagiologia do crescimento do tumor 4T1 e progressão in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="973">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Ami-X Imaging System&#160;</td>
			<td style="width:437px;">Spectral Instruments Imaging Inc. Tucson, AZ</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">IsoTec SurgiVet</td>
			<td style="width:437px;">Anesthesia Service &#38; Equipment , Inc., Atlanta, GA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">AutoClip Physicians Kit</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">9 MM AutoClip Applier&#160;</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">9 MM AutoClip Remover</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">9 MM AutoClip Wound Clip</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:437px;">Fisher</td>
			<td style="width:119px;">8940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Fine-Pointed Forceps</td>
			<td style="width:437px;">Fisher</td>
			<td style="width:119px;">8875</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">1/2 CC 27G1/2 tuberculin syringe&#160;</td>
			<td style="width:437px;">Becton Dickinson and Co. NJ&#160;</td>
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an orthotopic mouse model of spontaneous breast cancer metastasis

A metástase é a principal causa de mortalidade de pacientes de cancro da mama. O mecanismo subjacente a metástase de células de câncer, incluindo metástase do câncer de mama, é em grande parte desconhecido e é um foco na pesquisa do câncer. Vários modelos de cancro da mama espontâneos rato metástase foram estabelecidas. Aqui, nós relatamos um procedimento simplificado para estabelecer tumor primário do cancro da mama transplantado ortotópico e resultante metástase espontânea que imitam a metástase do câncer de mama humano. Combinado com a imagiologia de tumores bioluminescência vivo, neste modelo de ratinho permite o crescimento do tumor e cinética de progressão para ser monitorizada e quantificada. Neste modelo, uma dose baixa (1 x 10 4 células) de células de cancro da mama 4T1-Luc foi injectada em ratinhos BALB / C mamário de ratinho almofada de gordura utilizando uma seringa de tuberculina. Os ratinhos foram injectados com luciferina e fotografada em vários pontos de tempo, utilizando um sistema de imagiologia bioluminescente. Quando os tumores primários cresceu para o limite de tamanho como no protocolo IACUC-aprovado (aproxoximately 30 dias), os ratinhos foram anestesiados sob um fluxo constante de 2% de isoflurano e oxigénio. A área do tumor foi esterilizada com etanol a 70%. A pele do murganho em torno do tumor foi excisada para expor o tumor, que foi removido com um par de tesouras estéreis. A remoção do tumor primário estende-se a sobrevivência dos ratos portadores de tumor 4T-1, durante um mês. Os ratinhos foram em seguida repetidamente fotografada para propagação de tumores metastáticos para órgãos distantes. Os agentes terapêuticos podem ser administrados para suprimir a metástase do tumor neste ponto. Este modelo é simples e ainda sensível em quantificar o crescimento de células de câncer de mama no sítio primário e cinética progressão para órgãos distantes, e, portanto, é um excelente modelo para o estudo do crescimento e progressão do cancro da mama, e para testar agentes anti-metástase terapêuticos e imunoterápicos in vivo .

Estabelecimento de Ortotópico Breast Cancer Modelo rato 4T1 é uma linha de células de carcinoma da mama agressivo. A injecção de tão pouco como 1 x 10 4 células para a almofada de gordura mamária pode levar à criação de um único nódulo de tumor no local da injecção (Figura 2A). Portanto, o tumor primário imita carcinoma mamário humano. Quase 100% ratinhos desenvolvem o tumor ortotópico. O tamanho do tumor pode ser quan...

Watch this Scientific Journal Video about An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis at JoVE.com

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Um modelo de tumor primário do cancro da mama ortotópico e remoção cirúrgica do tumor primário para prolongar a vida do rato para gerar metástase espontânea são descritos. O crescimento do tumor e progressão são monitorados e quantificado por imagens de fluorescência da luciferase.

Um modelo de camundongo ortotópico de câncer de mama espontânea metástase

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

an-orthotopic-mouse-model-of-spontaneous-breast-cancer-metastasis

Research

JoVE Journal

Medicine

30.5K visualizações.  Augusta University.  Um modelo de tumor primário do cancro da mama ortotópico e remoção cirúrgica do tumor primário para prolongar a vida do rato para gerar metástase espontânea são descritos. O crescimento do tumor e progressão são monitorados e quantificado por imagens de fluorescência da luciferase. 

Vídeo: An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Um modelo ortotópico de câncer de bexiga murino

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Medicina

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Análise quantitativa de metástase de câncer utilizando um modelo de Embrião aviária

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

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 ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Na imagem de bioluminescência vivo Tumor de mama Dynamics Hipóxia metástase do cancro cerebral em um modelo de rato

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Radioterapia em uma combinação ortotópico modelo de tumor de cérebro de camundongos

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Um modelo de camundongo ortotópico de Carcinoma anaplásico da tiróide

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Um modelo de cancro da bexiga para Ortotópico Gene Estudos de entrega

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Modelo ortotópico bioluminescente de progressão do câncer de pâncreas

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Um Modelo murino de próstata humana Metástase Ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Início do Carcinoma de mama metastático por Segmentação do ductal Epithelium com Adenovírus-Cre: Um Modelo Novel de camundongo transgênico do Câncer de Mama

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...