Name: primary tumor and mef cell isolation to study lung metastasis
Uploaded: 2015-05-20T13:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about Primary Tumor and MEF Cell Isolation to Study Lung Metastasis at JoVE.com

We would like to thank NIH, LACaTS and Ochsner clinic foundation for the financial support.

Todos os experimentos foram realizados em ratos de acordo com protocolos aprovados pelo Animal Care Institucional e Comissões de Uso (IACUC) da LSU School of Medicine. Todos os instrumentos devem ser autoclavados antes da dissecção. 1. Preparação de células primárias de tumores de mama mouse <ol><li> Use um modelo de camundongo que foi geneticamente modificada com o mouse mamária Tumor poliomavï¿½us Oriente T-Antigen (mouse MMTV-PyMT-transgênica), que se desenvolvem espontaneamente tumores como descrito anteriormente 6. </li><li> Sacrificar os ratinhos com isoflurano (99,9%) e pulverizar etanol 75% para assegurar um ambiente estéril (Figura 1A). </li><li> Abra a pele a partir da área do orifício uretral para o pescoço com uma tesoura. Pin a pele para baixo para permitir fácil acesso aos órgãos (Figura 1B). </li><li> Isolar o tumor da mama mamária frio e lava-se com solução salina tamponada com fosfato estéril (PBS) (pH 7,4) (Figura 1C). </li><li> Transferência do tumor auma placa de cultura de 10 centímetros de tecido estéril e cortá-la com uma lâmina de barbear estéril e tesoura para aproximadamente 1 mm3 peças. Os passos subsequentes devem ser realizados numa câmara de fluxo laminar estéril. </li><li> Incubar o tecido tumoral picada com colagenase dissolvido em meio DMEM isento de soro com uma concentração final de 1 mg / ml num agitador rotativo durante 2 horas a 37 ° C. Centrifugar a suspensão do tecido do tumor durante 3 min a 200 x g. NOTA: A incubação da colagenase é essencial para a dissociação enzimática do tecido. </li><li> Descartar o sobrenadante e lavar o precipitado três vezes com PBS (pH 7,4) por centrifugação durante 3 minutos a 200 x g. Após a lavagem final, decanta-se o sobrenadante. </li><li> Ressuspender o tecido com 2 ml de EDTA de tripsina durante 5 min a 37 ° C, enquanto de nutação. Centrifugar a suspensão durante 3 minutos a 200 xg e repetir as lavagens. </li><li> Ressuspender o sedimento de tecido em meio de crescimento normal (DMEM, FBS a 10%, 2X pen / strep) numa placa de 10 centímetros. Mudançaa mídia todos os dias para os próximos 3 dias. Células de cultura de células até que o número desejado é obtido. NOTA: Aproximadamente 1x10 7 células serão isoladas a partir deste procedimento. Outros modelos tumorigénese mamário de ratinho, tal como MMTV-neu, dar menor rendimento de células quando comparado com MMTV-PyMT. Quando utilizando modelos que produzem um rendimento inferior, é benéfico para começar com um maior células tumorais e de cultura mais longos. </li></ol> 2. Rato Lung Extração de Visualize Lung Metástase <ol><li> Antes da extração do pulmão, do fígado e remover o diafragma. Desalojar as costelas sem perturbar os pulmões. </li><li> Com um par de tesouras finas, abrir a parte superior da traqueia, que está mais perto da veia jugular. </li><li> Usando um catéter, injectar um agente, tal como 1 ml de Z-fix ou Índia tinta através da traqueia para encher os pulmões (Figura 2A). Incorporar o Z-fix Pulmões em parafina. </li><li> De-manchar os pulmões injetados com nanquim em solução de Fekete(100 ml de álcool a 70%, 10 mL de formalina a 10% tamponada, e 5 ml de ácido acético glacial), durante 5 min. Definir nódulos tumorais em locais secundários com um ≥ 0,5 mm de diâmetro, determinar os nódulos como metástase. NOTA: Devido ao bloqueio brônquios e bronquíolos pelo tumor, nódulos tumorais não pode absorver 15% da tinta da Índia, e, assim, tecido pulmonar normal será em preto e nódulos tumorais será na cor branca 7. Isto irá permitir a visualização de nódulos tumorais individuais (Figura 2B). </li></ol> 3. Preparar mouse embrionárias fibroblastos (MEFs) a partir de embriões de camundongos <ol><li> Sacrifício camundongos grávidas com isoflurano (99,9%) e spray de etanol de 75% para garantir um ambiente estéril. </li><li> Abra a pele a partir da área do orifício uretral para o pescoço com uma tesoura e isolar os embriões do útero. </li><li> Tenha cuidado para não perturbar os embriões ao abrir a pele. </li><li> Remova os cornos uterinos e enxaguá-los em ste friorile PBS (pH 7,4). </li><li> Separa-se a partir de embriões placenta sua muito cuidadosamente e embriões lugar numa placa de 10 centímetros contendo PBS gelado (pH 7,4). </li><li> Remover a cabeça, fígado e intestino a partir do embrião e colocar a parte restante em uma placa de cultura de tecidos de 12 poços contendo PBS (pH 7,4). </li><li> Transferir o corpo do embrião para uma outra placa de 12 poços contendo EDTA de tripsina e cortados em pedaços pequenos (~ 1 mm3) utilizando uma lâmina de barbear esterilizada e tesoura. Incubar o material cortado com 5 ml de EDTA de tripsina durante 5 min a 37 ° C. </li><li> Inactivar a tripsina por adição de 1 ml de Soro Fetal Bovino (FBS) para os embriões picados. Centrifugar a suspensão a 200 xg velocidade durante 10 minutos, remover o sobrenadante cuidadosamente, ressuspender o sedimento com 1 ml de meio DMEM morno e transferir todo o conteúdo para um prato de 10 centímetros contendo 10 ml de DMEM (com 20% de FBS e 2X de Penicilina / Estreptomicina ). </li><li> Mudar a forma todos os dias durante dois dias. Cultura das células até célula desejadanúmero é alcançado. </li></ol>

