We thank Nguyen H. Nguyen for her technical help and Audrey O&#8217;Neill for support with the Zeiss Cell Observer spinning-disk confocal microscope. This work was supported by a Department of Defense postdoctoral fellowship (W81XWH-11-01-0139) and the Weizmann Institute of Science-National Postdoctoral Award Program for Advancing Women in Science (to V.P.).

Todos os procedimentos descritos devem ser realizados de acordo com as diretrizes e regulamentos para o uso de animais vertebrados, incluindo a aprovação prévia por parte do Care local Institucional Animal e Comitê de Uso (IACUC). 1. Geração de metástases pulmonares por ex vivo Imagens ao vivo (Transgenic ou veia caudal Injection) NOTA: As metástases pulmonares pode ser gerado através da utilização de modelos de ratinho geneticamente modificadas ou por injecção intravenosa (iv) a injecção de células cancerosas. <ol><li> Gerar metástases pulmonares para a imagem latente do cruzamento de um modelo de rato tumor geneticamente modificado em um rato repórter transgênicos, por exemplo, atravessar o modelo do rato do cancro da mama, rato antígeno do vírus do tumor mamário terminal longa do meio repeat-polioma T (MMTV-PyMT) 11 em ACTB-ECFP modelo do rato 12. NOTA: O modelo ACTB-ECFP expressa reforçada ciano proteína fluorescente (ECFP) sob a β-actno promotor de modo a que todas as células fluoresce na, canal PCP azul. No entanto, as células cancerosas são, de longe, o mais proeminente e aparecem como uma massa de células eCFP-positiva sob o microscópio. O modelo de rato de MMTV-PyMT desenvolve uma doença progressiva, em que o crescimento do tumor mamário está associada com a difusão de células de cancro para a periferia, especialmente para os pulmões. Em ratinhos MMTV-PyMT no FVB / N fundo, micrometástases pode ser observada cerca de 10-11 semanas de idade. Geralmente, estes progressos para macrometástases em cerca de 14 semanas de idade de 13 anos. OU </li><li> Gerar metástases experimentais utilizando células primárias ou linhas de células singeneicas. Utilização em células de tumor primário in vitro manipulado ou linhas de células (por ex., Transdução), seguido por injecção IV 14. <ol><li> Resumidamente, neste protocolo, injetar uma proteína verde fluorescente (GFP) -expressing (+) linha de células MMTV-PyMT em ratos fluorescentes Repórter (ACTB-ECFP) ou ratinhos de tipo selvagem. Depois,visualizar essas células referidas como células VO-PyMT 15 usando o verde, canal de GFP. NOTA: A linha celular VO-PyMT original foi derivada no Vanderbilt Ortopedia em Nashville, TN. VO significa Vanderbilt Ortopedia. </li><li> A seguir à injecção de 10 6 células (em 200 uL), observar o extravasamento de células de cancro imediatamente e até algumas horas após a injecção; observar micrometástases entre 1-3 semanas após a injecção e detectar macrometástases 3 semanas após a injecção 15. NOTA: menor número de células pode ser injectado para prolongar o tempo de injecção para o crescimento metastático. </li></ol></li></ol> 2. Rotulagem de componentes de interesse na metastático Microenvironment (Transgenic e / ou injetáveis) NOTA: A rotulagem pode ser conseguida por murganhos transgénicos e / ou por vários injectáveis. Certifique-se usar diferentes cores fluorescentes para a rotulagem dos vários tipos de células. <ol><li> Os componentes Label do microambiente metastático utilizando camundongos transgênicos. Cruzar o modelo de tumor de rato anteriormente mencionado (por exemplo., MMTV-PyMT x ACTB-ECFP) em um modelo de rato transgénico, em que as células estromais de interesse são marcados por uma proteína fluorescente que não é ECFP, por exemplo., C-fms-EGFP 4,16. NOTA: Além de visualização de células cancerosas no canal PCP, este permite a visualização de células mielóides no canal de GFP 4. E / OU </li><li> Rotular vários componentes do microambiente metastático em ratinhos usando injectáveis ​​repórter fluorescente ou transgénicos (não fluorescentes) ratinhos de tipo selvagem. NOTA: Vários compostos podem ser injectados para marcar vários componentes do microambiente metastático, por exemplo, um anticorpo conjugado AF647-Gr-1 é aqui utilizada para rotular neutrófilos e alguns monócitos