We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

Todos os protocolos de pesquisa descritas foram revistos pela Universidade de Revisão Institucional Chicago Board (IRB). O consentimento informado foi obtido de cada paciente antes da cirurgia e do estudo foi aprovado pela Universidade de Chicago IRB. Um gabinete de segurança biológica do tipo 2 e as luvas devem ser usados ​​ao manusear tecido humano para proteção e para reduzir o risco de contaminação de células. 1. Isolamento e Cultura de células estromais não transformadas primárias <ol><li> Coleta de tecido humano e preparação. <ol><li> Obter amostras de omento humano, 2 cm 3, removida durante uma cirurgia abdominal, e mergulhar imediatamente no tecido RT solução salina tamponada com fosfato (PBS) (Figura 2A). Um pedaço de omento 3 cm x 2 cm normalmente produz 1 milhão de células primárias humanas mesoteliais (HPMC) e 200.000 fibroblastos humanos primários ou fibroblastos omentais normais (NOF). </li><li> Girar o PBS o tecido foi mergulhado em a 0,5 g durante 3min o mais rapidamente possível após a colheita (&lt;2 h em PBS) e transferir para o tecido fresco 20 mL de PBS num tubo de 50 ml. Corta-se o tecido em 5mm 3 peças por picagem com bisturis em um 15 cm de diâmetro, estéril prato de cultura (Figura 2B). </li></ol></li><li> Isolamento de células mesoteliais humanas primárias 8 <ol><li> Para isolar HPMC, transferir o tecido moído em 50 ml de tubos cónicos e esperar 1 min para os pedaços sólidos a flutuar para o topo (Figura 2C e D). HPMC e glóbulos vermelhos (RBC) estará presente no líquido na parte inferior. Usar uma pipeta para retirar o líquido a partir do fundo do tubo e para um novo tubo cónico. Girar o líquido para baixo, para 0,5 xg durante 3 min e aspirar o sobrenadante do pellet de HPMC / RBC. <ol><li> Repita o processo três vezes: adicionar 20 ml de PBS ao tecido picada, permitir que o tecido a subir, remover o líquido do fundo com uma pipeta e no tubo de HPMC / RBC, girar para baixo, e retire asobrenadante. Depois de todas as rotações são concluídos e o sobrenadante é aspirado, o sedimento conterá HPMC e RBC. </li></ol></li><li> Placa de todas as células a partir do sedimento num frasco de 75 cm 2 em 15 ml de meio de crescimento completo (DMEM com soro a 10% de bovino fetal [FBS], 1% de vitaminas MEM [93 mg / L], 1% de MEM aminoácidos não essenciais ácidos [81,4 mg / l], 1% de penicilina estreptomicina [Pen-Strep, 100 U / ml de penicilina e 100 ug / ml de estreptomicina]). </li><li> Realize uma lavagem PBS secundário para isolar HPMCs adicionais. <ol><li> Para lavar o secundário PBS, agitar o tecido sólido restante a 200 rpm, 37 ° C durante 10 min em 20 ml de PBS, em seguida, esperar um minuto para o tecido a flutuar para o topo. Remover o líquido a partir do fundo do tubo para um novo tubo, centrifugar a 0,5 xg durante 3 min, e o sobrenadante aspirado. </li><li> Placa toda a HPMC / RBC do PBS secundário lavar em um frasco separado 75 cm 2 em 15 ml de meio de crescimento completo. </li></ol></li><li> Para isolar qualquer remaining HPMC, agitar o tecido moído a 200 rpm, 37 ° C graus durante 10 min em 20 ml de uma mistura 1: 1 0,25% de tripsina 25 mM de EDTA: solução de PBS em volume. Permitir que o tecido a subir, recolher o líquido na parte inferior do tubo em um novo tubo de 50 ml, e a rotação para baixo de 0,5 gx 3 min. <ol><li> Aspirar o sobrenadante e placa toda a HPMC / RBC em um frasco separado 75 cm 2 em 15 ml de meio de crescimento completo. </li></ol></li><li> Cultura