Name: an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation
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1. Ensaio clonogénico <ol><li> Preparar um único suspensões de células de linhas celulares do cancro da mama dependentes de estrogénio de células MCF-7 e T47D utilizando os passos descritos abaixo <ol><li> Aspirar o meio de cultura (DMEM / 10% inactivado pelo calor soro fetal de vitelo / glutamina e Pen / Strep) a partir de 10 cm do tecido placa de cultura, que é não confluentes mais de 50% com células T-47D MCF-7 ou. Lavar com PBS. Incubar com tripsina a 0,25% / EDTA 2,21 mM dissolvido em DMEM de alto teor de glucose a 37 ° C durante 1-4 min. </li><li> Confira as células em um mínimo de intervalos sob um microscópio de contraste de fase para garantir uma distribuição única célula. Volte a suspender as células com uma pipeta 2 ml por pipetagem cima e para baixo várias vezes para interromper contato célula-célula para atingir, um status de única célula quase invariável. </li><li> Continue a incubar células em tripsina a 37 ° C por até 4 min, se você observar aglomerados de células depois de apenas 2 min de incubação. Não use essas células para estudos clonogénicos se eles permanecem aderentes ao each outro após 4 min de tripsinização porque o erro será introduzido no rendimento número colônia. NOTA: Se as células são aglutinados, o número de colônias formadas irá reflectir o produto de menos células do que o número incubadas. Se as células são tratadas com tripsina excessivamente, o seu potencial clonogénico pode ser diminuída. </li><li> Prepara-se uma suspensão de células única de 1500 células / ml de meio de cultura para placas de 24 cavidades, ou menos, de (+ 500 células / ml, dependendo do tipo de célula ou o número de passagem), através de diluições em série em um tubo principal que contém todo o volume necessário para a todas as variáveis ​​no experimento. NOTA: O objectivo é uma densidade final de células de 800 células / cm2 (gama de aproximadamente 500 a 1100 células / cm2). O objectivo é produzir aproximadamente 100 + 50 colónias, que permite a contagem relativamente fácil, impede aglomeração e permite colónias suficientes para resultar em diferenças estatisticamente significativas quando as colónias são aumentadas ou diminuídas por perturba experimentalções. </li></ol></li><li> Incube as células a clonogénico de densidade usando etapas descritas abaixo <ol><li> Incubar as células em poços em quadruplicado em 24 placas revestidas com fibronectina a uma densidade de 1500 células clonogénicas / poço a partir de um tubo de suspensão de células individuais mestre de 1500 células / ml. Triturar o meio contendo células com uma pipeta 5 ml através da elaboração de 3 ml e 1 ml de meio de distribuição em cada um dos dois poços. NOTA: A fibronectina revestido placas devem ser compradas pré-revestido a partir de um fornecedor comercial. Placas de revestimento fora de uma qualidade controlada, processo resulta automatizados em uma superfície irregular inadequada para este ensaio. </li><li> Misture a suspensão por pipetagem cima e para baixo com uma pipeta de 5 ml, 3 ml elaborar de suspensão de células e preencher 2 poços com 1 ml cada. Encha apenas dois poços de uma só vez a partir de uma pipeta. Retorne o volume restante na pipeta à suspensão de células no tubo principal, voltar a suspender as células novamente pipetando cima e para baixo e elaborar outros 3 ml para encher outro 2 welsl com 1 ml cada. NOTA: mistura contínua do tubo principal é necessário porque as células irão sedimentar continuamente. Desenhando-se um volume suficiente para encher apenas dois poços é necessário adicionar o número de células semelhantes a cada poço, porque as células sedimentar na pipeta bem. </li><li> Trabalhar rapidamente para distribuir as grandes volumes de células porque as células que permitem a sentar-se em suspensão à temperatura ambiente e concentração de CO 2 vai modulam suas potenciais (observações não publicadas) clonogénicos. </li><li> Otimizar a distribuição espacial das células durante o ato de pipetar-los em poços para ensaios de colónias. Fazê-lo pipetando lentamente a suspensão contendo a concentração final de células no meio do poço. Não sujeite a placa para promover o movimento antes que as células se depositam no fundo. Não rodar a placa circular porque mistura irá eficazmente centrifugar as células para o perímetro das densidades celulares elevadas e assim criando colónias confluentes incontáveis ​​a 6 dias. NOTA: Não misture a menos que necessário uma vez que este é menos desejável que nenhuma mistura em tudo depois de introduzir o