<ol>
	<li>Minn, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+that+mediate+breast+cancer+metastasis+to+lung.">Genes that mediate breast cancer metastasis to lung.</a> Natur. 436 (7050), 518-524 (2005).</li><li>Poste, G., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+cancer+metastasis.">The pathogenesis of cancer metastasis.</a> Natur. 283 (5743), 139-146 (1980).</li><li>Wan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Metastasis+Moving+New+Biological+Insights+into+the+Clinic.">Tumor Metastasis Moving New Biological Insights into the Clinic.</a> Nat Med. 19 (11), 1450-1464 (2013).</li><li>Bergers, G., Benjamin, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenesis+and+the+angiogenic+switch.">Tumorigenesis and the angiogenic switch.</a> Nat Rev Cancer. 3 (6), 401-410 (2003).</li><li>Frese, K. K., Tuveson, 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=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nat Rev Cancer. 7 (9), 654-658 (2007).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12 (3), 954-961 (1992).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+Identification+of+Experimental+Pulmonary+Metastases.">Accurate Identification of Experimental Pulmonary Metastases.</a> J Natl Cancer Inst. 36 (4), 641-645 (1966).</li><li>Boczkowski, D., Nair, S. K., Nam, J. H., Lyerly, H. K., Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+tumor+immunity+and+cytotoxic+T+lymphocyte+responses+using+dendritic+cells+transfected+with+messenger+RNA+amplified+from+tumor+cells.">Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells.</a> Cancer Res. 60 (4), 1028-1034 (2000).</li><li>Vargo-Gogola, T., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nat Rev Cancer. 7 (9), 659-672 (2007).</li><li>Watts, A. M., Kennedy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+Tumor+Foci+in+an+Experimental+Murine+Tumor+Model+Using+Computer-Assisted+Video+Imaging.">Quantitation of Tumor Foci in an Experimental Murine Tumor Model Using Computer-Assisted Video Imaging.</a> Anal Bioche. 256 (2), 217-219 (1998).</li><li>Rosenberg, S. 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=Regression+of+Established+Pulmonary+Metastases+and+Subcutaneous+Tumor+Mediated+by+the+Systemic+Administration+of+High-Dose+Recombinant+Interleukin+2.">Regression of Established Pulmonary Metastases and Subcutaneous Tumor Mediated by the Systemic Administration of High-Dose Recombinant Interleukin 2.</a> J Exp Me. 161 (5), 1169-1188 (1985).</li><li>Fantozzi, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Re. 8 (4), 212 (2006).</li><li>Veer, L., 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=Gene+expression+profiling+predicts+clinical+outcome+of+breast+cancer.">Gene expression profiling predicts clinical outcome of breast cancer.</a> Nature. 415 (6871), 530-536 (2002).</li></ol>