das células plaqueadas a 37 ° C e 5% de CO2 num ambiente humidif içado. Alimentar as células banhado, adicionando 15 ml de meio de crescimento completo nos dias 3 e 5 sem a remoção dos meios gastos. HPMC podem ser cultivadas por 5-7 dias antes de se separarem. <ol><li> Use baixa passagem HPMC (até passagem 2) em todos os experimentos para minimizar desdiferenciação e modificação do fenótipo inicial. Use um microscópio de campo amplo de preferência com uma objetiva de 20x com capacidades de contraste de interferência diferencial plástico ou objetivo 4-20x com contraste de fase capabilities (filtros de fase de contraste no microscópio) para tomar todas as imagens das células, e uma objetiva de 10x () no campo claro para analisar imunohistoquímica 3,3&#39;-diaminobenzidina (DAB) coloração. </li></ol></li><li> Confirme que a HPMC são cubóide e expressar citoqueratina 8 e vimentina através da realização de imunohistoquímica 8 (Figura 3A, C, E). </li></ol></li><li> NOF isolamento 8 <ol><li> Preparar 10 ml de uma solução de estoque de colagenase de tipo III (10x) por combinação de uma mistura 1: 1 de Pen-Strep 100x:: 1 1.500 unidades de actividade / ml de colagenase de tipo III: Actividade de 714 unidades / ml de hialuronidase em PBS. </li><li> Agitar o tecido omento picada que permanece após HPMC isolamento a 200 rpm, 37 ° C durante 6 horas em 10 ml da solução 10x colagenase tipo III diluída em meio isento de soro (DMEM com 1% de vitaminas MEM [93 mg / L], MEM a 1% aminoácidos não essenciais [81,4 mg / l], 1% de penicilina estreptomicina [Pen-Strep, 100 U / ml de penicilina e 100 ug / ml de estreptomicina]). Tecido digerido vai olhar opaco e pode ter alguns detritos fibroso (Figura 2E). </li><li> Centrifugar a solução contendo as células NOF em suspensão em 0,5 xg durante 3 minutos à temperatura ambiente, aspirar o sobrenadante e o sedimento em placa num frasco de 75 cm 2 em 13 ml de meio de crescimento completo. </li><li> Remover a velha mídia e substituir com 15 ml de meio de crescimento completo fresco depois de 24 horas. NOF podem ser cultivadas durante 1-3 dias antes de se separarem. Proliferação nota da NOF cessará quando as células atingem a confluência. </li><li> Confirmar que os fibroblastos humanos primários, as células são alongadas planas e expressar vimentina mas não citoqueratina 8 8 através da realização de imuno-histoquímica (Figura 3B, D, F). Use NOF de baixa passagem para experiências (idealmente antes da passagem 3) para minimizar desdiferenciação e modificação do fenótipo inicial. Use um microscópio de campo amplo com uma objetiva de 20x com capacidades DIC de plástico para tomar todas as imagens de fase de células,e uma objectiva de 10x em campo brilhante para analisar a coloração imuno-histoquímica DAB. </li></ol></li></ol> 2. chapeamento o Organotípicas Cultura 8 <ol><li> NOF lançamento do balão de cultura de 75 cm por lavagem com 10 ml de PBS, seguido de 3 ml de tripsina a 0,25% / EDTA 25 mM, durante não mais do que 5 minutos. Neutralizar a tripsina com pelo menos 3 vezes o volume de meio de crescimento completo. </li><li> Transferir as células tripsinizadas em um tubo de 50 ml e girar para baixo em 0,5 gx 3 min. Remover o sobrenadante e trazer as células de volta em 5 ml de meio de crescimento completo. Utilizar um contador de células para contagem das células recuperadas a partir do balão de cultura. </li><li> Dilui-se as células no volume apropriado de meio de crescimento completo ao prato 2.000-4.000 NOF