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1. Preparação de lamelas de vidro para PAAs <ol><li> Limpos 12 lamelas mm com toalhetes baixos fiapos. </li><li> Chama as lamelas 12 milímetros e 14 milímetros a lamela na microplaca de cada 35 milímetros prato de vidro de fundo, passando-os através de uma chama do queimador de Bunsen, usando uma pinça. </li><li> Tratar os micropoços com 200 uL de NaOH 0,1 N durante 5 min à temperatura ambiente. </li><li> Aspirar e secar os micropoços para 30 min. </li><li> Tratar os micropoços com 50-100 ul de 3-aminopropiltrimetoxisilano, durante 10 min à temperatura ambiente, na hotte. Este produto químico reage com plástico; portanto, use pipetas de vidro e não preencher os micropoços completamente para evitar contato com o plástico prato. </li><li> Lave os micropoços com água ultrapura para cerca de 10 min até que o 3-aminopropiltrimetoxisilano se torna claro. </li><li> Lave os micropoços com água ultrapura duas vezes utilizando uma garrafa squeeze. </li><li> Lave os micropoços com 2 ml de água ultrapura umtemperatura ambiente t em um balancim fixado a uma velocidade média (~ 1 Hz), durante 10 min. </li><li> Aspirar e secar os micropoços para 30 min. </li><li> Tratar os micropoços com 2 ml de solução de glutaraldeído a 0,5% à temperatura ambiente num agitador rotativo ajustado a uma velocidade média (~ 1 Hz), durante 30 min. </li><li> Lave os micropoços com 2 ml de água ultrapura à temperatura ambiente num agitador rotativo à velocidade média (~ 1 Hz), durante 10 min. Repita duas vezes mais para um total de 30 min. </li><li> Micropoços seco a um ângulo inclinado (60º ou superior) por 30 min. Nota: Os pratos pode ser armazenada durante 2 meses num exsicador. </li></ol> 2. Preparação de PAAs para Invadopodia Ensaios <ol><li> Para uma solução de PAA suave (8% de acrilamida e 0,05% BIS) 1 ml, combinar 200 ul de 40% de acrilamida, 25 ul de 2% BIS, e 574 ul de água ultrapura (Tabela 1). </li><li> Para uma solução de PAA disco (8% de acrilamida e 0,35% BIS) 1 ml, combinar 200 ul de 40% de acrilamida, 175 &amp; #181; l de 2% BIS, e 409 ul de água ultrapura (Tabela 1). </li><li> Para uma solução de PAA rígida (12% de acrilamida e 0,60% BIS) 1 ml, combinar 300 ul de 40% de acrilamida, 300 ul de 2% BIS, e 169 ul de água ultrapura (Tabela 1). </li><li> Desgaseificar as soluções durante 15 min. Adicionar 200, 215, e 230 uL de 1 mg / ml de fibronectina de plasma humano em água ultrapura com as soluções de APA macios, duros, rígidos e, respectivamente. Para todas as soluções, adicionar 1 ml de 10 mg / ml de ácido acrílico N-hidroxissuccinimida (NHS), éster de 5 uL de um / ml de persulfato de amónio, 100 mg e 2 ul de N, N, N &#39;, N&#39;-tetrametiletilenodiamina (TEMED ; Tabela 1). Misture com pipetagem suave e evitar a criação de bolhas em soluções. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> PAA (1 ml) </td><td> 40% AA (L) </td><td> 2% (L) </td><td> Ultrapura Água (L) </td><td> 1 mg / ml FN (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS Éster (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / mL APS (L) </td><td> TEMED (L) </td><td> Elástico Módulo (Pa) </td></tr><tr><td> Macio </td><td> 200 </td><td> 25 </td><td> 574 </td><td> 200 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Duro </td><td> 200 </td><td> 175 </td><td> 409 </td><td> 215 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Rígido </td><td> 300 </td><td> 300 </td><td> 169 </td><td> 230 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22692 </td></tr></tbody></table> Tabela 1: Ingrediente volumes e resultando módulos elásticos para PAAs macios, duros e rígidos utilizados nos ensaios invadopodia. AA = acrilamida, FN = fibronectina, APS = persulfato de amónio. <ol start="5"><li> Pipetar 8,48 mL da solução de PAA sobre o centro de cada micropoço. Nota: Este volume teoricamente produz um PAA de 75 um de espessura. </li><li> Com cuidado, abaixe o lado inflamado da lamela 12 milímetros para a gota na microplaca, usando uma pinça. </li><li> Permitir que a solução de PAA ensanduichada polimerizar durante 15-30 min. Verifique polimerização, verificando a solução restante. </li><li> Adicionar 2 ml de 1x PBS a cada