e 13 diferentes dextranos de peso molecular são usadospara rotular capilares pulmonares. Para a preparação desses injetáveis ​​consulte a etapa 4. </li></ol> 3. Preparação de Materiais Antes Dissecação <ol><li> 2% de agarose <ol><li> Pesar 0,2 g de agarose e adicionar 10 ml de 1 x PBS. Aquece-se a solução para dissolver a agarose. Agarose irá solidificar à TA, assim mantê-lo em um banho de água a 37 ° C até ser utilizado para a inflação. </li></ol></li><li> CO 2 e controlador de temperatura <ol><li> Verifique DDH 2 O na câmara de umidificação. Reabastecer quando necessário. Inserir a placa de configuração no suporte da placa de estágio de temperatura (câmara climática). Ligue o controlador de CO 2 e definir de CO 2 a 5%. Certifique-se a taxa de fluxo de ar é fixada em 0,4 Nl / min. </li><li> Abra o ar e o CO 2 válvulas. Ligue o controlador de temperatura. Certifique-se de que a temperatura da câmara climática e a tampa são definidas a 37 ° C. </li><li> Liberar a pressão do ar no CO 2 metros. Entrada de CO 2 a aumentar, equilibration pode levar até 30 min. </li></ol></li><li> disco giratório microscópio confocal NOTA: Os detalhes do microscópio set-up foram previamente descritos 4,17. <ol><li> Ligue os lasers (o laser de argônio para 488 nm de excitação e de estado sólido 405 nm, 561 nm e 640 nm lasers). Ligue o microscópio, a câmera, a unidade de controle disco giratório, o AOTF, a unidade de controle do laser e o controlador da câmara. </li><li> Abra o obturador microscópio, ligue o computador a executar o microscópio e abrir o software. </li></ol></li><li> Preparação das ferramentas e plataforma de dissecção. <ol><li> Ligue o esterilizador talão quente e deixá-la atingir 250 ° C. Limpos 2 pares de tesouras cirúrgicas e pinças com água e sabão. Esterilizar as ferramentas durante pelo menos 30 seg. Deixe as ferramentas refrescar. Utilize uma tampa de poliestireno como plataforma de dissecção. Cubra-o com um pedaço de imersão laboratório. </li></ol></li></ol> 4. Preparação de injecções NOTA: Dependendo da meia-vida ea resposta preferida, injetar fluorescente etiquetado anticorpos e / ou moléculas fluorescentes ou imediatamente antes do sacrifício animal ou um par de horas a dias antes. <ol><li> Para neutrófilos imagem Gr1-positivos e monócitos, preparar uma seringa com 7 ul de anticorpo conjugado com AF647-Gr-1 da (1 mg / ml) em 100 ul de PBS estéril sob o capô. Coloque um G ½ agulha 27 na seringa. </li><li> Para capilares pulmonares imagem, preparar uma segunda e terceira seringa com 100 ul de quer 70 kD rodamina conjugada com dextrano (4 mg / ml) ou dextrano 10 kD AF647-conjugado (4 mg / ml). Coloque um G ½ agulha 27 nas seringas. </li><li> Injectar a AF647 conjugado com solução de anticorpo iv 5 horas antes da excisão &#39;pulmões. </li><li> Injectar uma ou ambas as soluções de dextrano iv imediatamente anteriores à excisão &#39;pulmões. </li></ol> 5. Preparação de Pulmões por ex vivo Imagens ao vivo NOTA: Tente trabalhar como estéril e cuidadosa quanto possível para evitar a desnecessária desafios de células imunes dentro dos pulmões. <ol><li> Injectar o rato por via intraperitoneal (ip) com uma sobredosagem letal de um anestésico permitida pelo protocolo animais aprovados pelo IACUC, por exemplo., 1 ml de Avertin 2,5%. Aguarde até que o mouse para parar de respirar e ser completamente não-responsivos a estímulos nocivos (pata traseira pitada). NOTA: deslocamento cervical e os eutanásia dióxido de carbono deve ser evitado, pois pode afectar negativamente a viabilidade das células do pulmão. </li><li> Imobilizar o rato em uma placa de dissecção e esterilizar o mouse com 70% de etanol. </li><li> Use tesouras cirúrgicas primeiro fazer uma incisão epigástrica transversal através da pele, seguido por uma incisão semelhante através do peritoneu. Segure a placa de dissecção na posição vertical e cortou a aorta descendente, de modo que as piscinas de sangue para baixo no abdômen e não na cavidade torácica. </li><li> Sbeliscar uma pequena abertura no diafragma para libertar vácuo. Corte ao longo da nervura 10 e 12 para excisar o diafragma e tenha acesso visual para os pulmões. </li><li> Use uma tesoura cirúrgica para cortar