Este trabalho foi financiado pelo National Institutes of Health (NIMHD-G12MD007581), através do RCMI-centro de saúde ambiental da Universidade de estado de Jackson e o departamento E.U. de defesa, prêmio Idea 11-1-0205.

Nota: Este protocolo é descrito no McArdle et al . 11. um cronograma é fornecido na Figura 1.
1. preparação da placa de cultura
<ol><li>Lave o prato de 35mm cultura contendo um vidro de 10 mm-fundo bem com 1 mL de ácido clorídrico de 1 M (HCl) por 15 min abaixar a hidrofobicidade do vidro-fundo.</li>
	<li>Lave a placa de cultura duas vezes com 1 mL de solução salina tampão de fosfato (PBS) para se livrar de todo o HCl.</li>
	<li>Lave a placa de cultura duas vezes com 1 mL de etanol a 70%.</li>
	<li>Lave a placa de cultura com 1 mL de meio de cultura celular específica (aqui, uso α-MEM suplementado com 10% de calor inativada FBS e 1% não aminoácidos essenciais). Nota: A placa de cultura tratada contendo meio de cultura pode ser armazenada a incubadora de CO2 com um humidificador a 37 ° C até o dia seguinte.</li>
	<li>Crescem as células (MSCs e cultura co MCF-7) em vidro-fundo tratado bem do prato 35mm cultura a confluência de 100%. Co cultura 35.000 células MSC e 75.000 de células MCF7 por 24 h.</li>
</ol>2. preparação do gel de colágeno
<ol><li>Armazene as pontas de micropipeta usadas para pipetar o gel de colágeno no congelador para evitar a polimerização do colágeno em cima do contato. Nota: Este é o passo mais crítico.</li>
	<li>Determinar um volume de colágeno de cauda de rato eu deve ser usado com base na concentração do estoque de colágeno. Prepare-se 100 µ l de gel de colágeno para este ensaio. Nota: Alta concentração de colágeno de cauda de rato que armazeno funciona melhor. Determine o volume de 10 x meio modificado águia de Dulbecco a ser usado para diluir o colágeno em conformidade.</li>
	<li>Combine, colágeno de cauda de rato gelo I (3.8 µ g/mL caldo), médio modificado águia de 10 x Dulbecco e 1 x celular cultura suplementado com 2% de soro fetal bovino e o apropriado quimiotático para uma concentração final de colágeno de 2,4 mg/mL.</li>
	<li>Adicione à mistura 4.5% da solução de bicarbonato de sódio para neutralizar o gel de colágeno. Nota: O volume de solução de bicarbonato de sódio adicionado pode variar de acordo com o pH exato dos outros reagentes. É melhor para testar a mistura com tiras de pH para garantir solução neutra, ou usar um indicador de pH do meio de cultura.</li>
	<li>Disponha as células dentro do vidro-fundo bem do prato cultura 80 µ l da mistura de colágeno.</li>
	<li>Permitir que o gel de colágeno polimerizar durante 30 min numa incubadora de CO2 com um humidificador a 37 ° C.</li>
	<li>Cubra o gel de colágeno com 2 mL de meio de celular com soro reduzido (2%) e incube as celulas a 37 ° C, uma incubadora de CO2 com um umidificador para 24, 48 ou 72 h. Atenção: A placa deve ser manipulada com cuidado para evitar que o gel de colágeno desanexação da parte inferior do vidro.</li>
</ol>3. exemplo de preparação de colágeno gel usando um estoque de colágeno de cauda de rato de 3.8 µ g /mL
<ol><li>No gelo, combine 114.5 µ l de colágeno de cauda de rato, 16,6 µ l de 10 x DMEM e 33,1 µ l de meio de cultura contendo o quimiotático.</li>
	<li>Neutralize a mistura de colágeno com 14,0 µ l de bicarbonato de sódio.</li>
	<li>Continue como na etapa 2.5.</li>
</ol>4. imagem latente das células
<ol><li>Remova cuidadosamente a mídia do prato.</li>
	<li>Lave suavemente o gel com 1 mL de PBS.</li>
	<li>Fixe as células com 1 mL de paraformaldeído 4% por 15 min.</li>
	<li>Lave as células duas vezes com 1 mL de PBS.</li>
	<li>Manche as células com solução DAPI (100 ng/mL) por 20 min em temperatura ambiente.</li>
	<li>As células usando um microscópio confocal em locais diferentes e tomar 400 µm z-pilha imagens de cada local em 16 µm passos da imagem. O microscópio usado tem um disco de unidade que permite que a imagem latente confocal usando uma luz branca, fonte de excitação do arco de digitalização. Use um 20 X lente utilizada com abertura numérica de 0,75.</li>
	<li>Imagens de reconstituir.<ol>
<li>Abrir imagens para um determinado gel (aproximadamente 25 imagens) e criar uma pilha de imagem clicando em imagem → pilhas → imagens de pilha. A primeira imagem que correspondia à parte inferior do gel (z = 0 µm), a segunda imagem correspondeu à primeira etapa na direção z (z = 16 µm), a terceira imagem correspondia à segunda etapa na direção z (z = 32 µm). Avaliar cada núcleo em cada profundidade de imagem e designar a profundidade na qual o núcleo foi em foco mais nítido como a profundidade de invasão para essa célula específica.</li>
	</ol>
</li>
</ol>