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Este protocolo é para isolar células primárias de um tumor fresco mouse. Os lisados ​​preparados a partir de este método são úteis para a proteína, ADN e análise de ARN. As células cancerosas de tumor da mama primário frequentemente metastizar para o cérebro, pulmão e osso 1. Inclui-se uma técnica para extrair o pulmão para a detecção de metástases. A coloração do pulmão com uma mancha de tinta vai permitir a visualização dos nódulos tumorais como pode ser visto na Figura 2B. Que fixa os pulmões em solução Z-fix nos permitirá fazer secções de pulmões, e manchar com anticorpos que são apropriados para estudar mecanismos metastáticos. Também está incluído um protocolo para o isolamento de fibroblastos embrionários de ratinho. Isto é útil para comparar a expressão de genes das células de tumor primárias com o fibroblasto embrionário no mesmo tipo de animal. Na preparação de células primárias e MEFs, a etapa mais crítica é a trituração do material. Sem repartição adequada, loo número de células W será alcançado. Se uma baixa contagem de células é conseguida, o protocolo pode ainda ser seguido, mas as células terão de ser cultivadas durante um longo período de tempo .. Na extracção do pulmão, a coloração adequada e descoloração é essencial para permitir a visualização do nódulo de tumor. Para extrair RNA / proteína a partir de pulmões, que vai ser bom para usar pulmões que são imaculado e não fixo. No entanto, o RNA pode ser extraído a partir de tecidos fixos, bem 8. Desde a fase metastática do câncer de mama representa a maior ameaça para o indivíduo afetado 2, é importante visualizar todos os componentes de tumorigênese da mama. Um desafio importante na compreensão dos mecanismos de progressão do câncer de mama é a disponibilidade de modelos que recapitulam a progressão da doença 9. A elucidação dos mecanismos moleculares da tumorigénese da mama e de metástases são alcançados através da utilização de modelos animais, tais como ratinhos 9, 10. As formas mais comuns para induzir compue tumores mamários são manipular geneticamente os ratos com mutações de oncogenes ou supressores de tumor pré-disposição, a introdução de substâncias cancerígenas, e injetando células cancerosas altamente invasivos 10-12. Tumores obtidos podem depois ser ainda analisados ​​quanto à expressão do gene. Para a investigação do cancro da mama, perfil de expressão gênica é importante, uma vez que prevê a evolução clínica do paciente 13. Algumas limitações existem com o modelo do rato de estudar tumorigênese mamária. Embora ratinhos partes moleculares semelhanças fisiológicas e com seres humanos, eles nem sempre recapitular todos os aspectos de doenças humanas 5. Isto deve ser tomado em consideração quando se utiliza certos ratos geneticamente modificados. No geral, modelos de ratos proporcionam uma forma mais realista para analisar mamário de ratinho tumorigenesis sobre outros modelos atuais como cultura de tecidos e tumores humanos isolados. Tomados em conjunto, este protocolo permite a uma investigação mais aprofundada dos diferentes aspectostumorigenesis de mama.

Metastasis is the final step in the progression of breast cancer 1. It involves a series of steps in which malignant cells are released from the primary tumor and disseminated to other organs 2. The cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system 3. Each step in tumorigenesis is tightly controlled by cells through various genetic and epigenetic changes 4. To study the mechanisms of breast cancer tumorigenesis, mouse model systems have been utilized due to their short lifespan, small size and fast breeding time. Some major advantages of using mouse models in cancer research are the molecular and physiological similarities they share with humans 5. 
Established mouse and human cell lines have been used extensively in breast cancer research. Although we can obtain some information about the steps in metastasis using these cells, genetically manipulated animal models with tumor suppressors/oncogenes will give far more information about tumor development and metastasis starting from the embryonic stage. Thus, with these mouse models, we can obtain genetic and developmental regulation of tumor growth. Furthermore, the tumor microenvironment also plays an important role in tumor cell gene expression and it can be difficult to mimic the same scenario with studies using established cell lines. Primary human mammary tumors can also be used to study mouse mammary tumorigenesis. The main disadvantage of using isolated breast tumors is that this newly implanted tumor does not have the mouse&#8217;s vasculature and lymphatic tissue. Whereas the genetic mouse model systems for studying mammary tumorigenesis will allow us to monitor the steps of metastasis, analyze the tumor microenvironment, and access to the entire body 5.
The overall goal of this protocol is to study breast tumorigenesis in mice. It is known that breast cancers typically metastasize to the brain, lung and bone. This protocol allows for the isolation of primary mammary tumor cells, as well as, studying breast cancer metastasis to the lung. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. Together, these techniques are useful in studying mouse mammary tumorigenesis.