a pele até à traqueia através da caixa torácica, mas deixar a caixa torácica intacta. Separar a pele da caixa torácica. Expor a traqueia através da remoção do tecido conjuntivo circundante, tomando cuidado para não danificar o próprio (Figura 1A) traqueia. </li><li> Cortar uma pequena abertura aproximadamente 1 mm de diâmetro na traqueia exposta paralela aos anéis cartilaginosos, tão perto quanto possível da laringe (Figura 1B). Tenha cuidado para não cortar completamente através da traquéia. </li><li> Dê uma agulha G 20 e gentilmente inserir a agulha 4-5 mm na traqueia, sem qualquer força contrária (Figura 1D). A extremidade da agulha deverá ser visível através da traqueia (Figura 1C). Use uma pinça para estabilizar a agulha na traqueia. Alternativamente, um fio de sutura pode ser amarrado Around a traqueia para manter a agulha no lugar. NOTA: Através da inserção muito profunda, a carena pode ser traumatizada ou apenas um lado dos pulmões pode ser insuflado. </li><li> Encher uma seringa com 400 ul de 37 ° C de agarose de ponto de fusão de baixa temperatura a 2% (feita directamente a partir de um banho de temperatura constante). Verifique se a placa de dissecção está de pé e, lentamente, incutir a agarose quente através da agulha para os pulmões, usar ~ 400 ul para inflar os pulmões. NOTA: Assista os pulmões inflar dentro da caixa torácica. Não excesso de inflar o pulmão como ele irá se romper. </li><li> Uma vez que os pulmões são inflados, enchendo ~ ⅔ da caixa torácica, retire a seringa e manter a agulha no interior da traqueia para evitar qualquer agarose vazem. </li><li> Pour aproximadamente 50 ml de PBS 20 ° C ao longo dos pulmões inflados para permitir que a agarose dentro dos pulmões para definir e solidificar. retirar a agulha devagar e fechar a traqueia com uma pinça para evitar qualquer não-solidificou agarose vazem. &lt;/ Li&gt; <li> Expor os pulmões através da realização de uma esternotomia e subsequentemente excisar os pulmões. Para a excisão dos pulmões, segurar a traqueia, enquanto cortando a traqueia completamente. Puxe cuidadosamente a traqueia para cima, cortar o tecido conjuntivo e do esôfago enquanto puxa os pulmões para fora da cavidade torácica, até os pulmões é separado do rato (Figura 1E). </li><li> Mergulhe os pulmões em warm RPMI-1640 para lavar excessiva de sangue e delicadamente separar os lóbulos usando tesouras e pinças para cortar brônquio principal dos lobos na hilo (Figura 1F). </li><li> Colocar os lóbulos, com a superfície plana virada para baixo para maximizar a superfície de imagem, em um poço de uma placa de imagem de 24 poços (Figura 1G). Adicionar 100 ul de 37 ° C, meio RPMI-1640 por cima dos lóbulos. Coloque várias 15 mm tampa desliza microscópio circular em cima dos lóbulos para evitar que ele flutua. </li><li> Despeje quente PBS nos poços ao redor para evitar a mídia RPMI-1640 de evaporating. Inserir a placa de 24 poços na câmara climatizada equilibrada e manter os lobos pulmonares a 37 ° C com ar e 5% de CO 2. Inserir a câmara climatizada na fase do microscópio confocal. NOTA: Outras misturas de gases (por exemplo, 5% de O2, 5% de CO 2 em N 2 para examinar o comportamento das células sob condições de hipoxia / oxigénio inferior) também poderia ser considerado. </li></ol><img alt="figura 1" src="/files/ftp_upload/53741/53741fig1.jpg" /> Figura 1. Protocolo para a preparação dos pulmões para geração de imagens ao vivo. (A) A exposição da traquéia após a preparação do mouse. (B) pinas pequena feita na traqueia exposta paralela aos anéis cartilagíneos. (C) 20 G agulha inserida 4-5 mm na traqueia. (D) A instilação de 400 ul de 2% de baixo ponto de fusão à temperatura de agarose para os pulmões. (E) Inflpulmões ated separado do mouse. (F) Lobes separados após a inflação. (G) lóbulos colocado em um poço de uma placa de imagem de 24 poços. <a href="https://www.jove.com/files/ftp_upload/53741/53741fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 6. Aquisição e Análise de Imagens NOTA: As imagens podem ser adquiridas com uma variedade de fiação de disco microscópios confocal apoiados por vários programas de software. Neste protocolo, quer μManager com um giro de disco microscópio