This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

1. Cultura tridimensional de células de câncer de mama em Membrana Basal Matrix (A Técnica Embedment) <ol><li> Manuseio matriz membrana basal Matrigel: Descongele no gelo durante a noite a 4 ° C. Matriz da membrana basal é líquido a baixa temperatura mas solidifica à temperatura ambiente. Mantenha a matriz da membrana basal em gelo (Figuras 1A-B). </li><li> Cobrir a No.1 prato confocal com fundo de vidro com 50 ul de matriz da membrana basal por meio de difusão da matriz, utilizando a ponta de uma P-200 Pipetman num padrão em espiral (Figura 1C). Tenha cuidado ao espalhar a matriz da membrana basal para evitar a formação de bolhas de ar. Da mesma forma, evitar a propagação da matriz para fechar as fronteiras do prato fundo de vidro para evitar a formação de menisco. Se a matriz de membrana basal manuseamento inexperiente, em seguida, pré-arrefecido a louça, P-200 Pipetman e pipetas pode ser em gelo (em alternativa, deixado durante a noite num frigorífico a 4 ° C). Este passo ofertastempo adicional para espalhar a matriz da membrana basal antes de solidificação. Colocar a cápsula (s) numa incubadora de cultura de células (a 37 ° C com 5% de CO 2) durante pelo menos 30 minutos para permitir que a matriz da membrana basal para solidificar (Figura 1D). </li><li> Enquanto a matriz é submetido a solidificação, trypsinize confluente de 100 mm de placa de células de 70-80%. Uma vez que as células tenham começado a levantar para fora da placa, ressuspender-los em 10 ml de meio RPMI 1640 contendo 10% (v / v) de soro fetal bovino (FBS), para inactivar a tripsina (Figura 1E). Em seguida, transferir as células ressuspensas para um tubo cónico de 15 ml (Figura 1F). </li><li> Rodar as células (presente no tubo cónico) a 100 xg durante 3 minutos em uma centrífuga de cultura celular dedicado (Figuras 1G-H). </li><li> Enquanto as células estão a ser girado para baixo, alíquota de 50 ul de matriz para um tubo de microcentrífuga de 1 ml (nota: cada prato de vidro de fundo é atribuído um tubo de microcentrífuga, portanto, sea experiência requer três pratos, então segue-se que três tubos de microcentrífuga também são necessários), e, em seguida, colocar o tubo em gelo (Figura 1B). </li><li> Aspirar o meio do tubo cónico da etapa 1.4, mas deixando a pelota não perturbadas (Figura 1I). Suspenda as células (que foram giradas para baixo) em 1 ml de meio RPMI suplementado com FBS (Figura 1J). </li><li> Uma vez que as células são contadas utilizando um hemocitómetro (Figura 1K), ou um contador de partículas alíquota de células de 2,5 x 10 4 células para dentro do tubo de microcentrífuga e ainda por cima utilizando os meios adequados, de modo a obter um volume total de 50 ul (Figura 1D). </li><li> Misturar as células do passo 1.7 (25.000 células em 50 mL) com o tubo de microcentrífuga contendo matriz a partir do passo 1.5, em uma proporção de 1:1; o volume final será de 100 ul (Figura 1M). </li><li> Suavemente prato 100 ul da matriz: mistura de células a partir do passo 1.8 para a solidified membrana basal revestida de matriz prato a partir do passo 1.2 (Figura 1 N). Isto permite a células a ser incorporado na matriz de membrana basal. </li><li> Colocar a cápsula (s) para a incubadora de cultura de células (a 37 ° C com 5% de CO 2) e permitir que a matriz de: mistura de células para solidificar, pelo menos, 30 min. </li><li> Uma vez que a matriz: mistura de células é solidificado, adicionar 2 ml de meio RPMI suplementado com FBS para