Os autores agradecem Chalid Ghadban pela sua excelente assistência técnica, Andreas Wodarz e Stephan Heermann para os seus conselhos técnicos sobre confocal e microscopia de lapso de tempo. Não há conflito de interesses por qualquer dos autores. Este trabalho é financiado pelo Conselho de Pesquisa Alemã (DFG) no Projeto 2 de Forschergruppe 942 (FOR942 BI 703/3-1), pelos Dres. Bayer-Stiftung (Baden Württembergischer Krebspreis, Alemanha) e pelo Programa de Pesquisa da Faculdade de Medicina, Georg-August-Universidade de Göttingen, na Alemanha.

Este novo modelo é uma adaptação de uma abordagem fatia do cérebro do hipocampo organotípica publicado anteriormente 9-12. As modificações e adições foram introduzidas para otimizar as interações de tecido de câncer de células do cérebro e para garantir a reprodutibilidade. O estudo foi analisado e aprovado pelo comitê de ética local. Os animais foram tratados com cuidado de acordo com as diretrizes para cuidados com os animais na Medicina da Universidade de Göttingen. A co-cultura organotípicas fatia do cérebro pode ser subdividida em duas etapas. O primeiro passo é a preparação da fatia do cérebro organotípica. Passo dois compreende a preparação de células de tumor e de deposição no modelo de co-cultura. 1. Organotípicas Cérebro Slice <ol><li> Preparar o meio de dissecção consistindo em meio essencial mínimo (MEM), suplementado com 0,2 mM de glutamina, 100 U / ml de penicilina, 100 mg / ml de estreptomicina, e 4,5 mg / ml de glicose. </li><li> Decapitar os ratos a partir de toda a tensão do mouse entre o dia pós-natalseis e oito (P6-8). </li><li> Remover o cérebro rapidamente do crânio, sob condições assépticas, e transferi-lo para o meio de dissecção gelado. </li><li> Remover o pólo frontal e o cerebelo a partir da secção de todo o cérebro. </li><li> Fix e estabilizar o cérebro em um palco com cola crio e 5% de agarose. </li><li> Corte as secções cerebrais horizontalmente para uma espessura de 350 um, utilizando um vibratome. </li><li> Recolhe 4-6 inteiros fatias de cérebro de um único cérebro do rato, dependendo da espécie e idade. </li><li> Preparar o meio de cultura consistindo de 50% de MEM, solução de sal equilibrado de 25% de Hanks (HBSS), 25% de soro normal de cavalo (NHS), 0,2 mM de glutamina, 100 U / ml de penicilina, 100 mg / ml de estreptomicina (Sigma, Munique, Alemanha), e glucose 4,5 mg / mL. </li><li> Coloque cada fatia de cérebro organotípica num mM policarbonato transwell insert membrana 0.4 numa placa de seis poços com 1 ml de meio de cultura no poço inferior. </li><li> Cultura organotípicas fatias do cérebro durante a noite em um humidified atmosfera com 5% de CO 2, a 37 ° C incubadora. </li></ol> 2. O Modelo Slice Coculture <ol><li> Encaixe 10 5 de tumor transfectadas com GFP ou outras células (por exemplo, células MCF-7-GFP) em 20 mL de matriz de gel, consistindo em 15% de meio RPMI e 85% de gel de ECM. </li><li> Coloque a mistura de matriz de células MCF-7-gel em um espaçador metálico estéril (3,8 mm de diâmetro) directamente adjacente à região cortical do cérebro fatia organotípicas e incubar durante 2 horas. </li><li> Remover o espaçador e permitir o esferóide tumor 3D para co-cultura com a fatia organotípicas de 24-96 horas. </li><li> Altere o meio de cultura a cada dois dias. </li></ol> 3. Imunofluorescência Coloração de astrócitos e microglia no organotípicas cerebral Fatia Coculture <ol><li> Fixe a co-cultura organotípicas fatia do cérebro com paraformaldeído a 4% para 8 horas a 4 ° C. </li><li> Lava-se a co-cultura de fatia com PBST (PBS com 0,5% de Triton X-100) durante 5 min. </li><li> Bloquear as splos com soro normal de cabra em PBST (01:20) à temperatura ambiente durante 1 hora. </li><li> Manchar os astrócitos incubando a co-cultura de fatia de cérebro com fibrilar glial anti-anticorpo monoclonal de proteína ácida (GFAP, 1:200 em PBST) durante 36 horas a 4 ° C seguido por anticorpo de cabra anti-murganho-TRITC (1:100 em PBST) coloração durante 1 hora à temperatura ambiente. </li><li> Lavar as amostras com PBST três vezes, durante 5 min. </li><li> Corar as células microgliais com ILB 4-Alexa Fluor 647 (1:100 em PBST) durante 1 hora à temperatura ambiente. </li><li> Contracoloração cérebro fatia de co-cultura com DAPI (1:1000) durante 3 minutos à temperatura ambiente. </li><li> Monte e lamela cérebro fatia co-cultura com DAKO fluorescente meio de montagem. </li><li> Avaliar o grau de invasão do tumor com base no seguinte sistema de pontuação: 0 = nenhuma das células; + &lt;1/3; + + = 1/3 - 2/3; + + + ≥ 2/3 das células invadidas (primeiro medir o comprimento da seção de contato entre o plugue tumor e fatia, em seguida medir o fraccionamenton de contacto detectável pelas células invasoras). </li></ol> 4. Imagens ao vivo da interação entre a glia e células tumorais <ol><li> Realize o experimento em uma Leica invertido DMI 6000B microscópio 10X lente de ampliação e uma Leica DFC câmera CCD 350 FX. </li></ol>