volume com as células em suspensão. Se é necessário para misturar as células, fazê-lo em movimento a placa para trás e para a frente em direcções perpendiculares, enquanto que repousa sobre uma superfície plana. </li><li> Incubar as células a 37 ° C, 5% de CO 2, sem mudança de meio, durante 6 dias. O pequeno número de células em um poço após 6 dias não vai ter impacto significativo na composição de nutrientes ou de citoquinas nem o pH do meio original. </li><li> Design o decurso de tempo do ensaio do seguinte modo: Incubar as células em fibronectina substratos revestidos no dia 1. Substitua meio existente, com 1 ml de meio fresco ou meio fresco contendo 10 ng / ml no dia 0. As células mancha no dia 6 como abaixo de 2 FGF- em 1.4). Realizar quaisquer perturbações experimentais no dia 3, como abaixo de 1.3). </li></ol></li><li> Configurar Perturbations experimentais de sinalização molecular ou Moléculas de Adesão <ol><li> No dia 3, adicionar 10056; l de uma solução contendo 10x da concentração final pretendida do agente perturbador para a forma de 1 ml nos poços. Não misture. Continue-se a incubar as células a 37 ° C, 5% de CO 2 por 3 dias adicionais. NOTA: agentes Perturbando pode incluir uma variedade de agentes bloqueadores e inibidores de moléculas de adesão, receptores ou outras proteínas de superfície, os inibidores de vias de sinalização intracelulares, moléculas, factores, co-factores ou proteínas estruturais, que podem desempenhar um papel no apoio ao estado dormente. </li><li> Colónias mancha no dia 6, como segue. </li></ol></li><li> Colônias Mancha <ol><li> Células mancha após 6 dias em cultura com um recém-feitos 0,1% violeta de cristal em 2% de etanol / 10 mM borato de sódio solução (pH 9,0). Meios Aspirar e adicionar uma solução de violeta de cristal ml a cada poço durante 20 minutos. </li><li> Lavar as placas por imersão com as aberturas de poços virados para baixo em um ângulo agudo em um balde de gelo repleto de execução contínua da torneiraágua na pia. Incline a placa em um ângulo horizontal, uma vez debaixo de água, bem abrindo para baixo, e em seguida, incline para trás a um ângulo agudo ao remover em um movimento que flui suave. </li><li> Repetir a imersão 2 ou 3 vezes até que a água no fundo dos poços não é mais azul. Lavagens vigorosa pode remover as células ou as colónias que são menos aderente devido à intervenção experimental, adicionando significativamente ao erro na contagem e os dados. </li><li> Chapas secas durante a noite, colocando-os de cabeça para baixo em toalhas na bancada adjacente às suas correspondentes tampas marcadas. </li></ol></li><li> Contagem de colónias <ol><li> Contar o número de colónias em crescimento e latentes em cada poço após 6 dias de incubação, a coloração e secagem. Contagem das colónias optimamente a 40X em um microscópio de contraste de fase invertida. Contagem de colônias de&gt; 30 células como crescer e colônias de 12 ou menos células, com o aspecto morfológico de tamanho muito grande em comparação com células em crescimento, grande Expacitoplasma NDED com grande citoplasma para núcleo rácios, mostrado na Figura 1 7,15. NOTA: Conjuntos de 13-29 células normalmente não são contados como eles não são muito frequentes em ensaios de dormência simples. Eles podem ser contadas se perturbações deslocar o potencial de crescimento da utilização de células em crescimento ou dormentes. Estes resultados terão então de ser correlacionado com significado biológico. </li></ol></li></ol> 2. clonogénico Incubação de Estudos de imunofluorescência <ol><li> Ajustar o número de células de aproximadamente 7,500-8,000 células / poço em placas de 6 cavidades, para corresponder à célula números de área de superfície / análogo a 24 experiências bem. </li><li> Coloque uma rodada, estéril, tampa de deslizamento revestido de fibronectina em cada base poço de placas de 6 poços para estudos de imagem antes da adição de células. Pipetar as células em volumes de 3 mL em cada poço 2 poços de uma vez em concentrações descritas, tal como descrito para as experiências clonogénicas acima em 1.2). </li><li> Incubar as célulasdurante 6 dias, como acima em 1.2.4 e 1.2.5. Adicionar factores perturbadores no dia 3 a 10X a concentração em volumes de 300 ul, tal como descrito nos procedimentos de ensaio de colónia em 1.3). </li><li> Células mancha no dia 6, com anticorpos para moléculas de adesão celular tais como as integrinas α4, α5, α6, β1, β3, por exemplo, moléculas de adesão focal complexos FAK, paxilina e vinculina, por exemplo, proteínas envolvidas na motilidade, tais como α-tubulina, por exemplo, membros da via de sinalização, tal como