o prato (Figura 1O) e levar o prato de volta na incubadora onde vai ser armazenado para o resto da experiência (Figura 1P). </li><li> Mudar mídia todos os dias durante um período de 5 dias (ou conforme exigido para um ensaio, da duração do ensaio para MDA-MB-231 é de 5 dias de duração). </li><li> Usando um microscópio de luz, tomar contraste de interferência diferencial (DIC) imagens dos MDA-MB-231 colónias em suspensão na matriz da membrana basal (Figura 3A). Imagem 20 áreas representativas a 10X objetivo uma vez por diadurante cinco dias, para determinar a colónia morfogénese (Figura 3C). </li><li> Analisar imagens cegamente para determinar a formação de colónias de células estreladas (Figura 3B). Uma colónia é considerada estreladas se uma ou mais projecções de esferóide das células são percebidos. Para determinar o percentual de colônias estreladas invasivos, divida o número de colônias estreladas pelo número total de colônias de células por imagem adquirida, e, em seguida, a média das colônias estreladas percentagens dos 20 imagens para cada dia. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> Figura 1. Cultura tridimensional de MDA-MB-231 de cancro da mama na matriz da membrana basal (a técnica de acamamento). AB) Um esquema da montagem experimental (realizada na coifa). C) O bem do prato fundo de vidro revestidocom 50 ul de matriz de membrana basal D) do prato. (es) colocado numa incubadora de cultura de células (a 37 ° C com 5% de CO 2) para permitir que a matriz para solidificar, pelo menos, 30 min.) E As células foram tratadas com tripsina. F ) As células foram ressuspensas em um tubo de 15 ml. GH) (presente em células tubo cónico) foram centrifugadas a 100 xg durante 3 minutos em uma centrífuga de cultura de tecidos. I) da pelota celular. J) As células (que foram fiados para baixo) foram ressuspensas em 1 ml de meio RPMI suplementado com FBS. K) As células foram contadas utilizando um hemocitómetro. G) 2.5 x 10 4 alíquotas de células no tubo de microcentrífuga e com tampo utilizando os meios adequados, de modo a obter um volume total de 50 ul. M ) Misturar as células do passo 1.7 (25.000 células em 50 mL) com o tubo de microcentrífuga contendo matriz a partir do passo 1.5, em uma proporção de 1:1; o volume final será de 100 μ.; L N) Gently placa de 100 mL da matriz: mistura de células a partir do passo 8 para o prato revestido de matriz solidificada a partir do passo 1.2 O) Uma vez que a matriz: mistura de células é solidificado, adicionar 2 ml de meio RPMI suplementado com FBS a. o prato. P) Coloque o recipiente na incubadora, onde serão armazenados para o resto da experiência. 2. Exame de Características morfogênicas de culturas 3D com Imunofluorescência <ol><li> Configurar uma bandeja de gelo, fosfato frio solução tamponada (PBS), 20% de acetona: 80% de metanol (a solução fixadora) e 3% de albumina de soro bovino (BSA; solução de bloqueio) (Figura 2A) para retirar a cápsula (es) da incubadora. </li><li> Colocar a cápsula (s) no suporte de tabuleiro de gelo e aspirado, em seguida, lavar 3x com 2 ml de PBS frio (Figura 2B). </li><li> Depois da última lavagem de PBS é aspirado, adicionar 2 ml de 20% de acetona: solução de metanol a 80% para o prato (s) (Figura 2C </stRong&gt;) para fixar as células durante 20 min a 4 ° C (ou em gelo ou num refrigerador) (Figura 2D). </li><li> Uma vez que a 20 min de fixação é terminada, os pratos trazer de volta à temperatura ambiente, aspirar o fixador, e, subsequentemente, lavar 3x com PBS. Uma vez que a