<table><tbody><tr><td>DMEM</td><td>HyClone</td><td>SH30243.01</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life&amp;nbsp; technologies</td><td>25300-062</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Collagenase Type IV</td><td>Sigma-Aldrich</td><td>C5138</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H3506</td><td>Store at &amp;ndash; 20 &amp;deg;C</td></tr><tr><td>Pen Strep</td><td>Life&amp;nbsp; technologies</td><td>15140-122</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gemini Bio-Products</td><td>100-106</td><td>Store at &amp;ndash; 80 &amp;deg;C</td></tr><tr><td>Z-fix</td><td>ANATECH</td><td>#174</td><td>Store at RT</td></tr><tr><td>India Ink</td><td>Yasutomo</td><td></td><td>Store at RT</td></tr><tr><td>AERRANE (Isoflurane)</td><td>Baxter</td><td>NDC 10019-773-60</td><td>Store at RT</td></tr><tr><td>Ethanol</td><td>EMD</td><td>AX0441-3</td><td>Store at RT</td></tr></tbody></table>

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.

A fase de progressão do tumor em que a sacrificar o animal depende do estudo particular e orientações IACUC. Na Figura 1A, um velho C57BL / 6 do mouse 100 dias com o mouse mamária Tumor poliomavï¿½us Oriente T-Antigen (MMTV-PyMT) foi sacrificado em três meses. A pele foi aberta a partir da área do orifício uretral para o pescoço com as tesouras e a pele foi fixado para baixo para permitir o fácil acesso a órgãos como mostrado na Figura 1B. Isto permitiu um fácil acesso ao tumor que foi obtido na Figura 1C. As células primárias obtidas a partir dos tumores são visualizados na Figura 1D. Uma vez que muitos genes medeiam a metástase do cancro da mama para o pulmão 1, extraiu-se que os pulmões (Figura 2A) para estudar o processo metastático. O pulmão foi inflado com Z-fix e corados com nanquim (Figura 2B) para visualizar os nódulos tumorais. A Figura 3 mostra rato FIBR embrionárias<html> oblasts isolado a partir de embriões de camundongos FVB tensão. <img alt="Figura 1" src="/files/ftp_upload/52609/52609fig1.jpg" /> Figura 1. Preparação de células primárias de tumores de mama. (A) A idade do rato fêmea com o Polioma Oriente T-Antigen (PyMT) 100 dias foi sacrificado. (B) A pele foi fixado para baixo para permitir fácil acesso ao tumor mamário. (C) O tumor de mama foi cortado do animal (D). As células primárias isoladas foram cultivadas em Dulbecco Modified Eagle Médium (DMEM) suplementado com 10% de soro bovino fetal e antibióticos (barra de escala: 100? M). As imagens mostram as células no dia 3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> fazer upload / 52.609 / 52609fig2.jpg &quot;/&gt; Figura 2. extracção de pulmão do rato para visualizar a metástase pulmonar. (A) O pulmão foi inflado com Z-fix e (B) corada com tinta nanquim para visualizar nódulos tumorais (barra de escala: 500 mm). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52609/52609fig3.jpg" /> . Figura 3. Preparar mouse embrionárias fibroblastos (MEFs) a partir de embriões de camundongos MEF células foram extraídas de um embrião de rato FVB (barra de escala: 100 M). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52609/52609fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Primary Tumor and MEF Cell Isolation to Study Lung Metastasis at JoVE.com

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

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 ...

primary-tumor-and-mef-cell-isolation-to-study-lung-metastasis

Universidad San Sebastian

Research

JoVE Journal

Medicine

15.8K visualizações.  Louisiana State University Health Sciences Center. 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.

Vídeo: Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Ensaio de recrutamento de células de Tumor de pulmão

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Pesquisa do câncer

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Ensaio Metástase Experimental

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medicina

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

Considerações práticas em estudar a colonização de pulmão metastático em osteossarcoma usando o ensaio de metástase pulmonar

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

O estabelecimento de um ensaio de colonização de pulmão para visualização de células de Tumor nos tecidos do pulmão de circulação

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Modelo de pulmão acelular e celular para estudar o Tumor metástase

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

Geração e Expansão das Linhas tumorais primárias e malignas do mesotelioma

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

Examinando a disseminação de células tumorais de metástases pulmonares

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Autor em destaque: Decodificando a semeadura de metástase em metástase usando uma nova técnica in vivo para rastrear a disseminação do câncer de mama 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Rastreando a Disseminação de Células Tumorais a partir de Metástases Pulmonares Usando Fotoconversão

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

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

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