Este trabalho foi financiado, em parte, pelo National Institutes of Health R01 CA131990 (BFS e MRR). Live-célula de imagem foi realizado no Núcleo de Recursos imagens de microscopia, e Citometria suportado, em parte, pelo NIH Centro de concessão P30 CA22453 ao Karmanos Cancer Institute, Wayne State University e pelo Poder Pesquisa Perinatologia do Instituto Nacional de Saúde Infantil e Desenvolvimento , Wayne State University.

1. Prepare DQ-substratos <ol><li> Permitir liofilizada DQ-substratos a aquecer até à temperatura ambiente antes de frascos de abertura, preparar solução stock de 1 mg / ml de DQ-substrato em água desionizada, dividem-se em alíquotas de 50 ul e armazenar a 4 ° C. </li></ol> Pode ser necessário agitar DQ-substrato num banho de água ultra-sons durante 5 min ~ e calor a 50 ° C para facilitar a dispersão. <ol start="2"><li> RBM descongelamento em gelo durante a noite a 4 ° C; RBM devem ser manuseados em gelo em todos os momentos. </li><li> Para fazer com que o fosfato de 10X tampão salino (PBS), dissolver 80 g de NaCl (1,37 M), 2 g de KCl (0,027 M), 14,4 g de Na 2 HPO 4 (1 M), e 2,4 g de KH 2 PO 4 (0,02 M) em 800 ml de água ultrapura. Ajustar o pH da solução-tampão PBS a 7,4 com 1,0 M de HCl. Traga volume de 1 litro, esterilizar por filtração. </li><li> Preparar solução de colagénio I em gelo: 8 partes de colagénio refrigeradas I para 1 parte de refrigerados PBS 10X. Ajustar o pHde mistura para 7,2-7,6 usando estéril 0,1 M de NaOH. Verificar o pH com tiras de pH e ajustar o volume final para 10 partes de água estéril. </li><li> Preparar DQ-colagénio I: colagénio I matriz diluindo DQ-colagénio I, em solução de colagénio I, em um tubo previamente arrefecido a uma concentração final de 25 ug / ml e 2,4 mg / ml, respectivamente. </li><li> Preparar DQ-colagénio IV: membrana basal reconstituída (FRP) da matriz através da diluição de DQ-colagénio IV em RBM (Cultrex ou Matrigel) em um tubo previamente arrefecido a uma concentração final de 25 ug / ml e 12-15 mg / ml, respectivamente. Misture no gelo usando pipetagem suave para evitar a criação de bolhas. </li></ol> 2. Prepare MAME coculturas Ver Figura 1 para um diagrama esquemático de coculturas. <ol><li> O local de duas lamelas de plástico rectangulares (esterilizado em álcool a 70% durante 10 min e seco ao ar) em um prato de cultura de 35 mm ou utilizar um IbiTreat 35-mm μ louça. Directions aqui são para ambos os lamínulas e u-Pratos, que usamos para alunosmorre em um microscópio vertical. Para os estudos sobre um microscópio invertido, usamos apenas u-Pratos. </li><li> Misture o número desejado de fibroblastos com DQ-colágeno I: colágeno tipo I da matriz. Para os coculturas de longo prazo ilustrados nas Figuras 2 e 3, usamos 500 fibroblastos em 10 uL de meios mais 60 uL de DQ-colagénio I: colagénio I da matriz. </li><li> Usando uma micropipeta 100-uL, cuidadosamente pipeta e espalhar 70 uL de fibroblastos: DQ-colagénio I: colagénio I matriz sobre toda a superfície de cada lamela e deixar a 37 ° C numa incubadora humidificada sem CO 2 durante 30 min a solidificar. </li><li> Transferência de 35 mm pratos contendo lamelas de 37 ° C incubadora com 5% de CO 2 durante 10 min para equilibrar. </li><li> Remover do incubador e deixar os pratos de 35 mm sob a capa até que atinjam a temperatura ambiente. </li><li> Adicionar 60 ul de DQ-colágeno IV: RBM matriz em cima do solidificou DQ-colágeno I: colágeno I matriz com fibroblastos incorporados. Com pipeta de ponta, carefully espalhar DQ-colágeno IV: RBM matriz uniformemente, evitando coçar o fibroblasto: DQ-colágeno I: colágeno tipo I da matriz. </li><li> Transferência de 35 mm pratos contendo lamelas de 37 ° C incubadora com 5% de CO 2 durante 10 min a solidificar. Enquanto o IV DQ-colágeno: RBM matriz está