<ol>
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Não temos nada a divulgar.

Investigações histológicas anteriores de metástase cerebral demonstrou mudanças rápidas e drásticas das células gliais residentes, especialmente de astrócitos e microglia 13. Para estudar essas mudanças e interações com as células de carcinoma, este sistema de co-cultura romance é bem adequado. Outros campos de pesquisa já tem experiência de longa data com organotípicas fatias do cérebro do hipocampo. Uma vantagem é que as culturas organotípicas do hipocampo do cérebro de fatia são viáveis ​​e eficazmente conservados durante dias ou semanas, tornando-o adequado para as experiências de longo prazo. Desde a introdução de Stoppini do sistema de fatia de hipocampo organotípicas em 1991, tem sido amplamente utilizada, por exemplo, na pesquisa de doenças degenerativas. Assim, este sistema de co-cultura de inovar representa uma modificação de um método bem estabelecido com uma variedade de aplicações em biologia tumoral 9,11. A modificação nos ofereceu um modelo reproduzível para avaliar o grau de invasão tumoral afterwards e culturas cultivadas pelo método de interface são idealmente adequado para experiências que requerem uma estrutura tridimensional. Várias técnicas têm sido utilizadas para co-cultura de fatias de hipocampo organotípicas com outras células. Estes incluem um sistema indirecto entre as células de macrófagos e a fatia de cérebro organotípica 14, e um co-cultura directa de duas fatias diferentes da região do hipocampo 15. Agregados glioma também foram co-cultivadas com fatias de cérebro 16. Estes modelos podem ser utilizados para analisar eventos celulares e moleculares nas fatias do cérebro, mas não permitem o contato direto, fisiológica entre as células tumorais, microglia e parênquima cerebral. Além disso, este método permite a observação da microglia, sem contaminação de osso derivadas de medula periféricas monócitos / macrófagos. A utilização do modelo de ratinho transgénico CCR2 e CX3CR1 é uma melhoria fundamental devido ao facto de que é difícil de distinguir da microglia residentes invadam monócitoscom base em suas propriedades semelhantes 17,18,19. Embora a injecção intracerebral de células de carcinoma permite investigar a progressão do tumor, não se pode dizer tanto quanto a saber se as células semelhantes a macrófagos rodeadas originam a população residente microglia do cérebro ou da medula óssea derivadas de monócitos / macrófagos periféricos 17. A equipe de Kettenmann introduziu um modelo fatia do cérebro organotípica que envolveu a inoculação de células de glioma em fatias de cérebro com um micromanipulador 20. No entanto, gliomas malignos primários diferem em muitos aspectos de carcinomas metastáticos. Primeiro, gliomas malignos são de origem mesenquimal, não metástase fora do sistema nervoso, e migrar / invadir células individuais como sem fronteira entre tumor e tecido cerebral. Por outro lado, o crescimento infiltrativa é uma característica típica patológico, e tais carcinomas geralmente migram / invadir como coortes. Em segundo lugar, os carcinomas frequentemente tentam reconstruir estruturas epiteliais no cérebro, ao passo que as células gliaistente separar o tumor a partir do tecido do cérebro por um (pseudo) da cápsula. Considerando-se características biológicas e morfológicas, glioma maligno e metástase dos carcinomas não são realmente comparáveis. Por estas razões, é modificado e desenvolvemos um sistema de co-cultura em que não co-cultura de injectar mas um tampão de células de carcinoma adjacente à fatia cerebral. Além disso, observou-se acumulação de microglia e astrócitos, na fronteira do bujão do tumor, o que significa que a microglia inserir a ficha do tumor e pode ser facilmente detectada por microscopia de campo brilhante e confirmados posteriormente por meio de microscopia confocal. As células cancerosas invadir na fatia do cérebro, que é comparável à do real em situação vivo e uma observação feita por Baumert e seus colegas, que encontraram uma zona de infiltração em 63% dos casos de autópsia com metástases cerebrais 21. Por causa da falta de perfusão de sangue, existem apenas residente macrófagos / microglia