prato e remover as lamelas de 12 milímetros, com uma pinça. Nota: 10x PBS é feita em laboratório e composto de 0,011 M de KH 2 PO 4, 1,54 M de NaCl, e 0,056 M de Na 2 HPO 4. </li><li> Lave os micropoços com 2 ml de 1x PBS à temperatura ambiente durante 5 min. Repita duas vezes mais para um total de 15 min. </li></ol> 3. Preparação de PAAs para TRACCÇÃO Ensaios <ol><li> Para uma solução de PAA suave (8% de acrilamida e 0,05% BIS) 1 ml, combinar 200 ul de 40% de acrilamida, 25 ul de 2% BIS, e 566 ul de água ultrapura (Tabela 2). </li><li> Para uma solução de PAA disco (8% de acrilamida e 0,35% BIS) 1 ml, combinar 200 ul de 40% de acrilamida, 175 ul de 2% BIS, e 401 ul de água ultrapura (Tabela 2). </li><li> Para uma solução de PAA rígida (12% de acrilamida e 0,60% BIS) 1 ml, combinar 300 ul de 40% de acrilamida, 300 ul de 2% BIS, e 161 ul de água ultrapura (Tabela 2). </li><li> Desgaseificar as soluções durante 15 min. Adicionar 200, 215, e 230 uL de 1 mg / ml de fibronectina do plasma humano para as soluções de APA macios, duros, rígidos e, respectivamente. Sonicate 8 l de 200 nm microesferas vermelhas fluorescentes (excitação / emissão de 580/605 nm) para 30 seg. Para cada solução, adicionar 8 l de 200 microesferas fluorescentes vermelhas nm,1 uL de 10 mg / ml de ácido acrílico, éster NHS 5 ul de um / ml de persulfato de amónio a 100 mg, e 2 ul de TEMED (Tabela 2). Misture com pipetagem suave e evitar a criação de bolhas em soluções. </li></ol><table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td style="width:40px"> PAA (1 ml) </td><td> 40% AA (L) </td><td> 2% BIS (L) </td><td style="padding-left: 4px; padding-right: 4px;"> Ultrapura Água (L) </td><td> 1 mg / ml FN (L) </td><td> As micro-esferas (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 10 mg / ml NHS Éster (L) </td><td style="padding-left: 4px; padding-right: 4px;"> 100 mg / mL APS (L) </td><td> TEMED (L) </td><td> Elástico Módulo (Pa) </td></tr><tr><td> Macio </td><td> 200 </td><td> 25 </td><td> 566 </td><td> 200 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 1023 </td></tr><tr><td> Duro </td><td> 200 </td><td> 175 </td><td> 401 </td><td> 215 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 7307 </td></tr><tr><td> Rígido </td><td> 300 </td><td> 300 </td><td> 161 </td><td> 230 </td><td> 8 </td><td> 1 </td><td> 5 </td><td> 2 </td><td> 22692 </td></tr></tbody></table> Tabela 2:. Ingrediente volumes e resultando módulos elásticos para PAA macios, duros, rígidos e utilizados nos ensaios de força de tracção AA = acrilamida, FN = fibronectina, APS = persulfato de amónio. <ol start="5"><li> Pipetar 8,48 mL da solução de PAA sobre o centro de cada micropoço. Nota: Este volume teoricamente produz um PAA de 75 um de espessura. </li><li> Com cuidado, abaixe o lado inflamado de the 12 milímetros lamela sobre a gota na microplaca, usando uma pinça. </li><li> Permitir que a solução de PAA ensanduichada polimerizar durante 15-30 min. Verifique polimerização, verificando solução restante. </li><li> Adicionar 2 ml de 1x PBS a cada prato e remover as lamelas de 12 milímetros, com uma pinça. </li><li> Lave os micropoços com 2 ml de 1x PBS à temperatura ambiente durante 5 min. Repita duas vezes mais para um total de 15 min. </li></ol> 4. Preparação de ECM para Invadopodia Ensaios <ol><li> Aquece-se a solução de gelatina (gelatina a 1% e 1% de sacarose) a 37 ° C. </li><li> Tratar o SAP com 150 ul da solução de gelatina, durante 1 min, em seguida, cuidadosamente aspirado a partir do fundo dos micropoços ao segurar os pratos de vidro de fundo num ângulo de 45 °. </li><li> Seca-se a camada fina remanescente de gelatina sobre o SAP nos micropoços em um ângulo agudo (60 ° ou maior) para optimizar a secagem durante 60 minutos. </li><li> Tratar os micropoços com 2 ml de uma solução de glutaraldeído a 0,5% refrigerado em ice durante 15 minutos, seguido por temperatura ambiente durante 30 min. </li><li> Lave os micropoços com 2 ml de uma 1x PBS durante 5 min. Repita duas vezes mais para um total de 15 min. </li><li> Tratar os micropoços com 2 ml de uma solução de boro-hidreto de sódio durante 1 min. Bater levemente pratos na bancada para remover as bolhas que se formam sobre