solidificando, trypsinize e contagem de células epiteliais. </li><li> Colocar 50 ul de uma suspensão de células epiteliais / tumor em lamelas revestidas com DQ-colágeno IV: RBM matriz. Colocar 35-mm pratos contendo as lamelas numa incubadora a 37 ° C. Permitir 40-60 min para as células para anexar DQ-colagénio IV: RBM matriz. Para os coculturas de longo prazo ilustrados nas Figuras 2 e 3, usamos 2500 células epiteliais ou tumor. </li><li> Adicionar 2 ml de meio de cultura contendo 2% RBM para cada prato de 35 mm. Se fazer todos os tratamentos de drogas, as drogas devem ser adicionados no momento. </li><li> Incubar durante período de tempo desejado antes de imagem. Para culturas a longo prazo, a mídia deve ser trocado a cada 3-4 dias. As células podem ser mantidasem seu meio de cultura normal. Para MAME coculturas de linhas de células de mama, usamos Meio Basal Epitelial (MEBM-PRF) suplementado com SingleQuots MEGM. </li></ol> 3. Execute 4D (3D + tempo) imagens ao vivo de MAME coculturas por microscopia confocal <ol><li> Para os estudos de prova de princípio ilustrados, MAME coculturas foram marcadas com um corante fluorescente de células de rastreamento de acordo com o nosso processo publicado 1. Após lavagem com PBS, coculturas foram incubadas com 5 Laranja CellTracker uM em meio MEGM durante 45 min em um incubador a 37 ° C, lavou-se novamente com PBS e incubadas com MEGMmedium pré-aquecido durante 30 min em 37 ° C com 5% de CO 2 incubadora antes de imagem . </li><li> Imagem MAME coculturas viver em um pequeno aumento (lente 10X de água, mergulhando) em um microscópio Zeiss LSM META-510 confocal. </li><li> Secções ópticas são capturados em intervalos ao longo de toda a profundidade das estruturas. Para os nossos estudos, podemos capturar secções ópticas em 10intervalos uM. </li><li> Use secções ópticas para reconstruir imagens em 3D. Nós usamos software Volocity para gerar reconstruções em 3D. </li><li> Faça filmes das reconstruções em 3D de modo que estruturas da célula,: interações celulares e interações com as matrizes circundantes podem ser visualizados. Fizemos filmes com QuikTime Player 7. </li></ol> 4. Os resultados representativos O MAME coculturas que desenvolvemos são um modelo tratável experimental para live-célula de imagem em tempo real das interações entre os vários constituintes celulares que compõem um tumor de mama e seu microambiente. Aqui demonstramos a capacidade de modelos MAME recapitular celular: interações celulares durante a progressão do câncer de mama e de live-célula de imagem para acompanhar essas interações ao longo do tempo. Nós ilustramos a capacidade de interações entre uma imagem pré-invasivo linha celular humana MCF10.DCIS de mama e um câncer humano de mama associado à linha de fibroblastos, WS-12TI, sobreum período de mais de três semanas em cultura. O coculturas foram fotografadas em 3, 16 e 23 dias. Secções ópticas através de todo o volume do coculturas foram reconstruídos em exemplos 3D e representativo são apresentados para cada um dos três pontos temporais na Figura 2. A fluorescência vermelha na Figura 2 representa a MCF10.DCIS e WS-12TI células. Para discriminar entre os dois tipos de células e para a longo prazo coculturas, pode-se transfectar ou transduzir tipos de células individuais com as proteínas fluorescentes antes de estabelecer as culturas. Um co-cultura de MAME MCF10.DCIS e WS-12TI células que são diferencialmente marcadas com proteínas fluorescentes para a identificação é ilustrado na Figura 3. Por substratos que incorporam, neste caso DQ-colagénios, no coculturas MAME, podemos avaliar e quantificar as mudanças ao longo do tempo em proteólise associada com a transição para um fenótipo invasivo. Nós estabelecemos métodos para a quantificação dos produtos de clivagem fluorescentes