nesta cultura. Além disso, por causa da mcélulas Issing T e, portanto, ausentes alo-reactividade, as células de carcinoma humano pode ser utilizado para a co-cultura, mesmo com fatias de cérebro de ratos imunocompetentes como em ratos (NMRI, B6, ou Wistar). Tal pode servir como uma alternativa para o modelo do ratinho nu. Desde 4-5 fatias pode ser obtido a partir de cada ratinho, são necessários números significativamente reduzidos de animais, em comparação com os modelos de injecção existentes. Além disso, os animais não sofrem durante um longo período de doença metastática e não submetidos a procedimentos operatórios repetitivamente 22. Com este sistema de co-cultura, que demonstraram a activação da microglia por células cancerosas e a capacidade de promover a invasão de células de cancro. Além disso, esta é a primeira vez, para o nosso conhecimento, microglia foram encontrados para transportar activamente células de carcinoma 23. Apesar de todas estas vantagens, o sistema de co-cultura tem, de fato, também limitações. É ainda um in vitromodelar faltando os passos de metástase antes da colonização. Por causa da falta de perfusão, não é possível estudar o extravasamento. Assim, o caminho alternativo para estudar o extravasamento é usar o modelo in vivo de injecção ou o sistema de câmara de Boyden modificada com a matriz extracelular, as HUVEC e astrócitos para imitar a barreira sangue-cérebro 22,24. O método de co-cultura fatia do cérebro é ainda um modelo confiável e reprodutível com muitas vantagens e um potencial para uma grande variedade de aplicações, tais como a análise da colonização, em particular, com um foco sobre o papel das células residentes. A combinação com outras técnicas estabelecidas (como a imuno-histoquímica, microscopia confocal e microscopia de lapso de tempo) apoia a investigação de interações diretas célula-célula. É uma alternativa e complementação de outras técnicas fáceis e oferece acesso à investigação das pistas e efeitos como impostas pelo microambiente metastático.

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco, Darmstadt,Germany</td>
			<td>24020</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco, Darmstadt , Germany</td>
			<td>32360</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>PAA Laboratories Inc., C&#246;lbe, Germany</td>
			<td>E15-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (PBS)</td>
			<td>Pan Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal horse serum (NHS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>10091148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>B. Braum, Melsungen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine-Penicillin-Streptomycin solution</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica SM 2000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane</td>
			<td>BD Falcon, Heidelberg,Germany</td>
			<td>353090</td>
			<td>transwell membrane insert (0.4 &#956;m pore size)</td>
		</tr>
		<tr>
			<td>ECM gel</td>
			<td>Trevigen, R&#38;D, Wiesbaden-Nordenstadt, Germany</td>
			<td>3432-005-01</td>
			<td>Basement membrane extract</td>
		</tr>
		<tr>
			<td>Metallic spacer</td>
			<td>Kig GmbG, Kirkel, Germany</td>
			<td>DIN 433</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-lapse microscope and camera</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>DMI 6000B microscope and a DFC 350 FX CCD camera</td>
			<td></td>
		</tr>
		<tr>
			<td>Anatomy microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>Stemi SV11</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1.04005.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG, F(ab&#39;)2- TRITC</td>
			<td>Santa Cruz, Heidelberg, Germany</td>
			<td>SC3796</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 647 conjugate</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>132450</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6&#39;-diamidino-2-phenylindole dihyfrochloride)</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent mounting medium</td>
			<td>DAKO, Glostrup, Denmark</td>
			<td>S3023</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture system with an organotypic brain slice and 3d spheroid of carcinoma cells