a superfície da gelatina. </li><li> Aspirar e lavar os micropoços com 2 ml de uma 1x PBS durante 5 min. Repita duas vezes mais para um total de 15 min. </li><li> Dilui-se uma solução de fibronectina de plasma humano marcada com FITC a 50 ug / ml com um 1x PBS e centrifugar a 175.000 x g a 4 ° C durante 15 min. Ou comprar comercialmente disponível fibronectina rotulados ou rotulá-la da seguinte forma: <ol><li> Dissolve-se 5 mg de fibronectina em 10 ml de tampão borato (170 mM de tetra-hidrato de metaborato de sódio e 40 mM de NaCl) e o lugar em tubagem de diálise. </li><li> Rotular a fibronectina, colocando o tubo em 200 ml de tampão de borato contendo 6 mg de FITC dissolvido à temperatura ambiente num agitador magnéticodefinido na configuração mais baixa para 1,5 horas no escuro. </li><li> Dialisar com 500 ml de uma 1x PBS num agitador magnético definido no nível mais baixo durante 3 dias num quarto escuro frio (4 ° C) com dois volumes muda de um dia. </li><li> Calcular a concentração de fibronectina marcada com FITC com base na densidade óptica de aliquotas diluídas (1: 200) a 280 nm e 493 nm como [OD 280 - (0,36 x DO 493)] / 1,4 vezes o factor de diluição de 2 vezes 200 (que corresponde para a concentração de glicerol a fibronectina no passo seguinte), resultando em unidades de mg / ml. </li><li> Dialisar durante a noite numa solução de glicerol a 50% num agitador magnético definido no nível mais baixo durante a noite numa câmara fria a 4 ° C). </li></ol></li><li> Tratar os micropoços com 150 ul de solução de fibronectina marcada com FITC à temperatura ambiente durante 60 min no escuro. </li><li> Aspirar cuidadosamente a solução de fibronectina marcada com FITC a partir do fundo dos micropoços ao segurar os pratos de vidro de fundo emum ângulo de 45 ° e, em seguida, encher os pratos de vidro de fundo com solução de etanol a 70% à temperatura ambiente durante 10 min, a uma capa de cultura de células para a esterilização escuro. Preencha também tampas de vidro prato fundo ou limpar com lenços lint baixa embebido em álcool 70%. </li><li> Lavar pratos com fundo de vidro, preenchendo-os com 1x PBS por 5 min. Repita duas vezes mais com 2 ml de PBS 1x para um total de 15 min. </li></ol> 5. Preparação e imagem das Invadopodia Ensaios <ol><li> Adicionar 25.000 células cancerosas em 2 ml de meio invadopodia (1: 1 de DMEM e meio RPMI 1640 com 5% de soro de baixo teor em proteína, 10% de soro fetal de bovino, e 20 ng / ml de factor de crescimento epidérmico adicionada de fresco) para os pratos de vidro de fundo e incubar durante a noite. </li><li> Aspirar e tratar os micropoços com 2 ml de uma solução de paraformaldeído a 3,7% à temperatura ambiente no escuro durante 20 minutos para fixar as células. </li><li> Após duas lavagens rápidas com 2 ml de PBS 1x, tratar os micropoços com 2 ml de Triton X-100 a 0,1% na solução tempe quartoratura no escuro durante 5 min para permeabilizar as células. </li><li> Depois de uma lavagem rápida com 2 mL de PBS 1x, tratar os micropoços com 3% de albumina de soro bovino solução à temperatura ambiente de bloqueio no escuro durante 60 min para bloquear as células. </li><li> Incubar os micropoços com 150 ul de anticorpo de ratinho anti-cortactina (1: 750) em solução à temperatura ambiente no escuro durante 60 minutos de bloqueio. </li><li> Lave os micropoços com 2 ml de 1x PBS durante 5 min. Repita duas vezes mais para um total de 15 min. </li><li> Incubar os micropoços com 150 ul de anticorpo de cabra anti-rato de anticorpo 633 (1: 500) e 546 faloidina (1: 750) em solução à temperatura ambiente no escuro durante 60 minutos de bloqueio. </li><li> Lave os micropoços com 2 ml de 1x PBS durante 5 min. Repita duas vezes mais para um total de 15 min. </li><li> Adicione seis gotas de meio de montagem para preencher cada micro e cubra com 22 x 22 lamelas. </li><li> Cortactina Imagem e actina para identificar invadopodia usando filtros de excitação e emissão de 632/647 nm e556/570 nm, respectivamente, utilizando uma lente de imersão em óleo de elevada NA, 40X sobre uma ampla microscópio de campo invertido. Da mesma forma, a fibronectina imagem para identificar a degradação de ECM utilizando um filtro de excitação e