<ol><li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li>
<li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+architecture%3A+the+ultimate+regulator+of+breast+epithelial+function%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li>
<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumour-cell+invasion+and+migration%3A+diversity+and+escape+mechanisms%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li>
<li>Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+cell+migration+assay+using+scratch+wound+healing%2C+a+comparison+of+image-based+readout+methods%5BTitle%5D)+AND+%22BMC+Biotechnol%22%5BJournal%5D)">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21(2004).</li>
<li>Wolf, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compensation+mechanism+in+tumor+cell+migration%3A+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li>
<li>Sahai, E., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho%2FROCK+signalling+and+extracellular+proteolysis%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li>
<li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198(2008).</li>
<li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays%5BTitle%5D)+AND+%22Int+J+Exp+Pathol%22%5BJournal%5D)">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li>
<li>Poujade, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li>
<li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis-induced+cancer+cell+fusion%3A+a+mechanism+of+breast+cancer+metastasis%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li>
<li>McArdle, T. J., Ogle, B. M., Noubissi, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types%5BTitle%5D)+AND+%22J+Cancer%22%5BJournal%5D)">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li>
<li>Noubissi, F. K., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+Cell+Fusion%3A+Mechanisms+Slowly+Unravel%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Cancer Cell Fusion: Mechanisms Slowly Unravel.</a> Int J Mol Sci. 17, (2016).</li>
<li>Meyvantsson, I., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+culture+models+in+microfluidic+systems%5BTitle%5D)+AND+%22Annu+Rev+Anal+Chem%22%5BJournal%5D)">Cell culture models in microfluidic systems.</a> Annu Rev Anal Chem. 1, 423-449 (2008).</li>
<li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+cell+migration+in+three+dimensions%5BTitle%5D)+AND+%22Methods+In+Enzymol%22%5BJournal%5D)">Tumor cell migration in three dimensions.</a> Methods In Enzymol. 406, 625-643 (2006).</li>
<li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+memory+and+dosing+influence+stem+cell+fate%5BTitle%5D)+AND+%22Nat+Materials%22%5BJournal%5D)">Mechanical memory and dosing influence stem cell fate.</a> Nat Materials. 13, 645-652 (2014).</li>
</ol>

Os autores declararam que nenhum interesse de concorrente existe.

Aqui, descrevemos um ensaio simples invasão vertical invertida que é conveniente para uma variedade de tipos de células, incluindo células que são raras e/ou dependente seu microambiente. Neste ensaio 3D invasão, as células permanecem em seu microambiente original em são cobertas pelo gel de colágeno contendo um quimiotático. As células então migram e invadem em colágeno. Neste ensaio, as células podem ser manchadas e facilmente monitoradas dentro do gel. A simplicidade e a reprodutibilidade deste teste o t...