confocal feito por encomenda ou Zen com um disco giratório microscópio confocal comercialmente disponível é utilizada para a aquisição de imagem, enquanto Imaris é usado para edição de filmes e análise. <ol><li> Adquirir imagens usando μManager. Um detalhado passo a passo protocolo para a aquisição de imagens usando software μManager é descrito anteriormente 18. OU </li><li> adquirir imagensusando a imagem de software de análise, tais como Zen (ver figura S1). <ol><li> Clique na guia &#39;Localizar&#39;, e escolha objetiva (10x ou 20x) no &quot;Caminho de Luz&quot; ferramenta (Figura S1A, caixa vermelha). Posteriormente, clique em &#39;Eyes - DAPI&#39; olhar para o canal PCP através da ocular (Figura S1A, caixa azul). Localize a amostra manualmente usando o microscópio. Clique em &#39;All Off&#39;after o tecido é o centro do campo de visão. </li><li> Clique na guia &quot;Aquisição&quot; para definir todos os parâmetros para aquisição de imagem. </li><li> Na ferramenta &quot;canais&quot;, clique no botão &#39;+&#39; (Figura S1B, caixa vermelha). Um menu pop-up aparece e procurar o corante (s) presente na amostra no &#39;Dye banco de dados&#39; (Figura S1B). Selecione o corante e clique em &quot;Adicionar&quot;. NOTA: O programa irá definir todos os filtros de ser otimizado. Um corante pode ser suprimidoselecionando-o seguido, clicando no botão da lixeira (Figura S1B, caixa amarela). </li><li> No menu &quot;Modo de Aquisição&quot;, set &#39;Binning&#39; para 5x5. Clique duas vezes no ECFP no menu de canais para a seleccionar. Diminuir a potência do laser para 20% assim que a amostra não será branqueada ao configurar os parâmetros para aquisição de imagem. </li><li> Marque a caixa &quot;Azulejo &#39;na seção&#39; Experiment Manager &#39;e a ferramenta de telhas aparece no grupo de ferramentas&quot; Multidimensional Aquisição &quot;(Figura S1C). Clique no botão &#39;Configuração Avançada&#39; para ver a imagem ao vivo da câmera. Clique no botão &quot;Adicionar&quot; na seção &quot;Posições&quot; para adicionar 4 a 6 posições para o experimento. Para excluir uma posição, selecione a posição e clique no botão de lixeira. </li><li> No grupo de ferramenta &quot;Aquisição Parâmetro &#39;, abra a ferramenta&#39; estratégia de foco &#39;, e selecione&#39; Absolute fixa Z-position &#39;na lista suspensa. </li><li> Marque a caixa Z-Stack na seção &#39;Experiment Manager&#39; ea ferramenta Z-Stack aparece no grupo de ferramentas a «Aquisição Multidimensional &#39;(Figura S1D). Clique duas vezes em uma das posições na secção &quot;Posições&quot; e pressione &#39;Live&#39;. definir manualmente primeiro e definir última posição do campo de imagem. Defina o intervalo para 4 mm. NOTA: O programa irá determinar o número de fatias para a gama escolhida e do intervalo. Idealmente, 5-7 fatias são convenientes para permitir a visualização suficiente e aquisição de imagem rápida. </li><li> Marque a caixa &quot;Série Time &#39;na seção&#39; Experiment Manager &#39;. Set desejado &#39;Duração&#39; e os tempos de &quot;intervalo&quot; na ferramenta &#39;Time Series &quot;, que apareceu no grupo de ferramentas&#39; Multidimensional Aquisição&quot; (Figura S1E). </li><li> No &#39;AcquisiModo ção &#39;de menu, defina &quot;Binning&#39; para 2x2. Dê um clique duplo em um fluoróforo no menu de canais para selecioná-lo e aumentar a potência do laser a 100%. Pressione &#39;Live&#39; e ajuste o &#39;Tempo de Exposição &quot;. Repita esse procedimento para cada fluoróforo. </li><li> Marque a caixa &quot;Ativar Auto Save&quot;. Selecione uma pasta e digite o nome do arquivo. Todas as imagens adquiridas serão salvas automaticamente nesta pasta. </li><li> Clique em &#39;Start Experiment&#39; na seção &#39;Experiment Manager&#39; para começar a aquisição de imagem. </li></ol></li><li> Após a aquisição da imagem, compilar os dados brutos em software Imaris. Converter imagens para .IMS arquivos e ajustes podem ser feitos. Um detalhado passo a passo protocolo para a conversão de arquivos, fazendo ajustes e filmes salvar usando Imaris é descrito anteriormente 18. </li><li> Ao salvar o filme, defina o &quot;Frame Rate&quot; 5 quadros por segundo (fps). </li></ol>