fosfo-Akt, fosfo-ERK, fosfo-p38, fosfo-JNK, por exemplo, ou qualquer outra proteína que é o alvo de investigação para o seu papel em dormência, utilizando técnicas padrão para directa ou indirecta imunofluorescência. <ol><li> Retirar as lâminas com uma pinça dia 6. Fix em acetona / metanol 1: 1 a -20 ° C durante 20 min e o ar seco. Um fixador alternativo, tal como paraformaldeído, pode ser utilizado, se necessário. Permeabilizar as células com 0,1% de Triton X-100, 0,1% de citrato de sódio durante 2 min fou detecção de antigénios intracelulares. Lavar as células com PBS. </li><li> Para a coloração indirecta immunofluorecence, bloco desliza durante 1 h à temperatura ambiente com BSA a 5% ou com 10% de soro pré-imune das espécies em que o anticorpo secundário foi gerado. </li><li> Incubar durante a noite a 4 ° C com os anticorpos primários para a célula moléculas de adesão, moléculas complexas focais, a sinalização da via de membros ou qualquer outra proteína que é o alvo de investigação para o seu papel em dormência diluído para as diluições específicas recomendadas pelo fabricante em PBS 0,1% de Triton X -100. </li><li> Lavar 3 vezes com PBS. Incubar lamelas com anticorpos conjugados com fluoróforo à temperatura ambiente durante 2 h. Como um exemplo, Alexa Fluor 488 de burro anti-IgG de ratinho de anticorpos podem ser utilizados para detectar anticorpos primários monoclonais de murino. Mount lamelas lado célula para baixo em lâminas de vidro, utilizando um agente antifade com DAPI. Selar os perímetros com unha polonês. </li><li> Para imunofluorescência direta, realizar a BSAbloqueio como acima no ponto 2.4.2), incubar com o anticorpo primário conjugado com fluoróforo durante a noite a 4 ° C. Lavar 3 vezes com PBS. Incubar as lâminas com um agente antifade e Dapi e selo com unha polonês. </li><li> Bandejas tampa deslizante com folha de alumínio e armazenar a 4 ° C para geração de imagens ea fotografia a qualquer hora até várias semanas depois. Veja fotos e células utilizando quaisquer sistemas de imagens microscópicas de fluorescência equipado com uma câmera de 1000X ampliação. </li><li> Para a coloração de actina fibrilar, bloco desliza em 1% de albumina de soro bovino (BSA) durante 30 min e incubar em BODIPY FL-Phallacidin (verde) ou rodamina-faloidina (vermelho) à temperatura ambiente durante 20 min. Adicionar um agente antifade e selar como acima. </li></ol></li></ol> 3. clonogénico Incubação de Estudos Moleculares <ol><li> Western Blots <ol><li> Incubar ER + do câncer de mama células MCF-7 ou T47D em densidades clonogénicos de 20.000 células / placa de 60 mm e 50.000 células / placa 100mm em fibronecplacas a 37 ° C em 5% de CO 2 revestido de estanho. </li><li> Incubar as células em densidades ligeiramente superiores de 75.000 células / 100 mm placa para estudos moleculares que exijam montantes mg para proteína de lisados. Use até dez placas por ponto experimental para coletar proteína suficiente para estudos moleculares utilizando Western blot ou para isolamento de RNA de transferência de Northern. </li><li> O número de células de gel com base de carregamento é um complemento necessário para comparar a expressão da proteína em células de cultivo e dormentes vastamente diferentes tamanhos. Recolher as células por trypsinizing e contagem de uma alíquota num hemocitómetro em 0,2% de Azul de Tripano, para preparação de lisados ​​de Western blot em vez de raspar as células com uma única lâmina de borda. </li><li> Células de centrífuga a 10.000 xg durante 2 minutos, retirar o suporte por aspiração, adicionar 200 ul de tampão de lise, sonicar as células em tampão de lise e determinar a concentração de proteína. </li><li> Calcula-se a quantidade de proteína por célula, dividindo a produção de proteína ao número de célulasque gerou esse montante. Carregar o lisado para cada poço de géis de poliacrilamida separadas que representa tanto a) quantidades equivalentes de proteína e b) as quantidades de proteína que representam o número de células equivalentes. Pelo menos 25 mg de proteína deve ser carregado em cada poço. NOTA: Isto irá permitir comparações entre crescimento e dormentes células, que são significativamente diferentes em tamanho e conteúdo de proteína (Figura 1). </li></ol></li><li> Citometria de Fluxo <ol><li> Recolher as células a partir de placas de 100 mm por tripsinização, como em 3.1 e analisar por células activadas por fluorescência (FACS padrão) usando protocolos de coloração por imunofluorescência primário ou secundário tanto para antigénios intracelulares ou extracelulares, como descrito em 2.4. NOTA: Desanexando células a partir das placas de cultura de tecidos por tripsinização não afecta a concentração de proteínas de membrana, conforme determinado por marcação com anticorpos. </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+temporal+and+spatial+gene+expression+of+fibroblast+growth+factor+family+members+during+mouse+mammary+gland+development.">Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development.