última lavagem de PBS é aspirado, adicionar 2 ml de BSA a 3% para o prato (s) para bloquear durante pelo menos 30 min à temperatura ambiente (Figura 2E). </li><li> Durante o período de bloqueio preparar diluições de primário (laminina V 1:100) e anticorpos secundários (a uma diluição adequada) dissolvidos em 3% de albumina sérica bovina (BSA), um tampão de bloqueio. Por favor, note que a 400-500 mL da solução &quot;BSA + anticorpo primário &#39;deve ser adicionado diretamente na matriz: mistura de células. </li><li> Uma vez a 30 minutos de bloqueio ter expirado, adicionar os anticorpos primários e incubar durante pelo menos uma hora à temperatura ambiente (Figura 2F). Por favor, note que não lavagens PBS deve ser realizada após a 30 min blockinperíodo g. </li><li> Após a conclusão do passo 2.6, remover a solução de BSA + anticorpo primário &quot;, e lavar 3x com PBS. </li><li> Adicionar o anticorpo secundário dissolvido em BSA a 3% (a uma diluição adequada) directamente sobre a matriz de: mistura de células, cobrir os pratos e incubar durante 1 hora à temperatura ambiente (Figura 2G). </li><li> Remover a solução de BSA + anticorpo secundário &quot;, e lavar 3x com PBS. </li><li> Adicionar 2 ml de Hoechst 33258 (1:10.000) dissolvidos em PBS para a coloração de núcleos, e incubar durante 5 minutos, sob uma folha de alumínio (ou cobrir com um recipiente opaco para limitar a fotodegradação) (Figura 2H). </li><li> Uma vez que a 5 min de incubação com Hoechst 33258t ter expirado, lave o prato (s) de 5x com PBS. </li><li> Adicionar meio de montagem diretamente na matriz: mistura de células no prato confocal e cobrir com lamela de vidro com cuidado para evitar induzir quaisquer interrupções para a integridade das colônias no matri membrana basalx: mistura de células (Figura 2I). </li><li> Colocar a placa (s) para secar durante a noite (ou 24 horas) à temperatura ambiente (Figura 2I). Depois de seco, o prato (s) pode ser armazenada a -20 ° C, mas é altamente recomendado que eles devem ser trabalhada, tão rapidamente quanto possível. </li><li> Adquirir imagens utilizando um microscópio de fluorescência com comprimentos de onda de laser adequados de anticorpos utilizados (Figuras 3D-E). </li></ol><img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> Figura 2. Exame de culturas em 3D, por imunofluorescência. . A) Um esquema do arranjo experimental B) Dish (es) colocado na bandeja de gelo e mídia aspirado; subsequentemente, as células foram lavadas três vezes com 2 ml de PBS frio C) 2 mL de acetona a 20%:. solução de metanol a 80% adicionado no prato (s) D) para corrigir as células.20 min a 4 ° C (ou em gelo ou num refrigerador). E) 2 ml de BSA a 3% adicionado ao prato (s) para bloquear durante pelo menos 30 min à temperatura ambiente. F) Uma vez que a 30 min de bloqueio ter expirado, adicionar os anticorpos primários e incubar durante pelo menos uma hora à temperatura ambiente G) os anticorpos secundários dissolvidas em BSA a 3% (a uma diluição adequada) foram adicionadas directamente sobre a matriz de membrana basal:. mistura de células; . subsequentemente pratos coberta e incubada durante 1 hr à temperatura ambiente H) 2 ml de Hoechst (1:10.000; dissolvida em PBS) adicionado ao dishe (es) e as células incubadas durante 5 minutos sob uma folha de alumínio I) meio de montagem adicionado directamente. sobre a matriz: mistura de células no prato confocal e o prato é coberta com lamela de vidro. Prato (s) deixa-se secar durante a noite (ou 24 horas) à temperatura ambiente.