Os pacientes com metástase cerebral de carcinomas têm um prognóstico pobre. No entanto, o processo no local metastático tem sido pouco investigada, em particular, o papel das células estromais (residente). Estudos em carcinomas primários demonstrar a influência do microambiente em metástase, mesmo no prognóstico 1,2. Especialmente os macrófagos associados ao tumor (TAM) migração apoio, invasão e proliferação 3. Curiosamente, os principais locais alvo de metástase possuem macrófagos de tecidos específicos, tais como células de Kupffer do fígado ou da microglia no sistema nervoso central. Além disso, os locais metastáticos também possuem outras células de tecidos específicos, tais como os astrócitos. Recentemente, os astrócitos foram demonstradas para promover a proliferação e persistência de células de câncer 4,5. Portanto, as funções destes tipos de células específicas de tecidos parece ser muito importante no processo de metástase cerebral 6,7. Apesar dessas observações,no entanto, até agora não há adequado modelo in vivo / in vitro disponíveis para visualizar diretamente as reações gliais durante a formação de metástase cerebral, em particular por meio de microscopia de campo claro. Recentes in vivo de imagens ao vivo de células de carcinoma demonstrou seu comportamento colonização cerebral 8. No entanto, este método é muito trabalhoso, dispendioso e tecnicamente complexa. Além disso, estes tipos de experimentos com animais são restritos a pequenas séries e vêm com um esforço substancial para os animais (pela implantação da placa de vidro, a injeção de células tumorais, a anestesia repetitiva e fixação de longa duração). Além disso, a imagem in vivo é, portanto, muito limitado para a visualização de células de carcinoma, ao passo que as interacções com células residentes ainda não foram ilustradas. Finalmente, as investigações de células de carcinoma humanos dentro animais imunocompetentes são impossíveis 8. Por estas razões, foi estabelecido um sistema de co-cultura consipicada de uma fatia de cérebro do rato organotípica e células epiteliais embutido em matrigel (esfera celular 3D). As esferas de carcinoma de células em 3D foram colocados ao lado da borda fatia do cérebro, a fim de investigar a invasão do tecido cerebral vizinho. Isso nos permite visualizar as alterações morfológicas e interações entre as células da glia e células de carcinoma de fluorescência e até mesmo por microscopia de campo claro. Após a experiência de co-cultura, o tecido cerebral, ou os esferóides de células em 3D podem ser recolhidos e utilizados para outras análises moleculares (por exemplo, de qRT-PCR, IHC, ou immunoblot), bem como para investigações através de microscopia confocal. Este método pode ser aplicado para monitorizar os eventos dentro de um tecido cerebral vivo para dias sem efeitos deletérios para as fatias do cérebro. O modelo também permite uma supressão selectiva e substituição das células residentes por células de um tecido dador para determinar o impacto distinto de um dado genótipo. Finalmente, o modelo de co-cultura é uma alternativa viávelpara in vivo se aproxima ao testar manipulações farmacológicas alvejados.