de emissão de 492/518 nm. </li></ol> 6. Preparação e imagem das TRACCÇÃO Ensaios <ol><li> Adicionar 15.000 células cancerosas em 2 ml de meio invadopodia para os pratos de vidro de fundo e incubar durante a noite. </li><li> Aspirar o meio em pratos de vidro de fundo e substituir com 2 ml de meio L-15, com os mesmos suplementos (5% de soro de baixo teor em proteína, 10% de soro fetal de bovino, e 20 ng / ml de factor de crescimento epidérmico) adicionado de fresco, pré -warmed a 37 ° C. </li><li> Incubar as placas de fundo de vidro em uma câmara ambiental em um microscópio invertido pré-equilibrado a 37 ° C, com humidade elevada durante 1 h. </li><li> Tome quatro conjuntos de imagens usando uma NA alta, 40X lente em um microscópio de campo amplo invertido com um estágio automatizado para marcar posições celulares: <ol><li> Células de imagens sobre ap dos PAAs usando contraste de fase (imagem de &quot;fase&quot;). </li><li> Imagem microesferas fluorescentes de excitação e emissão filtros de 560/645 nm, concentrando-se ligeiramente sob as células até a primeira camada de microesferas no topo dos AAP entra em foco (&quot;estressado&quot; imagem). </li><li> Além disso, o foco para o fundo do PAA (usando as microesferas como marcadores) e tomar uma imagem (imagem de &quot;fundo&quot;). Nota: A finalidade desta imagem é para registrar a posição z para obter a espessura PAA real em cada posição para cálculos de força de tração, conforme descrito em Resultados representativos. </li><li> Depois dessas imagens foram adquiridas em múltiplas posições, misturar suavemente em 220 ul de solução a 10% de Triton-X para remover as células. Microesferas de imagem no topo do SAP em cada posição conforme anteriormente descrito (imagem &quot;nula&quot;). </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology in Adhesion Protein Protocols. 1046, 171-189 (2013).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples).">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples).</a> Physics in medicine and biology. 52, 1565-1576 (2007).</li><li>Wang, J. H., Lin, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+traction+force+and+measurement+methods.">Cell traction force and measurement methods.</a> Biomechanics and modeling in mechanobiology. 6, 361-371 (2007).</li><li>Dembo, M., Oliver, T., Ishihara, A., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+traction+stresses+exerted+by+locomoting+cells+with+the+elastic+substratum+method.">Imaging the traction stresses exerted by locomoting cells with the elastic substratum method.</a> Biophysical journal. 70, 2008-2022 (1996).</li><li>Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stresses+at+the+cell-to-substrate+interface+during+locomotion+of+fibroblasts.">Stresses at the cell-to-substrate interface during locomotion of fibroblasts.</a> Biophysical journal. 76, 2307-2316 (1999).</li><li>Engler, A. J., Rehfeldt, F., Sen, S., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+elasticity:+measurements+by+atomic+force+microscopy+and+its+influence+on+cell+differentiation.">Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation.</a> Methods Cell Biol. 83, 521-545 (2007).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in cell biology. 83, 29-46 (2007).</li><li>Leach, J. B., Brown, X. Q., Jacot, J. G., Dimilla, P. A., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+and+branching+of+PC12+cells+on+very+soft+substrates+sharply+decreases+below+a+threshold+of+substrate+rigidity.">Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity.</a> J Neural Eng. 4, 26-34 (2007).</li><li>Zhou, J., Kim, H. Y., Wang, J. H., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+stiffening+of+embryonic+tissues+via+microtubules,+RhoGEF+and+the+assembly+of+contractile+bundles+of+actomyosin.">Macroscopic stiffening of embryonic tissues via microtubules, RhoGEF and the assembly of contractile bundles of actomyosin.</a> Development. 137, 2785-2794 (2010).</li><li>Buxboim, A., Rajagopal, K., Brown, A. E., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+deeply+cells+feel:+methods+for+thin+gels.">How deeply cells feel: methods for thin gels.</a> J Phys Condens Matter. 22, 194116 (2010).</li></ol>