que resultamda DQ-colágeno degradação 2,3. Nós normalizar produtos de degradação em todo o volume 3D da coculturas MAME numa base por célula por marcação e contando os núcleos das células. Além disso, pode-se localizar a degradação de compartimentos extracelulares e intracelulares e quantificar os totais, produtos de degradação intracelular e extracelular como já descritos anteriormente 2. A fluorescência verde nas Figuras 2 e 3 representa produtos de clivagem dos dois DQ-colagénio substratos. Neste momento, colagénios diferencialmente-rotulados não estão disponíveis, que permitiria fazer a distinção entre a degradação do colagénio de tipo IV e da degradação de colagénio tipo I. As interações no coculturas MAME são dinâmicas e podem mudar ao longo do tempo. Para se obter informação temporal e dinâmico, o mesmo campo de visão pode ser imaginadas secções ópticas e tomadas através de toda a amostra do volume repetidamente ao longo de um período de tempo de minutos a semana,assim, a coleta de dados em 4-D. Na Figura 2, ilustramos mudanças bastante dramáticas que ocorrem mais de 23 dias: mudanças no número de celular, migração celular e interações celulares, volumes de estrutura, formas de estrutura, desvios das estruturas de esfericidade, excrescências invasivos e proteólise. Além disso, também demonstram uma mudança global do volume devido à coculturas MAME degradantes seu meio circundante matriz extracelular ao longo deste período de tempo. Aos três dias, conforme ilustrado na Figura 2A, o volume é 110.000 iM 3. Em contraste, o volume do coculturas MAME menos 16 e 23 dias é apenas 46.250 iM 3, conforme ilustrado nas Figuras 2B e C. <img alt="A Figura 1" src="/files/ftp_upload/3661/3661fig1.jpg" /> Figura 1. Diagrama esquemático do MAME coculturas. Lamínula de plástico é revestido com colágeno tipo I, contendo DQ-colágeno I e fibroblastos (magenta). A 2 ª camada da membrana basal reconstituída (RBM) contendo DQ-collagen IV é adicionado. As células tumorais (vermelho) são colocadas em cima e RBM 2% em meios de cultura é sobreposta (pontos brancos) a cada mudança de mídia. O verde representa os produtos de dissociação fluorescentes de DQ-colágeno I e IV. <img alt="A Figura 2" src="/files/ftp_upload/3661/3661fig2.jpg" /> Figura 2. MAME coculturas de células mamárias humanas MCF10.DCIS e WS-12TI fibroblastos humanos da mama. Os fibroblastos foram incluídos em colágeno I / DQ-colágeno I e revestida com RBM contendo DQ-colágeno IV. As células CDIS foram semeadas na RBM e revestida com mídia contendo RBM 2%. Ao longo dos 23 dias em co-cultura ilustradas aqui, houve a proliferação celular, a degradação de DQ-colagénio IV e I (verde) e matrizes de colagénio, tal como indicado pela redução no volume do coculturas. As células foram localizados através de marcação com laranja CellTracker in situ, antes de imagem (vermelho). Os mesmos coculturas ao vivo foram observadas durante os 23 dias, com imagens captadas por cmicroscopia onfocal menos 3, 16 e 23 dias de co-cultura. As fatias resultantes ópticos foram reconstruídos em 3D com software Volocity. Ampliação, 10X. <img alt="A Figura 3" src="/files/ftp_upload/3661/3661fig3.jpg" /> Figura 3. MAME 16 coculturas dia de células mamárias humanas MCF10.DCIS e WS-12TI fibroblastos humanos da mama que expressam RFP (vermelho) e YFP (branco), respectivamente. Coculturas foram estabelecidos e analisados ​​como na Figura 2. Os dois tipos de células mostradas aqui tinha, no entanto, sido diferencialmente rotulados de modo que pudessem ser distinguidas uma da outra. As imagens maior ampliação nesta figura, em comparação com aqueles na Figura 2, ilustram a proteólise de DQ-colagénio IV, à superfície das células DCIS e proteólise difusa de DQ-colagénio I, em áreas próximas dos fibroblastos. Ampliação, 20X.