</a> Molecular Endocrinology. 8 (2), 218-229 (1994).</li><li>Plath, 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=Expression+and+localization+of+members+of+the+fibroblast+growth+factor+family+in+the+bovine+mammary+gland.">Expression and localization of members of the fibroblast growth factor family in the bovine mammary gland.</a> J Dairy Science. 81 (10), 2604-2613 (1998).</li><li>Lavandero, S., Chappuzeau, A., Sapag-Hagar, M., Oka, T. <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+and+in+vitro+evidence+of+basic+fibroblast+growth+factor+action+in+mouse+mammary+gland+development.">In vivo and in vitro evidence of basic fibroblast growth factor action in mouse mammary gland development.</a> FEBS letters. 439 (3), 351-356 (1998).</li><li>Sinowatz, F., Schams, D., Plath, A., Kolle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+localization+of+growth+factors+during+mammary+gland+development.">Expression and localization of growth factors during mammary gland development.</a> Adv in Exp Med &amp; Biol. 480, 19-27 (2000).</li><li>Korah, R., Sysounthone, V., Scheff, E., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+FGF-2+promotes+differentiation+in+T47-D+breast+cancer+cells.">Intracellular FGF-2 promotes differentiation in T47-D breast cancer cells.</a> Biochem Biophys Res Comm. 277 (1), 255-260 (2000).</li><li>Korah, R., Das, K., Lindy, M. E., Hameed, M., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinate+loss+of+FGF-2+and+laminin+5+expression+during+neoplastic+progression+of+mammary+duct+epithelium.">Co-ordinate loss of FGF-2 and laminin 5 expression during neoplastic progression of mammary duct epithelium.</a> Human Pathology. 38 (1), 154-160 (2007).</li><li>Wieder, R., 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=Overexpression+of+basic+fibroblast+growth+factor+in+MCF-7+human+breast+cancer+cells:+lack+of+correlation+between+inhibition+of+cell+growth+and+MAP+kinase+activation.">Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation.</a> J. Cellular Physiology. 177, 411-425 (1998).</li><li>Brunner, G., Gabrilove, J., Rifkin, D. B., Wilson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+release+of+basic+fibroblast+growth+factor+from+human+bone+marrow+cultures+as+a+biologically+active+complex+with+a+phosphatidylinositol-anchored+heparan+sulfate+proteoglycan.">Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.</a> J Cell Biol. 114 (6), 1275-1283 (1991).</li><li>Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., Gabrilove, 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=Basic+fibroblast+growth+factor+stimulates+myelopoiesis+in+long-term+human+bone+marrow+cultures.">Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures.</a> Blood. 77 (5), 954-960 (1991).</li><li>Gupta, P., McCarthy, J. B., Verfaillie, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblast+heparan+sulfate+is+required+for+cytokine-mediated+ex+vivo+maintenance+of+human+long-term+culture-initiating+cells.">Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.</a> Blood. 87 (8), 3229-3236 (1996).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Farnesyl+transferase+inhibitor+treatment+of+breast+cancer+cells+leads+to+altered+RhoA+and+RhoC+GTPase+activity+and+induces+a+dormant+phenotype.">Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype.</a> Int J Cancer. 129 (1), 61-69 (2011).</li><li>Korah, R., Sysounthone, V., Golowa, Y., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+confers+a+more+differentiated+phenotype+in+MDA-MB-231+human+breast+cancer+cells.">Basic fibroblast growth factor confers a more differentiated phenotype in MDA-MB-231 human breast cancer cells.</a> Cancer Res. 60 (3), 733-740 (2000).</li><li>Dontu, 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+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes &amp; Dev. 17 (10), 1253-1270 (2003).</li><li>Tivari, S., Lu, H., Dasgupta, T., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+dormant+estrogen-dependent+breast+cancer+micrometastases+by+bone+marrow+stromal+injury+in+an+in+vitro+model+of+dormancy.">Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy.</a> Proceedings of the 105th Annual Meeting of the American Association for Cancer Research. , (2014).</li></ol>