Este trabalho foi financiado pelo Instituto Nacional do Câncer dos Institutos Nacionais de Saúde sob Award Número K25CA143412 (Parekh). Gostaríamos de reconhecer que o apoio adicional foi fornecido pelo Departamento de Otorrinolaringologia. O conteúdo é da exclusiva responsabilidade dos autores e não representam, necessariamente, a posição oficial do National Institutes of Health.

Apresenta-se um método para a fabricação de PAA que podem ser usados ​​como a base para invadopodia força de tracção e ensaios para correlacionar comportamentos celulares invasivos e contrácteis. Enquanto PAAs têm sido muito utilizados para analisar os efeitos da rigidez nas células e calcular forças de tração 18,24,27, este protocolo é o primeiro a desenvolver ensaios paralelos baseados em PAAs com os mesmos factores de rigidez para correlacionar comportamentos celulares invasivos e contrát...

A rigidez da ECM associado a tumor tem sido identificada como um factor significativo na condução comportamento maligno, aumentando a contractilidade actomiosina 1-3. Embora este efeito principalmente tem sido demonstrado com células de cancro da mama, a rigidez da matriz foi encontrado para alterar as propriedades invasivas das células derivadas de uma variedade de cancros o que sugere que 4-8 rigidez tumoral podem desempenhar um papel em outros tipos de cancros. Para penetrar tecidos reticulados durante a migração invasiva, as células cancerosas utilizam saliências adesivas ricas em actina conhecidos como invadopodia que localizar proteinases para degradar focally o ECM 9. Invadopodia são consideradas uma característica de células invasivas e têm sido implicados na invasão de células de tumores e metástases 10,11. Os trabalhos anteriores mostraram que a rigidez da matriz pode regular números invadopodia e associado ECM degradação 4,12 através da atividade da miosina II e proteínas mechanosensitive 12. Considerando acorrelação entre a densidade e a agressividade do tumor do cancro 13,14, estes resultados sugerem um mecanismo pelo qual as células cancerígenas podem responder aos tecidos rígidos de tumor para dirigir a invasão e metástases através de actomiosina contractilidade. Em rigidez MEC in vitro e em densidade do tecido vivo têm sido mostrados para regular o comportamento invasivo das células cancerosas 1,15-17. Enquanto actomiosina contractilidade parece ser importante neste processo, estudos actual conflito a respeito de se a capacidade metastática está correlacionada com o aumento ou a diminuição da força contráctil 6,18-20. Além disso, não se sabe se essas forças mediar diretamente a atividade invadopodia 21. Recentemente, descobriu que forças contráteis da célula cancerosa eram dependentes de rigidez da matriz e foram preditivos de degradação ECM por invadopodia 5. Estes resultados sugerem que as forças celulares podem desempenhar um papel importante na progressão do cancro pela mediação invadopodia acdade em resposta às propriedades mecânicas do microambiente do tumor. Para correlacionar propriedades invasivas e contrátil das células cancerosas, 5, modificamos um protocolo para a criação de PAAs com diferentes rigidez que anteriormente foi usado para investigar a atividade invadopodia dependente da rigidez 4,12,22. Por reticulação química fibronectina do plasma humano ao longo do PAA, estes hidrogéis modificadas podem ser utilizadas como a base para ambas invadopodia e ensaios de força de tracção para assegurar que as células experimentado as mesmas em ambas as experiências rigidez 5. Nos ensaios invadopodia, a fibronectina fornece um domínio de ligação natural para gelatina para ligar o ECM sobreposta para o SAP para detectar a degradação da matriz. Nos ensaios de força de tração, a fibronectina fornece um ligando para adesão celular direto para detectar deslocamentos de microesferas utilizadas para calcular as forças de tração celulares. Este método resulta em o que temos chamado macio, duro,e PAAs rígidas que são obrigados a pratos com fundo de vidro e têm módulos elásticos, E, de 1023, 7307 e 22692 Pa 5, que abrange a gama de propriedades mecânicas reportados para os tecidos normais e cancerosos 23.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-Aminopropyltrimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>281778</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic acid NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>A8060</td>