por 100 ul numa placa preta, clara de fundo, de 96 cavidades. Adicionar 0,5 ug / 100 ul de colagénio de cauda de rato-I da mistura de células antes do plaqueamento. Let células sentar a 37 ° C em 5% de CO2 num ambiente humidif içado, durante pelo menos 4 horas, ou até que as célulaspara aderir à superfície da chapa. </li><li> HPMC liberar a partir do frasco de cultura utilizando os mesmos métodos descritos nos passos 2.1 e 2.2. Dilui-se as células na quantidade apropriada de meio de crescimento completo ao prato 10.000-20.000 HPMC por 50 ul no topo da NOF já revestida com colagénio I, em placas de 96 poços. Permitir que as células se sentar a 37 ° C em 5% de CO2 num ambiente humidif içado durante pelo menos 18 horas antes de iniciar as experiências. </li><li> Cultura proteína fluorescente verde (GFP) -expressing HeyA8 células cancerígenas de ovário em meio de crescimento completo. HeyA8 células cancerosas do ovário está marcado de forma fluorescente por expressão de um vector lentiviral expressando copépodo cGFP (pCDH-CMV-MCS-EF1). HeyA8 células cancerosas do ovário deve ser 80-90% confluentes no dia da utilização (fase de crescimento logarítmica). Libertar as células a partir da placa de cultura e contagem usando os mesmos métodos descritos nos passos 2.1-2.2 para células primárias. </li><li> Permitir que as células de cancro do ovário para recuperar num meio de crescimento completo durante 15-20 min a 37 ° C numatubo cónico com a tampa solta selado. </li></ol> 3. Ensaio de Adesão 8 <ol><li> Após a recuperação, sedimentar as células do cancro do ovário por centrifugação durante 3 minutos a 0,5 xg e, em seguida, remover o sobrenadante e dilui-se as células no volume apropriado de meio isento de soro para alcançar uma concentração de 50.000 células / 100 pi. </li><li> Inverter as placas de 96 poços com as culturas organotípicas / NOF HPMC para remover meios gastos, e adicionar 100 uL da suspensão de células de câncer em meio isento de soro a cada poço. Incubar a placa a 37 ° C em 5% de CO2 num ambiente humidif içado, durante 30 min a 4 h. </li><li> Inverta a placa de 96 poços e meios para remover as células não aderentes. Use uma pipeta multicanal com baixo poder de cuidado e gentilmente pipeta 100 ul de PBS em cada poço e inverter placa novamente. Repita o PBS lavar mais uma vez. </li><li> Inverta a remover PBS e adiciona-se 100 ul de paraformaldeido a 4% a cada poço. Permitir que a placa de sentar-se por 20 min para corrigir as células. Após 20 min, inverter a placa e adiciona-se 100 ul de PBS. </li><li> Note-se que a fluorescência total dos poços pode ser reportada ou o número de células pode ser quantificada. Para a quantificação, a primeira placa de uma curva padrão em duplicado utilizando células HeyA8 a uma concentração de 1 milhão de células / ml. <ol><li> Fazer a curva padrão por fiação para baixo 1 milhão de células, removendo mídia, e permitindo-lhes para se sentar em 1 ml de paraformaldeído a 4% durante 20 minutos. </li><li> Células girar novamente e trazer em 1 ml de PBS (células recontagem para confirmar 1 milhão / ml de concentração). Placa de 50.000, 40.000, 30.000, 20.000, 10.000, 5.000, 1.000 e 0 células em cada cavidade, em duplicado, e adicionar PBS para um volume total final de 50 uL em cada poço. </li></ol></li><li> Inferior ler a placa de cultura em um leitor fluorescente placa (excitação = 488 nm, emissão = 528 nm), dimensionamento altos poços (50.000 na curva padrão). Use a curva padrão para calcular quantas células