Compatível com o Departamento de Defesa Grants DAMD17-01-C-0343 e DAMD17-03-1-0524, a Comissão Estado de Nova Jersey em Cancer Research 02-1140-CCR-E0 ea Ruth Goldberg Estrin Memorial para Pesquisa do Câncer (RW)

O nosso modelo é composta por vários elementos chave de dormência na medula óssea. É composto de estrogénio células sensíveis, que são do tipo susceptíveis de permanecer dormentes na medula por períodos prolongados 10, que consiste de fibronectina, um elemento estrutural chave da medula, o FGF-2, um factor de crescimento abundantemente sintetizado pelo estroma da medula óssea e fortemente depositado na matriz extracelular da medula óssea 31,32 e incubação de células clonogénicas, e...

Células de cancro da mama metastizar para a medula óssea antes de a doença é detectável 1, assim que os tumores se desenvolvem pequenos vasos sanguíneos 2,3. O processo metastático é rápida, mas ineficiente. Células entram os novos vasos sanguíneos rapidamente, a milhões por dia 4, mas poucos sobrevivem a viagem para órgãos distantes 5. No entanto, algumas micrometástases sobreviver no osso e pode ser encontrado como células individuais ou pequenos aglomerados de células nos aspirados de medula óssea de pacientes recentemente diagnosticados 1. Estas células resistem à quimioterapia adjuvante, que é administrado pela própria finalidade de eliminá-los 6. Esta resistência é dotado, substancialmente, por sobrevivência sinalização iniciada por interacções com o microambiente da medula óssea 7,8. Micrometástases pode ser encontrado em cerca de um terço das mulheres com câncer de mama localizado e representam um indicador independente de sobrevida quando analisados ​​por análise univariada 9. Alguns micrometástases são o crescimento iniciado, mas padrões de retorno depende do tipo de célula. Pacientes com câncer de mama triplo negativo tendem a recorrer entre 1 a 4 anos, sugerindo pobre controle sobre o estado de dormência. Outros tipos de células, incluindo ER / PR +, células podem permanecer dormentes por até 20 anos, com uma taxa constante e contínuo de recorrência 10. Embora as diferenças em dormência assinaturas de expressão gênica entre ER- linhas de células de mama e tumores ER + e refletem diferentes potenciais de dormência 11, interacções com estroma da medula óssea provavelmente representam um contributo significativo para a dormência. O estudo de dormência in vivo é excepcionalmente difícil porque micrometástases são raros e estão em desvantagem por células hematopoiéticas em mais de 10 vezes de 6. Assim, os modelos relevantes devem ser gerados que fornecem dados in vitro que pode sugerir mecanismos e gerar hipóteses testáveis ​​in vivo. Uma série de modelos de dormência, incluindo mathemodelos matical 12,13, modelos in vitro, in vivo 7,8,14,15 modelos de xenotransplante de 16, combinações de in vitro e modelos de xenotransplante de 17,18 e tumorais e metástases modelos espontâneos 19, têm rendido alguns insights sobre a dormência célula cancerosa 20 . Cada um desses modelos têm as suas próprias limitações e são em si principalmente útil para gerar hipóteses sobre a sinalização molecular e interações que regem a dormência a ser testada em modelos mais biologicamente relevantes. Com o objetivo geral de definir os mecanismos moleculares de dormência, as interações com o microambiente que resulta na paragem do ciclo, rediferenciação e resistência e os mecanismos terapêuticos que resultam em recorrência em células ER +, desenvolvemos um modelo in vitro que fornece selecionado elementos relevantes da estromal microambiente 7. Este modelo, embora relativamente escassa em seu componentes, é suficientemente robusto para permitir investigadores para derivar mecanismos moleculares específicos que afectam funções significativas de dormência. Estas experiências gerar hipóteses que podem ser directamente testados in vivo. O modelo baseia-se em alguns elementos-chave que demonstraram ser relevante em dormência. Eles incluem a utilização de células de cancro da mama dependentes de estrogénio, a cultura de células a uma densidade clonogénico onde a sua interacção é principalmente com o substrato e os componentes solúveis do meio, um substrato fibronectina e a presença do factor de crescimento básico de fibroblastos (FGF-2) no meio. Foram caracterizados os mecanismos que governam o sistema in vitro, incluindo a indução de paragem do ciclo celular por FGF-2 21, mediada através de TGFp 22, a sobrevivência a sinalização através de PI3-quinase ERK 7,8 e 8 e diferenciação morfogénica para um fenótipo epitelial, que dependia RhoA inactivação, integrina45; 5β1 regulação positiva e ligadura de fibronectina do estroma para a sobrevivência 7,15 (Figura 1). Os efeitos do ciclo celular in vitro de FGF-2 em células MCF-7 em concentrações começam pelo menos um log inferior a 10 ng / mL 21,23. A lógica baseou-se no controlo temporal da expressão de FGF-2 que regula a morfogénese ductal da mama, de expansão cíclica e recessão em um certo número de sistemas de mamíferos 24-27. Nós demonstramos que o FGF-2 induz a diferenciação, incluindo morfogénese ductal in cultura 3D 28, e que a expressão de FGF-2 são geralmente perdido com a transformação maligna de tumores humanos 29. A expressão de FGR1 permaneceu intacto em carcinomas da mama examinados 29 e MCF-7 de células continuam a expressar todos os quatro receptores de FGF 30. No contexto da dormência, o FGF-2 é exportada por e fortemente depositado em estroma de medula óssea 31,32 onde funciona na preservação de células-tronco hematopoiéticas 33. Nós demmonstrado que o FGF-2 induz um estado dormente em ER + do câncer de mama células cultivadas em substratos fibronectina, também abundantes na medula, onde induz a diferenciação morphogenic 7. No modelo, as células de cancro da mama são crescimento inibido, inactivar Rho A através do RhoGap Graf, redifferentiate a um fenótipo epitelial e re-express integrinas α5β1 lost com a progressão maligna. Ligam-se fibronectina através integrina α5β1 e activar a sobrevivência de sinalização que torná-las resistentes à terapia citotóxica 7,8,15 (Figura 1). A inibição de Rho GTPases classe tem sido demonstrado anteriormente para induzir um fenótipo dormente 34. Aqui vamos delinear os procedimentos específicos que permitam aos investigadores estabelecer o modelo e estudar os mecanismos moleculares e celulares específicas que regulam a dormência das células ER + do câncer de mama. Nas experiências aqui apresentadas para ilustrar o uso do modelo, Que orientada via PI3K (Figura 1B) com um inibidor Akt e um inibidor de PI3K e todos os membros da família Rho (Figura 1B) com um inibidor da pan-Rho e Rho cinase um (ROCK) inibidor.