			<td>prepare fresh in fume hood 10 mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 phalloidin</td>
			<td>Life Technologies</td>
			<td>A22283</td>
			<td>can also use rhodamine</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td>prepare fresh 10% solution in 1X PBS</td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>18606</td>
			<td>use six drops to fill microwells</td>
		</tr>
		<tr>
			<td>BIS (2%)</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>RPI</td>
			<td>A30075</td>
			<td>make 3% for blocking solution in 1X PBS and store in 4 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9777</td>
			<td>pre-equilibrate in borate buffer for 15-30 min</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>10-013-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>use to make acrylic acid NHS ester solution</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PHARMCO-AAPER</td>
			<td>E200</td>
			<td>dilute with ultrapure water to 70%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific</td>
			<td>SH30070.03</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>FITC</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Polysciences</td>
			<td>00639</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (35 mm coverslips)</td>
			<td>MatTek</td>
			<td>P35G-0-14-C</td>
			<td>coverslips are uncoated</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%)</td>
			<td>Polysciences</td>
			<td>01909</td>
			<td>dilute with 1X PBS to 0.5%</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa Fluor 633 antibody&#160;</td>
			<td>Life Technologies</td>
			<td>A21050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Life Technologies</td>
			<td>33016-015</td>
			<td>add 5 ml of ultrapure water to make 1 mg/ml; aliquot in volumes based on use to avoid excessive freezing and thawing cycles</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>PX-1565-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>mouse anti-cortactin 4F11 antibody&#160;</td>
			<td>EMD Millipore</td>
			<td>05-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>SX-0720-1</td>
			<td>use to make 10X PBS stock</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23020</td>
			<td>use to make 10X PBS stock and borate buffer</td>
		</tr>
		<tr>
			<td>NaOH (1 N)</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>dilute with ultrapure water to 0.1 N</td>
		</tr>
		<tr>
			<td>Nu-Serum (low-protein serum)</td>
			<td>BD Biosciences</td>
			<td>355500</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros</td>
			<td>416785000</td>
			<td>typically make 10% stock in 1X PBS, prepare in fume hood, and add a few ml of strong NaOH to dissolve paraformaldehyde easily then bring back to pH 7.4 with strong HCl)</td>
		</tr>
		<tr>
			<td>PBS (sterile)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>use for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Cellgro</td>
			<td>10-040-CV</td>
			<td>use to make invadopodia medium</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452882</td>
			<td>prepare fresh in fume hood 1 mg/ml in 1X PBS&#160;</td>
		</tr>
		<tr>
			<td>sodium metaborate tetrahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S0251</td>
			<td>use to make borate buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060</td>
			<td>typically make 10 ml of 1%sucrose/1% gelatin solution in PBS and store at 4 &#176;C (preheat PBS to dissolve gelatin easily)</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td>make 10% stock in 1X PBS and use as is for cell removal in traction force assay or dilute with 1X PBS for staining</td>
		</tr>
	</tbody>
</table>