<ol>
	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

Não temos nada a divulgar.

Como demonstrado, o coculturas MAME pode ser usado para vivo de células de imagem em tempo real das interacções entre os vários constituintes celulares que compreendem um tumor da mama e do seu microambiente. Em estudos em curso no nosso laboratório, utilizou-se MAME coculturas para identificar vias proteolíticas associados com a transição de CDIS carcinoma ductal infiltrante, bem como as interacções entre as vias proteolíticas e outras vias envolvidas nesta transição, tais como as vias de quimiocina citoci...

<table border="1"><tbody><tr><td> Reagente / Equipamento </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> Membrana basal reconstituída (Cultrex) </td><td> Trevigen </td><td> 3445-005-01 </td><td> Comparável a Matrigel (BD Biosciences) </td></tr><tr><td> Colágeno I </td><td> Laboratórios de Coesão </td><td> 5005-B </td><td></td></tr><tr><td> DQ-substratos (Colágeno I, IV) </td><td> Invitrogen </td><td> DQ-col I-D12060 DQ-col IV D12052 </td><td></td></tr><tr><td> 22-mm lamelas de plástico </td><td> Fisher Scientific Co. </td><td> 12-547 </td><td> Cortar pela metade, deixe em etanol 70% por 10 min e ar seco antes do uso. </td></tr><tr><td> Meio de cultura celular </td><td> Lonza </td><td> MEBM-PRF CC-3153 MEGM CC-4136 </td><td> Vermelho de fenol livre </td></tr><tr><td> ibiTreat μ Dish- </td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Software Volocity </td><td> Perkin-Elmer </td><td> Versão 5.5 </td><td> Outras marcas de software de imagem pode ser utilizada para gerar reconstruções 3D e filmes. </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