<table><tbody><tr><td>MCF-7 cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>T47D cells&amp;nbsp;</td><td>ATCC</td><td>HTB-123</td><td></td></tr><tr><td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td><td>Corning</td><td>354411</td><td></td></tr><tr><td>BD BioCoat Fibronectin 60 mm Culture Dishes</td><td>Corning</td><td>354403</td><td></td></tr><tr><td>BD BioCoat Fibronectin 100 mm Culture Dishes</td><td>Corning</td><td>354451</td><td></td></tr><tr><td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td><td>Corning</td><td>354088</td><td></td></tr><tr><td>6 Well tissue culture plate</td><td>CellTreat</td><td>229106</td><td></td></tr><tr><td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td><td>Corning Life Sciences</td><td>50-013-PB</td><td></td></tr><tr><td>Heat Inactivated, Fetal Bovine Serum</td><td>Serum Source International</td><td>FB02-500HI</td><td></td></tr><tr><td>0.25% Trypsin/2.21 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100X</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>L-Glutamine, 100x, Liquid</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Recombinant Human FGF&amp;nbsp;basic</td><td>R&amp;amp;D Systems</td><td>234-FSE-025</td><td></td></tr><tr><td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td><td>CalBiochem</td><td>124005</td><td></td></tr><tr><td>LY294002&amp;nbsp;</td><td>CalBiochem</td><td>19-142</td><td>Chenical PI3K inhibitor</td></tr><tr><td>C3 transferase&amp;nbsp;</td><td>Cytoskeleton</td><td>CTO3</td><td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td></tr><tr><td>Y-27632 dihydrochloride&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>129830-28-2</td><td></td></tr><tr><td>BODIPY FL-Phallacidin (green)&amp;nbsp;</td><td>Molecular Probes</td><td>B607</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>BODIPY FL-Rhodamine phalloidin (red)&amp;nbsp;</td><td>Molecular Probes</td><td>R415</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&amp;nbsp;</td><td>Molecular Probes</td><td>R37114</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI&amp;nbsp;</td><td>Molecular Probes</td><td>P-36931</td><td></td></tr></tbody></table>