Motilidade celular é um processo fisiológico e de desenvolvimento normal das células. No entanto, as mudanças no padrão e a capacidade das células para migrar e invadir estão associadas com condições patológicas, incluindo doenças inflamatórias e câncer metástase1,2,3. Metástase é, sem dúvida, o aspecto mais mal compreendido em câncer. Para metástase, as células cancerosas deve degradar a matriz extracelular (ECM), invadir o tecido local e intravasate. Uma vez em circulação, as células cancerosas vão divulgar o site distante, extravasate e formar tumores secundários. Portanto, a capacidade das células para degradar o ECM, para migrar e invadir está relacionada ao seu potencial metastático. Diferentes abordagens têm sido desenvolvidos para analisar e quantificar os recursos de migração e invasão de células. As abordagens mais utilizadas incluem a ferida cura ensaio, microscopia de lapso de tempo e o ensaio de câmara Boyden. A ferida cura ensaio4 e tempo lapso microscopia são ensaios simples e convenientes que dariam preliminar insight sobre migração celular. No entanto, ambos são ensaios 2D que não refletem o ambiente 3D, no qual existem células em vivo. Estudos têm mostrado que as células respondem de forma diferente para um ambiente 3D em comparação com um 2D5,6. Além disso, ensaios de cura ferida são difíceis de reproduzir e produzir resultados quantitativos7,8,9. O ensaio da zona de exclusão de célula, que é uma modificação 3D da ferida do ensaio de cura, é um ensaio mais fisiologicamente relevante, mas não é apropriado para células baixas no número como células híbridas resultantes de célula fusão eventos10,11 .
O ensaio de câmara Boyden é um ensaio 3-dimensional que fornece o microambiente relevantes para estudos celulares. No entanto, requer células para ser removido de seu microambiente original e depositados na câmara superior do sistema de ensaio de Boyden, migrar e invadir para baixo em direção a médio contendo quimiotático. Esta abordagem 3D não é apropriada ao analisar a capacidade de migração e invasão de células vulneráveis ao seu microambiente e/ou limitada em número de11,de10,12. É uma abordagem mais recente para analisar a invasão e a migração celular gradiente câmara microfluidic ensaio13. Embora adequados e fiáveis em estudos de migração e invasão, este ensaio é mais caro e pode ser tecnicamente desafiador para um laboratório típico. Descrevemos aqui um ensaio de invasão vertical invertida simples que é compatível com muitos tipos de células, incluindo células sensíveis ao seu microambiente e/ou limitados em número. Este ensaio foi adaptado a partir do protocolo proposto pela Hooper et al 14. neste ensaio, as células permanecem na sua original microambiente e sua migração e invasão é monitorado como eles se movem para cima no gel de colágeno contendo um quimiotático11. Este ensaio é reprodutível e foi otimizado e validado no prato 35mm cultura. Isso pode ser adaptado às condições de ensaio diferentes incluindo tamanhos de cultura de células diferentes, diferentes matrizes ou força de adesão e diferentes quimioatraentes. Este ensaio poderia servir como uma plataforma em vitro para triagem inicial no processo de descoberta de drogas.

<table><tbody><tr><td>MatTek P35G-1.5-10C</td><td>MatTek Corporation, Ashland, MA</td><td>P35G-1.5-10-C</td><td>mattek glass bottom dishes</td></tr><tr><td>Rat tail collagen I</td><td>Corning, Corning, NY, USA</td><td>354236</td><td>Collagen gel</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>H1758</td><td>HCl</td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>10010023</td><td>PBS</td></tr><tr><td>10X Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>D2429</td><td>10X DMEM</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Confocal Fluorescent microscope</td><td>Olympus America, Center Valley, PA, USA</td><td>Olympus IX81</td><td>Confocal&amp;nbsp; microscope</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td><td>SH30070.03</td><td>FBS</td></tr><tr><td>Ti:Sapphire multi-photon laser scanning microscope</td><td>Prarie Technologies, Sioux Falls, SD, USA</td><td>40x NA 0.8 objective</td><td>MPLSM</td></tr><tr><td>Imaris software</td><td>Bitplane Inc. Zurich, CH</td><td>Imaris 7.5.2</td><td>Imaris software</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>D9542</td><td>DAPI</td></tr><tr><td>Paraformaldehyde</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>50980487</td><td>PFA</td></tr><tr><td>&amp;alpha;-minimum essential medium</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>M0894</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC; Manassas, VA, USA</td><td>HBT-22</td><td></td></tr><tr><td>MSC</td><td>Trivedi and Hematti, 2007</td><td></td><td></td></tr><tr><td>20X lens UPLAPO</td><td>Olympus America, Center Valley, PA, USA</td><td>36-064</td><td>Olympus UPLSAPO 20X Objective</td></tr></tbody></table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