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J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic,+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy.">Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harb Protoc. (2), (2011).</li><li>Bonnans, C., Lohela, M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+myeloid+cells+dynamics+in+ApcMin/++intestinal+tumors+by+spinning+disk+confocal+microscopy.">Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.</a> J Vis Exp. (92), (2014).</li><li>Nakasone, E. S., Askautrud, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer cell. 21 (4), (2012).</li><li>Cheng, N., Lambert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+transplantation+of+stromal+cells+and+carcinoma+cells+in+C57BL/6J+mice.">Mammary transplantation of stromal cells and carcinoma cells in C57BL/6J mice.</a> J Vis Exp. (54), (2011).</li><li>Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., Muschel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+origin+of+metastasis+from+the+proliferation+of+endothelium-attached+tumor+cells:+a+new+model+for+metastasis.">Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.</a> Nat Med. 6 (1), 100-102 (2000).</li><li>Wong, C. W., Song, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+location+of+breast+cancer+cells+after+spontaneous+metastasis+to+the+lung.">Intravascular location of breast cancer cells after spontaneous metastasis to the lung.</a> Am J Pathol. 161 (3), 749-753 (2002).</li><li>Liang, C. -. C., Park, A. Y., Guan, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat protoc. 2 (2), 329-333 (2007).</li><li>Nelson, K., Bobba, C., Ghadiali, S., Hayes, D. J., Black, S. M., Whitson, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research.">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World J Exp Med. 4 (2), (2014).</li><li>Magness, S. T., Bataller, R., Yang, L., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+reporter+gene+transgenic+mouse+demonstrates+heterogeneity+in+hepatic+fibrogenic+cell+populations.">A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.</a> Hepatology. 40 (5), 1151-1159 (2004).</li><li>Motoike, T., Loughna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+GFP+reporter+for+the+study+of+vascular+development.">Universal GFP reporter for the study of vascular development.</a> Genesis. 28 (2), (2000).</li><li>Srivastava, M. K., Andersson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+suppressor+cells+and+immune+modulation+in+lung+cancer.">Myeloid suppressor cells and immune modulation in lung cancer.</a> Immunotherapy. 4 (3), (2012).</li><li>Craig, A., Mai, J., Cai, S., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+to+the+lungs+during+bacterial+pneumonia.">Neutrophil recruitment to the lungs during bacterial pneumonia.</a> Infect Immun. 77 (2), 568-575 (2009).</li><li>Kreisel, D., Nava, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proc Natl Acad Sci USA. 107 (42), 18073-18078 (2010).</li></ol>