de cancro do ovário aderiram ao organotypIC cultura em cada poço (Figura 4A-C). </li></ol> 4. Ensaio de Proliferação de 10 <ol><li> Após as células de cancro do ovário recuperar (ver passos 2.5 e 2.6, supra), sedimentar as células por centrifugação durante 3 minutos a 0,5 xg e depois dilui-se as células no volume apropriado de 1% de meio de FBS (DMEM com FBS a 1%, 1% MEM vitaminas [93 mg / L], MEM a 1% de aminoácidos não essenciais [81,4 mg / l], 1% de penicilina estreptomicina [Pen-Strep, 100 U / ml de penicilina e 100 ug / ml de estreptomicina]) para obter uma concentração de 4000 células / 150 ul. </li><li> Inverta a placa de 96 poços com as culturas organotípicas de HPMC / NOF para remover o meio gasto e adicionar 150 uL da suspensão de células do cancro em 1% de meio FBS a cada poço. Incubar a placa a 37 ° C em 5% de CO2 num ambiente humidif içado, durante 72 h. </li><li> Parte inferior da placa de cultura de ler num leitor de placas fluorescente, uma vez por dia (excitação = 488 nm, emissão = 528 nm) para avaliar a proliferação relativade células. Continue o ensaio durante 96 horas, mudando a mídia em 48 horas (Figura 5A-C). Tenha cuidado para não perturbar a cultura quando se muda de mídia (invertendo a placa é preferível). </li></ol> 5. Invasão Ensaio 8 <ol><li> Antes de chapeamento a cultura 3D, adicionar 7,5 g rato-cauda colágeno I em 200 ï¿½ de PBS a um 24 bem inserção placa de cultura (8 mm de tamanho de poro). Deixe a placa incubado O / N, em seguida, retire cuidadosamente a PBS com uma pipeta imediatamente antes do plaqueamento a cultura organotípico HPMC / NOF para as inserções revestidos com colagénio (etapa 2, acima). </li><li> Preparar as células de cancro do ovário como no passo 3.1. Ao prato as células de cancro do ovário para as culturas organotípicas sobre as inserções, utilize cuidadosamente uma pipeta para remover o excesso de mídia a partir do topo das inserções. Adicionar 100 ul da suspensão de células do cancro do ovário em meio isento de soro a cada poço. </li><li> Adicionar 750 ul de meio de crescimento completo na cavidade abaixo da inserção para induzir células no cancrovasion em culturas organotípicas. Incubar a placa a 37 ° C em 5% de CO2 num ambiente humidif içado, durante 24 horas. Nota: As células do cancro do ovário que invadem a cultura organotípica vai atravessar a inserção e permanecem no lado inferior da peça inserta. </li><li> Após 24 horas, segure a inserção com pinças e brevemente enxágüe-la num copo de PBS, em seguida, passar a inserção em um poço vazio. Esfregar a parte superior de cada inserto com dois lados de uma mecha de algodão para um total de 1 min. Rapidamente colocar o inserto para uma placa contendo 750 ul de paraformaldeido a 4% em cada poço. Permitir que cada inserção para sentar-se em paraformaldeído durante 10 minutos antes de transferir as inserções em poços cheios com 750 ï¿½ de PBS. </li><li> Ver as células invadidas (aderidas e fixas à parte inferior das inserções) com um microscópio com uma ampliação de 2,5 vezes. Quando todo o poço está dentro do campo de visão, para ajustar a ampliação de 10x e levar 5 imagens, um perto do centro e um em cada quadrante do poço, evitandoa tomada de imagens que são demasiado perto das bordas. Siga o mesmo padrão para cada poço. </li><li> Usar o programa do fabricante para contar o número de células em cada imagem para obter um número médio de células invadidas por campo em cada poço (Figura 6A-C) de acordo com o seu protocolo. </li></ol>