an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

<html> As experiências foram conduzidas de recapitular o ensaio. O curso do tempo da experiência é mostrado na Figura 2A. As células são incubadas a densidade clonogénico no dia -1, é adicionado FGF-2 em meio fresco no dia 0 e as células são cultivadas até ao dia 6, quando eles são coradas e as colónias são contadas. Quaisquer perturbações do sistema são administrados no dia 3 em volumes de 100 uL a 10X a concentração final desejada. A Figura 2B mostra a aparência típ...

Watch this Scientific Journal Video about An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation at JoVE.co

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Um<em&gt; In Vitro</em&gt; Dormência Modelo de câncer de mama estrogênio-sensíveis na medula óssea: Uma Ferramenta para Estudos Moleculares Mecanismo e Hipótese Geração

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

8.2K visualizações.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

Vídeo: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Desenvolvimento de um modelo humano pré-clínicos de Osteoclastogenesis de monócitos do sangue periférico co cultivados com linhas de células de câncer de mama

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Pesquisa do câncer

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Um In Vitro Sistema para Estudo de dormência Tumor ea Mude para o crescimento metastático

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Medicina

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Modelo de matriz extracelular óssea tridimensional para osteossarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Modelos de metástase óssea

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Transplante Ortotópico de Tumores mamários como Modelos Pré-Clínicos para o Câncer de Mama

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Isolamento e Avaliação Funcional de Células-Tronco de Câncer de Mama Humano de Amostras de Células e Tecidos

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Eferóides de células epiteliais e endoteliais como um modelo in vitro potencial funcional para pesquisa de câncer de mama

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injeção Orthotopic de células cancerosas da mama na mamária Fat Pad de Ratos para o Estudo de crescimento tumoral.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Um<em&gt; Ex vivo</em&gt; Modelo para estudar a ação do hormônio no peito humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Métodos para a cultura fémur humano explantes de tecido para o Estudo de Câncer de Mama Colonização celular do Niche metastático

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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