The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with IACUC approved protocols, UCSF.

Este manuscrito descreve um método detalhado para ex vivo imagens ao vivo da metástase pulmonar em modelos de ratos com metástases. Este protocolo de imagem fornece uma visualização direta das interações tumor de células-estroma dinâmicas e espaciais dentro do microambiente do pulmão. É um método relativamente fácil e rápido que permite imagens de confiança de metástases do pulmão durante pelo menos 4 h. Filmes adquiridos a partir destas experiências pode ser utilizada para controlar processos...

The deadliest aspect of cancer is metastasis, which accounts for more than 90% of cancer-related morbidity and mortality1. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular mechanisms that govern metastatic dissemination and growth are still elusive. To metastasize, tumor cells in the primary tumor must detach from their neighboring cells and basement membrane, cross through the extracellular matrix, intravasate, travel via blood or lymphatic vessels, extravasate at the secondary site, and finally, survive and establish secondary tumors. In addition to the properties of the tumor cells, the contribution from the microenvironment, which includes the adjacent stroma along with the normal counterparts of the cancer cells, is crucial for the seeding and establishment of metastatic lesions2.
Traditional methods to study metastatic seeding and growth examine static states, as tissues are excised and sectioned for histology. These data only generate a snapshot of this highly dynamic process. Although some useful information can be gained from these studies, the complicated process by which tumor and stromal cells interact during metastatic formation cannot be adequately assessed by these methods. Furthermore, it is not possible to gain insights into tumor or stromal cell migration patterns, which are important in establishing a colony at the distant site. In order to effectively study the metastatic process, it is essential to visualize various interactions between cancer cells and their microenvironment in a continuous manner and at real time. 
The lung is a common site for metastases from solid tumors as breast, colorectal, pancreatic cancer, melanoma and sarcoma3. Intravital imaging was previously used to study cell-cell interaction in various primary tumor and metastatic models4,5. Methods of lung imaging in mice, including intravital imaging, lung section imaging, and an ex vivo pulmonary metastasis assay have been published6&#8211;9. Intravital imaging of mouse lungs utilizes a thoracic suction window to stabilize the lungs6. This method is used for time-lapse imaging of the lung microcirculation and alveolar spaces. The anatomical location of the lungs poses a challenge to intravital imaging. In order to access the lungs, the chest cavity must be opened which leads to loss of negative pressure and collapsed lungs. This method only allows the visualization of a small part of the lungs and is technically demanding; an unnecessary complication in studies that examine processes that are independent of blood flow. Moreover, this method also requires gating out movement caused by breathing. This is done either by collecting images between breaths or during post image acquisition analyses10. The alternative ex vivo lung section imaging provides stability and depth, and also prepares lung parenchyma for immunostaining7. However, the lengthy sectioning process leads to an extensive delay between the time of animal sacrifice and the start of the imaging session. Moreover, the process of sectioning a mouse lung causes considerable amount of cell death8, thus interfering with the quality and quantity of imaging samples and perhaps needlessly altering tumor-stroma interactions. In order to technically bridge between the methods of intravital imaging and lung section imaging, while exploiting the advantages of the two techniques, a relatively fast and easy method for ex vivo lung imaging was developed. This method was achieved by imaging of non-sectioned whole lung lobes. 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.