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	<li>Siegel, R., Ma, J., Zou, Z., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J. Clin. 65 (1), 5-29 (2015).</li><li>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=Ovarian+cancer+development+and+metastasis.">Ovarian cancer development and metastasis.</a> Am. J. Pathol. 177, 1053-1064 (2010).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nat. Med. 19, 1423-1437 (2013).</li><li>Correia, A. L., 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+tumor+microenvironment+is+a+dominant+force+in+multidrug+resistance.">The tumor microenvironment is a dominant force in multidrug resistance.</a> Drug Resist Updat. 15, 39-49 (2012).</li><li>Hanahan, D., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+Functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: Functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 21, 309-322 (2012).</li><li>Nieman, K. 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=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nat. Med. 17, 1498-1503 (2011).</li><li>Daya, D., McCaughy, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+the+peritoneum:+A+review+of+selected+topics.">Pathology of the peritoneum: A review of selected topics.</a> Semin. Diagn. Pathol. 8, 277-289 (1991).</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.">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.</a> Int. J. Cancer. 121, 1463-1472 (2007).</li><li>Kenny, H. A., Kaur, S., Coussens, L. M., 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=The+initial+steps+of+ovarian+cancer+cell+metastasis+are+mediated+by+MMP-2+cleavage+of+vitronectin+and+fibronectin.">The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin.</a> J. Clin. Invest. 118, 1367-1379 (2008).</li><li>Kenny, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesothelial+cells+promote+early+ovarian+cancer+metastasis+through+fibronectin+secretion.">Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.</a> J. Clin. Invest. 124, 4614-4628 (2014).</li><li>Strobel, T., Cannistra, 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=&#946;1-integrins+partly+mediate+binding+of+ovarian+cancer+cells+to+peritoneal+mesothelium+in+vitro.">&#946;1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro.</a> Gynecol. Oncol. 73, 362-367 (1999).</li><li>Strobel, T., Swanson, L., Cannistra, 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=In+vivo+inhibition+of+CD44+limits+intra-abdominal+spread+of+a+human+ovarian+cancer+xenograft+in+nude+mice:+A+novel+role+for+CD+44+in+the+process+of+peritoneal+implantation.">In vivo inhibition of CD44 limits intra-abdominal spread of a human ovarian cancer xenograft in nude mice: A novel role for CD 44 in the process of peritoneal implantation.</a> Cancer Res. 57, 1228-1232 (1997).</li><li>Iwanicki, 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=Ovarian+cancer+spheroids+use+myosin-generated+force+to+clear+the+mesothelium.">Ovarian cancer spheroids use myosin-generated force to clear the mesothelium.</a> Cancer Discov. 1, 144-157 (2011).</li><li>Lessan, K., Aguiar, D., Oegema, T. R., Siebenson, L., Skubitz, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+and+&#946;1+integrin+mediate+ovarian+carcinoma+cell+adhesion+to+peritoneal+mesothelial+cells.">CD44 and &#946;1 integrin mediate ovarian carcinoma cell adhesion to peritoneal mesothelial cells.</a> Am. J. Pathol. 154, 1525-1537 (1999).</li><li>Ahmed, N., Riley, C., Rice, G., Quinn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+integrin+receptors+for+fibronectin,+collagen+and+laminin+in+the+regulation+of+ovarian+carcinoma+functions+in+response+to+a+matrix+microenvironment.">Role of integrin receptors for fibronectin, collagen and laminin in the regulation of ovarian carcinoma functions in response to a matrix microenvironment.</a> Clin. Exp. Metastasis. 22, 391-402 (2005).</li><li>Kaur, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;3-integrin+expression+on+tumor+cells+inhibits+tumor+progression,+reduces+metastasis,+and+is+associated+with+a+favorable+prognosis+in+patients+with+ovarian+cancer.">&#946;3-integrin expression on tumor cells inhibits tumor progression, reduces metastasis, and is associated with a favorable prognosis in patients with ovarian cancer.</a> Am. J. Pathol. 175, 2184-2196 (2009).