Embora a invasão 3D ensaios têm sido desenvolvidos, persiste o desafio de estudar células sem afetar a integridade do seu microambiente. Ensaios de 3D tradicionais tais como a câmara de Boyden exigem que as células são deslocadas do local cultura original e mudou-se para um novo ambiente. Não só faz isso atrapalhar os processos celulares que são intrínsecos para o microambiente, mas muitas vezes resulta em uma perda de células. Estes problemas são especialmente desafiadoras quando se lida com as células que são raros ou extremamente sensível ao seu microambiente. Aqui, descrevemos o desenvolvimento de um ensaio de invasão 3D que evita as duas preocupações. Neste ensaio, as células são banhadas dentro de um pequeno bem e uma matriz de ECM contendo um quimiotático é colocada no topo das células. Isto requer nenhum deslocamento de célula e permite que as células invadir para cima dentro da matrix. Neste ensaio, invasão da célula, bem como a morfologia da célula pode ser avaliada dentro do gel de colágeno. Usando este teste, podemos caracterizar a capacidade invasiva das células de raras e sensíveis; as células híbridas resultantes da fusão entre células de câncer de mama MCF7 e mesenquimais/multipotentes haste/estroma células (MSCs).

Nós usamos um prato de cultura 35mm com um vidro inferior e rato cauda de colágeno para desenvolver e otimizar uma invasão 3D em vitro ensaio protocolo. Usando este ensaio, nós mostramos aquele peito de células cancerosas MCF7 e MSC células poderiam invadir para o gel de colágeno a uma distância média de 0,04 ± 1,8 µm e 41,3 ± 76.0 µm respectivamente, após 48 h quando IGF1 (18.6 ng/mL) foi usado como quimiotático (Figura 2). Mais impor...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Este protocolo descreve um ensaio de invasão vertical invertida que poderia ser usado para quantificar os recursos de migração e invasão de células em uma configuração tridimensional, preservando o microambiente celular. Este ensaio é adequado para células raras e/ou sensíveis.

Se movendo para cima: Um simples e flexível em Vitro invasão tridimensional protocolo do ensaio

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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Universidad San Sebastian

Research

JoVE Journal

Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

6.0K visualizações.  University of Minnesota - Twin Cities. Este protocolo descreve um ensaio de invasão vertical invertida que poderia ser usado para quantificar os recursos de migração e invasão de células em uma configuração tridimensional, preservando o microambiente celular. Este ensaio é adequado para células raras e/ou sensíveis.

Vídeo: Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Ensaio esferóide para TGF-β Medida induzida Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicina

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 ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Matriz tumor-baseada inteiramente humana no ensaio tridimensional da invasão de spheroid

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Pesquisa do câncer

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Um In vitro FluoroBlok Assay invasão tumoral

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Biologia

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Em Vitro ensaios para avaliar a migração, invasão e proliferação de linhas de células humanas imortalizado primeiro trimestre trofoblasto

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Biologia do Desenvolvimento

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

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

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

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

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

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

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

A modificação<em&gt; In vitro</em&gt; Invasion Ensaio para determinar o possível papel dos hormônios, citocinas e / ou fatores de crescimento na Cancer Mediating Invasion celular

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Modelos 3D em Biologia do Câncer: Dinâmica de Invasão em Heterosferóides

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Autor em destaque: decifrando a diafonia entre câncer e células imunológicas durante a progressão do câncer de cabeça e pescoço

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Se movendo para cima: Um simples e flexível em Vitro invasão tridimensional protocolo do ensaio

Resumo

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