polyacrylamide gels for invadopodia and traction force assays on cancer cells

Tecidos tumorais rígidas têm sido fortemente implicado na regulação da migração e invasão de células de cancro. Migração invasivo através de tecidos de ligação cruzada é facilitada por saliências ricos em actina chamados invadopodia proteoliticamente que degradam a matriz extracelular (ECM). Atividade Invadopodia demonstrou ser dependente de rigidez ECM e contráteis da célula cancerosa forças sugerindo que sinais de rigidez pode regular estas estruturas subcelulares através actomiosina contratilidade. Propriedades invasivas e contrátil das células cancerosas podem ser correlacionados in vitro utilizando invadopodia e força de tração ensaios baseados em géis de poliacrilamida (PaaS) de diferentes factores de rigidez. Propriedades invasivas e contrátil das células cancerosas podem ser correlacionados in vitro utilizando invadopodia e força de tração ensaios baseados em géis de poliacrilamida (PaaS) de diferentes factores de rigidez. Enquanto existem algumas variações entre os dois ensaios, o protocolo aqui apresentado fornece um método para a criação de PAA thno pode ser usado em ambos os ensaios e são facilmente adaptáveis ​​às necessidades biológicas e técnicas específicas do usuário.

No ensaio invadopodia, invadopodia são tipicamente identificadas por uma co-localização de marcadores como actina e cortactina em estruturas puntiformes dentro do corpo celular (Figura 1). Ambos degradar de forma activa e não-degradante invadopodia pode ser contado e são diferenciados por se estas estruturas são co-localizada com as áreas pretas falta de sinal fluorescente no fibronectina marcada com FITC (Figura 1). Invadopodia são contadas manualmente, e a degradação de ECM ...

Watch this Scientific Journal Video about Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells at JoVE.com

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigidez mecânica no microambiente tumoral desempenha um papel crucial na condução de comportamento maligno, aumentando a atividade invadopodia e actomiosina contratilidade. Usando géis de poliacrilamida (PaaS), invadopodia e força de tração ensaios podem ser utilizados para estudar as propriedades invasivas e contrátil das células cancerosas em resposta a matriz rigidez.

Géis de poliacrilamida para Invadopodia e Tração Força Ensaios sobre as células cancerosas

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

polyacrylamide-gels-for-invadopodia-traction-force-assays-on-cancer

Research

JoVE Journal

Medicine

12.0K visualizações.  Vanderbilt University Medical Center.  Rigidez mecânica no microambiente tumoral desempenha um papel crucial na condução de comportamento maligno, aumentando a atividade invadopodia e actomiosina contratilidade. Usando géis de poliacrilamida (PaaS), invadopodia e força de tração ensaios podem ser utilizados para estudar as propriedades invasivas e contrátil das células cancerosas em resposta a matriz rigidez. 

Vídeo: Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Processamento de tecido primário do tumor cerebral para Ensaios de Células Tronco e Fluxo de Classificando

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Formação de Epithelium próstata humano utilizando Tissue Recombinação de Rodent Urogenital Mesênquima Sinus e células-tronco humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptação do Semi-Automática de circulação Tumor de Células (CTC) Ensaios para aplicações clínicas e pré-clínica de Pesquisa

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling de MicroRNAs estrogênio regulados em células do cancro da mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para obtenção de células do cancro do ovário primárias de amostras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Enriquecimento para resistente à quimioterapia do cancro do ovário células-tronco de linhas de células humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Um Sistema Organotípicas alta taxa de transferência para Caracterização de Drogas de sensibilidade de células de mieloma múltiplo primárias

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...