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <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+degradation+of+extracellular+matrix+by+human+ovarian+carcinoma+cells.">In vitro degradation of extracellular matrix by human ovarian carcinoma cells.</a> Clin. Exp. Metastasis. 5, 181-197 (1987).</li><li>Kanemoto, T., Martin, G. R., Hamilton, T. C., Fridman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synthetic+peptides+and+protease+inhibitors+on+the+interaction+of+a+human+ovarian+cancer+cell+line+(NIH:OVCAR-3)+with+a+reconstituted+basement+membrane+(matrigel).">Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (matrigel).</a> Invasion Metastasis. 11, 84-92 (1991).</li><li>Rieppi, 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=Mesothelial+cells+induce+the+motility+of+human+ovarian+carcinoma+cells.">Mesothelial cells induce the motility of human ovarian carcinoma cells.</a> Int. J. Cancer. 80, 303-307 (1999).</li><li>Barbolina, M. V., Adley, B. P., Ariztia, E. V., Liu, Y., Stack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+membrane+type+1+matrix+metalloproteinase+activity+in+ovarian+carcinoma+cells+via+collagen-induced+EGR1+expression.">Microenvironmental regulation of membrane type 1 matrix metalloproteinase activity in ovarian carcinoma cells via collagen-induced EGR1 expression.</a> J. Biol. Chem. 282, 4924-4931 (2007).</li><li>Burleson, K. 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=Ovarian+carcinoma+ascites+spheroids+adhere+to+extracellular+matrix+components+and+mesothelial+cell+monolayers.">Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.</a> Gynecol. Oncol. 93, 170-181 (2004).</li><li>Suzuki, 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=HMOCC-1,+a+human+monoclonal+antibody+that+inhibits+adhesion+of+ovarian+cancer+cells+to+human+mesothelial+cells.">HMOCC-1, a human monoclonal antibody that inhibits adhesion of ovarian cancer cells to human mesothelial cells.</a> Gynecol. Oncol. 95, 290-298 (2004).</li><li>Kishikawa, 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=Two+distinct+pattern+of+peritoneal+involvement+shown+by+in+vitro+and+in+vivo+ovarian+cancer+dissemination+models.">Two distinct pattern of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models.</a> Invas. Metast. 15, 11-21 (1995).</li><li>Casey, R. 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=Establishment+of+an+in+vitro+assay+to+measure+the+invasion+of+ovarian+carcinoma+cells+through+mesothelial+cell+monolayers.">Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.</a> Clin. Exp. Metastasis. 20, 343-356 (2003).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+human+ovarian+tumor+cells+with+human+mesothelial+cells+grown+on+extracellular+matrix.">Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix.</a> Exp. Cell Res. 160, 499-513 (1985).</li><li>White, E. A., Kenny, H. A., 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=Three-Dimensional+modeling+of+ovarian+cancer.">Three-Dimensional modeling of ovarian cancer.</a> Adv. Drug Deliv. Rev. 79-80, 184-192 (2014).</li><li>Kenny, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high+throughput+screening+using+a+primary+human+three-dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.</a> Nature Comm. , (2015).</li><li>Sawada, K., 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=c-Met+overexpression+is+a+prognostic+factor+in+ovarian+cancer+and+an+effective+target+for+inhibition+of+peritoneal+dissemination+and+invasion.">c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion.</a> Cancer Res. 67, 1670-1680 (2007).</li><li>Kenny, H. A., 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=MMP-2+functions+as+an+early+response+protein+in+ovarian+cancer+metastasis.">MMP-2 functions as an early response protein in ovarian cancer metastasis.</a> Cell cycle. 8, 683-688 (2009).</li><li>Sawada, K., 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=Loss+of+E-cadherin+promotes+ovarian+cancer+metastasis+via+alpha+5-integrin,+which+is+a+therapeutic+target.">Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target.</a> Cancer Res. 68, 2329-2339 (2008).</li><li>Mitra, A. K., 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=Ligand-independent+activation+of+c-Met+by+fibronectin+and+&#945;(5)&#946;(1)-integrin+regulates+ovarian+cancer+invasion+and+metastasis.">Ligand-independent activation of c-Met by fibronectin and &#945;(5)&#946;(1)-integrin regulates ovarian cancer invasion and metastasis.</a> Oncogene. 30, 1566-1576 (2011).</li></ol>