<table>
	<tbody>
		<tr>
			<td>MMTV-PyMT/FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>2374</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>ACTB-ECFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>c-fms-EGFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>1800</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>GFP+ VO-PyMT cells</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70,000 kDa Dextran, rhodamine-conjugated</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>10,000 kDa Dextran, Alexa Fluor 647 conjugated</td>
			<td>Invitrogen</td>
			<td>D22914</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse Gr-1 antibody Alexa Fluor 647</td>
			<td>UCSF Monoclonal antibody core</td>
			<td></td>
			<td>Stock 1mg/ml. Use 7 ug per animal.</td>
		</tr>
		<tr>
			<td>Anesthetic</td>
			<td></td>
			<td></td>
			<td>Anesthesia approved by IACUC, used for anesthesia and/or euthanesia</td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>UCSF cell culture facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, USP sterile&#160;</td>
			<td>Amresco INC</td>
			<td>K813-500ML</td>
			<td>Ultra pure grade for i.v. injection</td>
		</tr>
		<tr>
			<td>Styrofoam platform</td>
			<td></td>
			<td></td>
			<td>Will be used as dissection board</td>
		</tr>
		<tr>
			<td>Fine scissors sharp&#160;</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz Surgical Store</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr>
			<td>Air</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe without needle&#160;</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 1/2 needle&#160;&#160;</td>
			<td>BD</td>
			<td>305109</td>
			<td>for i.v. injection</td>
		</tr>
		<tr>
			<td>20 G x 1 needle, short bevel&#160;&#160;</td>
			<td>BD</td>
			<td>305178</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting-temperature agarose&#160;</td>
			<td>Lonza</td>
			<td>50111</td>
			<td>To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 &#176;C water bath until used for inflation.</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium without phenol red</td>
			<td>Life Technologies</td>
			<td>11835-030</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well Imaging plate&#160;</td>
			<td>E&#38;K scientific</td>
			<td>EK-42892</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slides, 15 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>22-031-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital CO2 and temperature controller</td>
			<td>Okolab</td>
			<td>DGTCO2BX</td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Climate chamber</td>
			<td>Okolab</td>
			<td></td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Carl Zeiss Inc</td>
			<td>Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCD camera</td>
			<td>Stanford Photonics</td>
			<td>XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal scan-head</td>
			<td>Yokogawa Corporation</td>
			<td>CSU-10b</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mManager</td>
			<td>Vale lab, UCSF</td>
			<td></td>
			<td>Open-source software</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of lung metastasis and their microenvironment