<ol>
	<li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nat Rev Cancer. 2, 563-572 (2002).</li><li>Kramer, N., 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+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> Mutat Res. 752, 10-24 (2013).</li><li>Shaw, K. R., Wrobel, C. N., Brugge, 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=Use+of+three-dimensional+basement+membrane+cultures+to+model+oncogene-induced+changes+in+mammary+epithelial+morphogenesis.">Use of three-dimensional basement membrane cultures to model oncogene-induced changes in mammary epithelial morphogenesis.</a> J Mammary Gland Biol Neoplasia. 9, 297-310 (2004).</li><li>Eccles, S. A., Welch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+recent+discoveries+and+novel+treatment+strategies.">Metastasis: recent discoveries and novel treatment strategies.</a> Lancet. 369, 1742-1757 (2007).</li><li>Poincloux, R., Lizarraga, F., Chavrier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+invasion+by+tumour+cells:+a+focus+on+MT1-MMP+trafficking+to+invadopodia.">Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia.</a> Journal of cell science. 122, 3015-3024 (2009).</li><li>Bissell, M. J., Labarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context,+tissue+plasticity,+and+cancer:+are+tumor+stem+cells+also+regulated+by+the+microenvironment.">Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment.</a> Cancer Cell. 7, 17-23 (2005).</li><li>Burgstaller, G., Oehrle, B., Koch, I., Lindner, M., Eickelberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Profiling+of+Cellular+Invasion+in+3D+Cell+Culture+Models.">Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models.</a> PLoS One. 8, (2013).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, 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=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30, 256-268 (2003).</li><li>Mroue, R., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cultures+of+mouse+mammary+epithelial+cells.">Three-dimensional cultures of mouse mammary epithelial cells.</a> Methods Mol Biol. 945, 221-250 (2013).</li><li>Vidi, P. A., Bissell, M. J., Lelievre, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+of+human+breast+epithelial+cells:+the+how+and+the+why.">Three-dimensional culture of human breast epithelial cells: the how and the why.</a> Methods Mol Biol. 945, 193-219 (2013).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Debnath, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+apoptosis+in+creating+and+maintaining+luminal+space+within+normal+and+oncogene-expressing+mammary+acini.">The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini.</a> Cell. 111, 29-40 (2002).</li><li>Weaver, V. M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+culture+models+to+study+mechanisms+governing+apoptosis+in+normal+and+malignant+mammary+epithelial+cells.">Functional culture models to study mechanisms governing apoptosis in normal and malignant mammary epithelial cells.</a> J Mammary Gland Biol Neoplasia. 4, 193-201 (1999).</li><li>Weaver, V. M., Howlett, A. R., Langton-Webster, B., Petersen, O. W., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+functionally+relevant+cell+culture+model+of+progressive+human+breast+cancer.">The development of a functionally relevant cell culture model of progressive human breast cancer.</a> Semin Cancer Biol. 6, 175-184 (1995).</li><li>Kenny, H. A., Krausz, T., Yamada, S. D., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+novel+3D+culture+model+to+elucidate+the+role+of+mesothelial+cells,+fibroblasts+and+extra-cellular+matrices+on+adhesion+and+invasion+of+ovarian+cancer+cells+to+the+omentum.">Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells to the omentum.</a> Int J Cancer. 121, 1463-1472 (2007).</li><li>Cougoule, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+leukocytes+and+macrophages+of+various+phenotypes+have+distinct+abilities+to+form+podosomes+and+to+migrate+in+3D+environments.">Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments.</a> European journal of cell biology. 91, 938-949 (2012).</li><li>Sung, K. E., 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=Understanding+the+Impact+of+2D+and+3D+Fibroblast+Cultures+on+In+Vitro+Breast+Cancer+Models.">Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models.</a> PLoS One. , (2013).</li><li>Li, T. T., 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=Beta-arrestin/Ral+signaling+regulates+lysophosphatidic+acid-mediated+migration+and+invasion+of+human+breast+tumor+cells.">Beta-arrestin/Ral signaling regulates lysophosphatidic acid-mediated migration and invasion of human breast tumor cells.</a> Mol Cancer Res. 7, 1064-1077 (2009).</li><li>Zajac, M., 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=GPR54+(KISS1R)+transactivates+EGFR+to+promote+breast+cancer+cell+invasiveness.">GPR54 (KISS1R) transactivates EGFR to promote breast cancer cell invasiveness.</a> PLoS One. 6, (2011).</li><li>Underwood, J. M., 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=The+ultrastructure+of+MCF-10A+acini.">The ultrastructure of MCF-10A acini.</a> J Cell Physiol. 208, 141-148 (2006).</li><li>Lelievre, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+phenotype+depends+on+reciprocal+interactions+between+the+extracellular+matrix+and+the+structural+organization+of+the+nucleus.">Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.</a> Proc Natl Acad Sci U S A. 95, 14711-14716 (1998).</li><li>Petersen, O. W., Ronnov-Jessen, L., Howlett, A. R., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+basement+membrane+serves+to+rapidly+distinguish+growth+and+differentiation+pattern+of+normal+and+malignant+human+breast+epithelial+cells.">Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.</a> Proc Natl Acad Sci U S A. 89, 9064-9068 (1992).</li><li>Weaver, V. M., 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=beta4+integrin-dependent+formation+of+polarized+three-dimensional+architecture+confers+resistance+to+apoptosis+in+normal+and+malignant+mammary+epithelium.">beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium.</a> Cancer Cell. 2, 205-216 (2002).</li><li>Cvetkovic, D., 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=KISS1R+Induces+Invasiveness+of+Estrogen+Receptor-Negative+Human+Mammary+Epithelial+and+Breast+Cancer+Cells.">KISS1R Induces Invasiveness of Estrogen Receptor-Negative Human Mammary Epithelial and Breast Cancer Cells.</a> Endocrinology. , 1999-2014 (2013).</li><li>Alemayehu, M., 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=beta-Arrestin2+regulates+lysophosphatidic+acid-induced+human+breast+tumor+cell+migration+and+invasion+via+Rap1+and+IQGAP1.">beta-Arrestin2 regulates lysophosphatidic acid-induced human breast tumor cell migration and invasion via Rap1 and IQGAP1.</a> PLoS One. , (2013).</li></ol>