Nós desenvolvemos modelos de co-cultura em 3D, que MAME prazo (m ammary um rchitecture e m icroenvironment ngineering e), e os usou para live-célula de imagem em tempo real da célula: interações celulares. Nosso principal objetivo foi desenvolver modelos que recapitulam a arquitetura das lesões mamárias pré-invasivo para estudar a sua progressão para um fenótipo invasivo. Especificamente, desenvolvemos modelos para analisar as interacções entre pré-malignas da mama variantes de células epiteliais e outros tipos de células de tumor que o microambiente têm sido implicados na melhoria ou reduzir a progressão de células de mama preinvasive epiteliais para carcinomas ductais. Outros tipos de células estudadas até à data são células mioepiteliais, fibroblastos, macrófagos e células do sangue e linfáticos microvasculares endoteliais. Além dos modelos Mame, que são concebidos para recapitular as interacções celulares dentro da mama duranteprogressão do câncer, desenvolvemos modelos comparáveis ​​para a progressão do câncer de próstata. Aqui temos ilustram os procedimentos para o estabelecimento das coculturas 3D, juntamente com a utilização de vivo de células de imagem e um ensaio de proteólise funcional a seguir a transição de coculturas de carcinoma da mama ductal in situ (CDIS) células e fibroblastos para um fenótipo invasivo ao longo do tempo, em Neste caso mais de vinte e três dias em cultura. O coculturas MAME consistem em camadas múltiplas. Os fibroblastos são incorporados na camada inferior de colagénio tipo I. Em que está colocada uma camada de membrana basal reconstituída (FRP) sobre o qual as células são semeadas DCIS. A camada final topo da RBM 2% incluído e repostos a cada mudança de mídia. Para a proteólise da imagem associada com a progressão para um fenótipo invasivo, usamos corante-temperados (DQ) proteínas fluorescentes matriz (dq-colagénio I misturado com a camada de colagénio I e DQ-colagénio IV misturado com a camada do meio de rBM) e observar culturas vivas utilizando microscopia confocal. Secções ópticas são capturados, processados ​​e reconstruído em 3D com o software de visualização Volocity. Ao longo de 23 dias coculturas em MAME, as células DCIS proliferar e coalescer em grandes estruturas invasivos. Fibroblastos migram e se incorporaram essas estruturas invasivas. Fluorescentes fragmentos proteolíticos dos colagénios são encontrados em associação com a superfície de estruturas CDIS, intracelularmente, e também dispersa por toda a matriz circundante. Drogas que proteolítica alvo de quimiocinas, / citocina e quinase vias ou modificações na composição celular do coculturas pode reduzir a capacidade de invasão, sugerindo que os modelos de MAME pode ser usado como telas de pré-clínicos para novas abordagens terapêuticas.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Nós desenvolvemos modelos 3D para co-cultura de células de imagem ao vivo em tempo real das interações entre as células tumorais de mama e outras células em seu microambiente que a progressão de impacto para um fenótipo invasivo. Estes modelos podem servir como telas pré-clínicos de medicamentos para atingir parácrina induzidas proteolíticas caminhos, quimiocinas / citocina e quinase envolvidos na invasão.

Modelos MAME para formação de imagens 4D Live-célula do tumor: Interações microambiente que a progressão de Impacto maligno

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

mame-models-for-4d-live-cell-imaging-tumor-microenvironment

Research

JoVE Journal

Medicine

14.3K visualizações.  Wayne State University. Nós desenvolvemos modelos 3D para co-cultura de células de imagem ao vivo em tempo real das interações entre as células tumorais de mama e outras células em seu microambiente que a progressão de impacto para um fenótipo invasivo. Estes modelos podem servir como telas pré-clínicos de medicamentos para atingir parácrina induzidas proteolíticas caminhos, quimiocinas / citocina e quinase envolvidos na invasão.

Vídeo: MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Ortotópico do xeno-Human-luciferase Tagged maligna dos nervos periféricos Células Tumorais Bainha para In vivo Teste de Agentes Terapêuticos candidatos

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Medicina

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Avaliação do Câncer de Migração Stem Cell Usando compartimentar dispositivos microfluídicos e imagens de células vivas

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ensaio in vitro de Apuramento mesotelial que os modelos dos primeiros passos da metástase do câncer de ovário

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotípica tridimensional<em&gt; In Vitro</em&gt; Modelagem de Estroma-epitelial de ovário Interações durante o início e progressão do câncer

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Viver de imagem de Respostas de drogas no microambiente tumoral em modelos do rato do cancro da mama

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

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

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

De alta frequência Ecografia de mouse linfonodos cervicais

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Um tumor cerebral / Slice organotípicas Co-cultura Sistema de Estudos de Tumor Microenvironment e alvejado droga Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Método Tumor Quantificação Baseada em bioluminescência para a progressão do tumor Monitoramento e efeitos do tratamento em modelos de ratos linfoma

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Ex vivo de imagens ao vivo de pulmão Metástase e seu microambiente

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...