A metástase é uma das principais causas de morbidade e mortalidade relacionada ao câncer. A metástase é um processo de várias etapas e, devido à sua complexidade, os processos celulares e moleculares exatos que governam disseminação metastática e crescimento são ainda imperceptíveis. imagens ao vivo permite a visualização das interações dinâmicas e espaciais das células e seu microambiente. Os tumores sólidos geralmente metástases para os pulmões. No entanto, a localização anatômica dos pulmões representa um desafio para imagiologia intravital. Este protocolo fornece um método relativamente simples e rápido para ex vivo imagens ao vivo das interações dinâmicas entre as células tumorais e sua estroma circundante dentro de metástase pulmonar. Usando este método, a motilidade de células cancerosas, bem como as interacções entre as células cancerosas e as células do estroma no seu microambiente podem ser visualizados em tempo real, durante várias horas. Ao utilizar ratinhos transgénicos repórter fluorescentes, uma linha de células fluorescentes, injectável marcado fluorescentementemoléculas e / ou anticorpos, vários componentes do microambiente do pulmão pode ser visualizado, tais como os vasos sanguíneos e células do sistema imunológico. Para imagem os diferentes tipos de células, foi utilizado um disco giratório microscópio confocal que permite imagens contínuo a longo prazo com rápida, de quatro cores de aquisição de imagem. filmes de lapso de tempo compilados a partir de imagens recolhidas ao longo de várias posições e planos focais mostram interações entre metastático ao vivo e células do sistema imunológico durante pelo menos 4 horas. Esta técnica pode ser também utilizada para testar a quimioterapia ou terapia-alvo. Além disso, este método pode ser adaptado para o estudo de outras patologias relacionadas com o pulmonares que podem afectar o microambiente do pulmão.

Utilizando microscopia confocal de disco rotativo, vários sistemas modelo rato e injectáveis, o microambiente metastático pode ser visualizado e rastreada ao longo do tempo. Usando um MMTV-PyMT; ACTB-ECFP; modelo de ratinho transgénico triplo EGFP c-fms, diferentes componentes celulares são marcado de forma fluorescente (Figura 2A, um filme). A estrutura típica do parênquima pulmonar pode ser visualizado no canal PCP uma vez que todas as células expressam ECFP ...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment at JoVE.com

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

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

Descreve-se um método relativamente simples para a imagiologia ex vivo, ao vivo das interacções de células de estroma tumoral dentro de metástase pulmonar, utilizando repórteres fluorescentes, em ratinhos. Utilizando microscopia confocal de disco rotativo, esta técnica permite a visualização de células vivas durante pelo menos 4 horas e pode ser adaptado para estudar outras condições inflamatórias do pulmão.

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

ex-vivo-live-imaging-of-lung-metastasis-and-their-microenvironment

Research

JoVE Journal

Medicine

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

12.3K visualizações.  University of California.  Descreve-se um método relativamente simples para a imagiologia ex vivo, ao vivo das interacções de células de estroma tumoral dentro de metástase pulmonar, utilizando repórteres fluorescentes, em ratinhos. Utilizando microscopia confocal de disco rotativo, esta técnica permite a visualização de células vivas durante pelo menos 4 horas e pode ser adaptado para estudar outras condições inflamatórias do pulmão. 

Vídeo: Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex Infecção Vivo de tecido vivo com Vírus Oncolíticos

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Medicina

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

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

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 ...

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 ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Profiling Molecular do microambiente do tumor invasivo em um modelo 3-Dimensional de células de câncer colorretal e<em&gt; Ex vivo</em&gt; Os fibroblastos

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Método de Isolado<em&gt; Ex Vivo</em&gt; Perfusão pulmonar em um modelo de rato: Lições aprendidas com o desenvolvimento de um Programa PPEV Rat

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validação de nanocorpo e Antibody Based<em&gt; In Vivo</em&gt; Experimentos Tumor Xenoenxerto NIRF-imagem em ratos usando<em&gt; Ex Vivo</em&gt; Citometria de fluxo e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Tumor primário e isolamento de células MEF para o Estudo de metástases pulmonares

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

Corte de precisão Fatias pulmonares como uma ferramenta eficiente para ex vivo estrutura de vasos pulmonares e estudos de contratibilidade

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

Culturas de fatias organotípicas como modelos pré-clínicos de microambiente tumoral em câncer de pâncreas primário e metástase

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Perfusão pulmonar ex situ com pressão negativa normotérmica: avaliação da função e metabolismo pulmonar

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...