The authors have nothing to disclose.

Um modelo organotípico do microambiente peritoneal foi criada para avaliar a função (s) individual e coletiva de ambos os componentes celulares e inatas do microambiente na disseminação do cancro do ovário. Os protocolos específicos para o plaqueamento e personalizar a cultura organotípico 3D para investigar ovário adesão célula cancerosa, proliferação e invasão são fornecidos. Células humanas primárias omentais mesoteliais e fibroblastos foram isoladas de pacientes e usado em uma passagem antecipada pa...

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2. 
The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16. 
Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

<table>
	<tbody>
		<tr>
			<td>1. Isolation and culture of primary cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>SH3001304</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-edged razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm culture dishes</td>
			<td>BD Biosciences</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass flask</td>
			<td>?</td>
			<td>?</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000044_3616914956</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with L-Glutamine</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning</td>
			<td>25-020-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker&#160;</td>
			<td>Thermo-Fisher</td>
			<td>MaxQ 4450</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo-Fisher</td>
			<td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA, 1x (0.25%)</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Worthington Biochemical</td>
			<td>LS002592</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Flasks</td>
			<td>BD Biosciences</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>T-175 Flasks</td>
			<td>BD Biosciences</td>
			<td>353112</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Rainin</td>
			<td>P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Corning</td>
			<td>Filtered tips P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>2. Plating 3D culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Invitrogen</td>
			<td>Countess</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10313</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate, clear bottom, black</td>
			<td>BD Biosciences</td>
			<td>353219</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>3. Adhesion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipet</td>
			<td>Eppendorf</td>
			<td>Xplorer 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Molecular Devices</td>
			<td>Minimax</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>5. Invasion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Inserts (8um, 24-well)</td>
			<td>BD Biosciences</td>
			<td>353097</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Q-tips</td>
			<td>cotton swabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200m</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Profiler</td>
			<td>public domain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>BD Biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>6. Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;V&#946;3 Antibody, clone LM609</td>
			<td>EMD Millipore</td>
			<td>MAB1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 1&#160;</td>
			<td>Oncosynergy</td>
			<td>OS2966</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha 5 [CD49e]</td>
			<td>ID Pharmingen</td>
			<td>555615</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 4 [CD104]</td>
			<td>EMD Millipore</td>
			<td>MAB 2058</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling the early steps of ovarian cancer dissemination in an organotypic culture of the human peritoneal cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

A cultura organotípica foi montada misturando primeiro fibroblastos humanos primários com colagénio do tipo I e, em seguida, sobrepondo esta cultura com 5 vezes o número de células de mesotélio. A cultura foi incubada durante, pelo menos, 18 horas antes das células de cancro do ovário foram adicionados a estudar a adesão, invasão ou proliferação. Cada ensaio foi repetido com múltiplos (n = 3-5) culturas em 3D obtidas de diferentes pacientes e numerosos poços foram testados em cada condição para a adesão...

Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modelando os passos iniciais do cancro do ovário Divulgação em uma cultura organotípicas do Peritoneal Cavity Humano

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

modeling-early-steps-ovarian-cancer-dissemination-an-organotypic

Research

JoVE Journal

Medicine

26.5K visualizações.  University of Chicago. Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation. 

Vídeo: Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelagem e imagem 3-Dimensional Invasion cela coletiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Medicina

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Pele da biópsia Explant Cultura para Derivação de primárias fibroblastos humanos

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ensaio in vitro de Apuramento mesotelial que os modelos dos primeiros passos da metástase do câncer de ovário

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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

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

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

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

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

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

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In Vivo e Ex Vivo Abordagens para o Estudo do cancro do ovário metastático Colonização de Estruturas Láctea natural em Peritoneal adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

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 ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

A quantificação da Intra-peritoneal Ovarian Cancer Metástase

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Citologia tubária do tubo de Falópio como uma ferramenta promissora para a detecção precoce do câncer de ovário

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Modelando os passos iniciais do cancro do ovário Divulgação em uma cultura organotípicas do Peritoneal Cavity Humano

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