Não temos nada a divulgar.

O desenvolvimento das técnicas de cultura de células em 3D tem permitido aos investigadores estudar a transformação de células epiteliais da mama, o que nos permite visualizar as alterações morfológicas drásticas. Além de analisar a invasão da célula, os esferóides epiteliais mamárias simples ou multicelulares pode ser utilizado para avaliar alterações na adesão celular, a proliferação, tamanho, polaridade e basal-apical. Em contraste com os métodos previamente descritos, onde as células são cobert...

Migração e invasão de células individuais ou coletivas são duas características de câncer, e necessário para a propagação metastática de células de câncer 1-4. A capacidade de células cancerígenas para iniciar metástase depende da sua capacidade para migrar e invadem o tecido vizinho usando invadopodia para degradar a membrana basal das células. Invadopodia são saliências de degradação da matriz dinâmica ricas em actina que permitem a degradação da matriz extracelular através da liberação de proteases que degradam a matriz 5. Invasão de células do cancro envolve a degradação da matriz seguindo-se a migração das células cancerosas e isto é acompanhado de uma reorganização do ambiente da matriz tridimensional (3D) 2. Assim, para penetrar através da matriz, uma célula deve transformar a sua forma e interagir com a matriz extracelular (ECM) 2. A manutenção da integridade do tecido mamário depende fortemente controlled arquitectura dos tecidos, uma vez junções célula-ECM e de adesão célula-célula influenciar a expressão genética e a interrupção da polaridade epitelial pode levar ao aparecimento de cancro 6-10. No entanto, a maioria dos in vitro de migração e invasão ensaios como transwell ensaios de câmara ou ensaios ferida-risco são bidimensionais (2D) e, portanto, estes negligenciar as interações complexas entre as células e seu meio ambiente adjacente 3,6,8,11-14. Diversidades morfológicas e funcionais consideráveis, incluindo variações na morfologia celular, diferenciação celular, adesões célula-matriz e os padrões de expressão gênica foram detectados através da cultura de células em culturas 3D que são comumente faltando em 2D ensaia 2,6,8,11. Assim, a utilização de testes de 3D ​​são significativamente benéfico em recapitulando a mais fisiológica em condições in vivo, levando a uma melhor tradução das descobertas revolucionárias em pesquisa básica para a clínica 6-10. No entanto, deve-se notar, Apesar das muitas vantagens obtidas com a utilização de culturas em 3D, este modelo não pode capturar todas as complexidades do microambiente do tumor in vivo, que inclui vários tipos de células. No entanto, é possível incorporar células estromais em modelos 3D (por exemplo, os fibroblastos, leucócitos e macrófagos) para estudar o efeito da interação tumor-estroma na adesão célula cancerosa e invasão 15-17. Células epiteliais de mama em cultura crescer de forma mais eficaz quando as proteínas da MEC, como laminina e colágeno estão presentes. Com esta conhecida, uma mistura de matriz comercialmente disponível foi derivado de Engelbreth-Holm-Swarm (EHS) de tumor murino e é conhecido como matriz da membrana basal 2,8 Matrigel. Um número de técnicas têm sido estabelecido para cultivar células epiteliais como colónias em 3D da matriz da membrana basal 2,8. O modelo da matriz da membrana basal 3D é eficaz para o estabelecimento de células da mama, tanto malignas e não malignascrescimento, assemelhando-se o que está ocorrendo no ambiente in vivo 18,19. MCF10A células são células epiteliais mamarias não malignas. Quando crescido na matriz da membrana basal, estas células apresentam características de in vivo de células da mama normais e submetidos a proliferação celular, a polarização celular, apoptose e para estabelecer o espaço lúmen 8,12,20 controlada. Além disso, a aparência de núcleos de células de células que formam MCF10A ácinos em culturas 3D mais se assemelham aos de células epiteliais mamárias de tecido do que os cultivados em monocamada de 21. Estudos por Bissell e colegas foram os primeiros a mostrar que as células malignas da mama podem ser diferenciadas a partir de células não-malignas da mama, quando cultivada em um ambiente rico em laminina, uma vez que as células apresentam um fenótipo maligno altamente desorganizada, a proliferação aumentada, diminuída da célula-a- a adesão celular, o aumento da expressão de marcadores mesenquimais e um aumento no número de estrutura invasiva formado 3,6,22. Anormalidades do ambiente celular pode influenciar a formação do tumor 20. O método de cultura em 3D podem ser utilizados para estudar a eficácia de comunicação que ocorre entre as células tumorais e o seu ambiente envolvente e determinar a expressão de proteínas tais influências 14,20,21,23 comunicação. Este artigo estabelece uma metodologia detalhada para crescer MDA-MB-231 de cancro da mama em culturas em 3D para analisar invasão e para estudar a perda de morfologia epitelial utilizando um marcador epitelial laminina, um componente da membrana basal de células 18,19,24, 25. Os procedimentos detalhados fornecem a capacidade de quantificar com precisão e de forma reprodutível estreladas (invasiva) de formação da estrutura por qualquer célula de cancro invasivo e não se limitando a linhas celulares de cancro da mama (comum, tais como MDA-MB-231, Hs578T, MCF-7, ou T47D ). Assim, este ensaio pode servir como uma plataforma para avaliação da forma como a expressão da proteína em células ou tratamento com pro-ou anti-compostos invasivos regulam a degradação da matriz extracelular, pelas células únicas ou múltiplas.