Em primeiro lugar, todas as células de carcinoma utilizados (humano: MCF-7 e murino: 410.4) invadiram a fatia do cérebro organotípica mouse. A invasão foi, por conseguinte, da espécie independentes (Figura 1A), que indica que uma vasta gama de aplicações de uma forma independente de espécies. Além disso, a microglia, bem como astrócitos acumuladas na interface conforme anteriormente descrito, in vivo e em amostras de paciente 13. Imaging Time-lapse durante um período prolongado de tempo, não só revelou a viabilidade da fatia, mas também ofereceu uma boa plataforma para observar as interações celulares. Através de experiências de lapso de tempo, as células microgliais foram gravadas para entrar no 3D esfera celular enquanto, por sua vez, as células cancerosas também invadiu a fatia do cérebro (Figuras 1B1-B2). Além disso, mostrámos previamente a capacidade de microglia para transportar células de carcinoma por um mecanismo ainda enigmática, assim, ajudar na invasão carcinoma. Usando técnicas de imunofluorescência, nósdescrito co-localizações de células tumorais e de células do estroma (por exemplo, microglia e astrócitos), tanto no tecido cerebral e no bujão de células de tumor (Figuras 1C-D), o que sugere uma interacção estreita entre estas células, durante o processo de invasão. Foram obtidos resultados comparáveis ​​quando fatias do cérebro do rato foram co-cultivados quer com humanos (Figuras 1C1-C3) ou murino (Figuras 1D1-D3) de células de carcinoma - que mostra a grande gama de aplicações para o estudo de células de diferentes espécies, e estirpes de genótipos. <img alt="Figura 1" src="/files/ftp_upload/50881/50881fig1.jpg" fo:content-width="4.75in" fo:src="/files/ftp_upload/50881/50881fig1highres.jpg" /> Figura 1. Cérebro modelo de co-cultura fatia com um esferóide 3D ​​de rato e células de carcinoma humanos. A) Quantificação da invasão de células de câncer em todo organotípicas coculturas fatia do cérebro com um rato de câncer de mamalinha celular de 410,4 ou uma linha celular de cancro da mama humano MCF-7. Os dados representam a percentagem de o grau de invasão de células na fatia de cérebro em cada grupo com n ≥ 38. Não houve diferença significativa entre os dois grupos (teste de Kruskal-Wallis). B) imagens de lapso de tempo de todo organotípicas coculturas fatia do cérebro com células MCF-7-GFP e imagens representativas de dia2 (B1) e dia 5 (B2) foram mostrado. imagens de microscopia CD) confocal mostrou coortes de carcinomas, astrócitos e microglia. Coloração dupla de astrócitos (anti-GFAP-TRITC, vermelho) e microglia (ILB4-Alexa Fluor 647, violeta) de toda a co-cultura com a grossa fatia organotípicas cérebro 350 mM ea ficha de células tumorais transfectadas-GFP (verde): 3D- MCF-7-GFP (C1-C3) e 3D-410.4-GFP (D1-D3). Traços brancos mostraram a borda da fatia de cérebro e da frente da invasão tumoral. As barras de escala representam 50 mm. Microglia, astrócitos umª tumores colocalizations (C1 e D1), astrócitos-tumores colocalizations (C2 e D2) e colocalizations microglia-tumorais (C3 e D3) na co-cultura fatia. <a href="https://www.jove.com/files/ftp_upload/50881/50881fig1highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a>

Watch this Scientific Journal Video about Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells at JoVE.com

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

A co-cultura de fatia cerebral organotípicas com células de carcinoma permite visualizar as alterações morfológicas por fluorescência, bem como o campo brilhante (vídeo) microscopia durante o processo de invasão de células de carcinoma de tecido cerebral. Este modelo de sistema também permite abordagens taxas de célula e de reabastecimento e oferece uma ampla variedade de manipulações e análises.

Sistema de co-cultura com uma fatia organotípicas Cérebro e esferóide 3D de carcinomas de células

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

coculture-system-with-an-organotypic-brain-slice-3d-spheroid

Research

JoVE Journal

Medicine

20.5K visualizações.  University of Göttingen.  A co-cultura de fatia cerebral organotípicas com células de carcinoma permite visualizar as alterações morfológicas por fluorescência, bem como o campo brilhante (vídeo) microscopia durante o processo de invasão de células de carcinoma de tecido cerebral. Este modelo de sistema também permite abordagens taxas de célula e de reabastecimento e oferece uma ampla variedade de manipulações e análises. 

Vídeo: Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

Transplante mamária de células estromais e células de carcinoma em camundongos C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Medicina

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Um modelo de camundongo ortotópico de Carcinoma anaplásico da tiróide

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Avaliação do cancro do ovário esferóide Apego e invasão de células mesoteliais em Tempo real

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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 ...

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 ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Um tumor cerebral / Slice organotípicas Co-cultura Sistema de Estudos de Tumor Microenvironment e alvejado droga Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

A Cancer Cell Spheroid Ensaio para avaliar a invasão em um ambiente 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Um modelo de cultura de fatia organotipica de glioblastoma humano para estudo de migração de células tumorais e efeitos específicos de pacientes de drogas anti-invasivas

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Symmetric Bihemispheric Postmortem Cérebro de corte para o Estudo de Saúde e Condições Patológicas cerebral em humanos

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.