<table border="0" cellpadding="0" cellspacing="0" width="1062">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

É agora bem conhecido que o microambiente celular e do tecido são reguladores críticos que influenciam a iniciação e progressão tumoral. Além disso, a matriz extracelular (ECM) tem sido demonstrado ser um regulador crítico do comportamento das células em cultura e na homeostase in vivo. A abordagem actual de cultura de células em duas dimensões (2D), superfícies de plástico resulta na perturbação e perda de interacções complexas entre as células e o seu microambiente. Através do uso de três dimensões (3D) de ensaios de cultura, são estabelecidas as condições para a interacção célula-microambiente que se assemelha ao microambiente in vivo. Este artigo fornece uma metodologia detalhada para cultivar células de câncer de mama em uma matriz de proteínas de membrana basal 3D, exemplificando o potencial da cultura 3D na avaliação da invasão celular no ambiente circundante. Além disso, iremos discutir como estes ensaios 3D tem o potencial para analisar a perda de sinalização Molecules que regulam a morfologia epitelial por imunomarcação procedimentos. Estes estudos ajudar a identificar detalhes importantes mecanicistas nos processos que regulam a invasão, necessários para a propagação do câncer de mama.

Um exemplo de MDA-MB-231 em células invasoras matriz 3D é ilustrada na Figura 3C. As células são incorporados na matriz (Dia 1) e começam a formar-(estreladas) estruturas invasoras no Dia 3, e invadir completamente para dentro da matriz por Dia 5 (Figura 3C). O número de colónias formadas estreladas são contadas, e expressas como uma percentagem do número total de colónias por placa (invasivos e não invasivos). Além disso, uma vez que as medições são feitas diariamente du...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

Este artigo proporciona metodologias detalhadas para a utilização de ensaios tridimensionais (3D), para quantificar a invasão das células do cancro da mama. Especificamente, vamos discutir os procedimentos necessários para a criação de tais ensaios, quantificação e análise de dados, bem como métodos para analisar a perda da integridade da membrana que ocorre quando as células invadem.

Quantificação de cancro da mama invasiva das células usando um modelo (3D) tridimensional

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

quantification-breast-cancer-cell-invasiveness-using-three

Research

JoVE Journal

Medicine

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

15.7K visualizações.  University of Western Ontario.  Este artigo proporciona metodologias detalhadas para a utilização de ensaios tridimensionais (3D), para quantificar a invasão das células do cancro da mama. Especificamente, vamos discutir os procedimentos necessários para a criação de tais ensaios, quantificação e análise de dados, bem como métodos para analisar a perda da integridade da membrana que ocorre quando as células invadem. 

Vídeo: Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Na imagem de bioluminescência vivo Tumor de mama Dynamics Hipóxia metástase do cancro cerebral em um modelo de rato

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Medicina

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal de imagem de angiogênese em um modelo de rato nu de Câncer de mama utilizando a ressonância magnética, tomografia computadorizada volumétrica e ultra-som

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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

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

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

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

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

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

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos Linhas tumor neuroendócrino de células como um modelo tridimensional para o estudo da terapia de tumores humanos Neuroendócrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Cultura de Tecidos modelo tridimensional para estudar primária da medula óssea humana e suas Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Início do Carcinoma de mama metastático por Segmentação do ductal Epithelium com Adenovírus-Cre: Um Modelo Novel de camundongo transgênico do Câncer de Mama

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

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 ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Três Culturas dimensionais: Uma ferramenta para o estudo normal Acináceo Arquitetura vs transformação maligna das células de mama

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensional modelo de co-cultura para Interaction Tumor estromal-

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensional (3D) Tumor Esferóide Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...