Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

NOTA: Não ética consideração, pois as células cancerosas em animais e humanos foram adquiridos ou gentilmente cedido para nós. 1. Fazendo Collagen Plugs (Tumor Pseudo-primário) <ol><li> Prepara-se uma dispersão de colagénio. Colágeno tipo I de tendões da cauda de rato (RTT) podem ser extraídos e esterilizados conforme relatado anteriormente 17, ou comprada. Dispersar colagénio liofilizado RTT (3,25-3,50 mg / ml em 0,02 N de ácido acético) usando um misturador (ajuste de alta velocidade; cinco mínimo de duas vezes), para uma mistura uniforme. </li><li> Colheita (tripsina-EDTA, geralmente) e usar a exclusão de azul de tripano para a contagem de células viáveis ​​utilizando um hemacitómetro. Ajustar a densidade celular desejada (5 x 10 4 culas por tampão). </li><li> Preparar todas as soluções (NaOH, soro de bovino fetal, meio DMEM 5x, NaHCO3) separadamente (Tabela 1) sob condições estéreis e mantê arrefecida em gelo. Nota: A ordem de adição dos vários soluções é importante para evitar choques osmóticos ou ácidas em células. </li><li> Realizar dispersão de células (1,25 x 10 6 células) na solução de colagénio final (5 ml) tão rapidamente quanto possível. Misture bem (por pipetagem para cima e para baixo), evitando bolhas de ar, e, em seguida, distribuir rapidamente 200 pi da solução pronta para uso, em cada poço de uma placa de 96 poços. Suavemente atacar a placa multi-poços na superfície da capa de cultura de célula a área de trabalho para remover as bolhas de ar e para espalhar a solução uniformemente dentro dos poços. </li><li> Depois de preencher todos os poços (esta etapa leva cerca de 15-20 minutos por placa de 96 poços), armazená-lo para a incubadora. </li><li> Incubar a placa a 37 ° C a partir de 2 horas até durante a noite. O colagénio de gelificação (ou seja, a fibrilogénese) ocorre dentro de 30 min. Adicionar meio de cultura (100 ul / cavidade) à cultura para realizar uma incubação de um dia para o outro. </li></ol> 2. primeira camada de fibrina Gel <ol><li> Fibrinogênio Preparação Solution. NOTA: O mesmo lote de gel de fibrina devem, idealmente, ser utilizado para Resul mais reprodutívelTS, como formação de gel de fibrina pode variar entre os diferentes lotes de fibrinogénio liofilizado comercial. <ol><li> Sempre use uma solução de fibrinogénio preparados na hora. Traga o fibrinogénio liofilizado até à temperatura ambiente antes de abrir o frasco para evitar a formação de cristais de hidrato. </li><li> Progressivamente dissolver o fibrinogénio em pré-aquecido (37 ° C) Solução Salina Equilibrada de Hank (HBSS) com Ca2 + / Mg2 + a uma concentração de trabalho de 3 mg / ml (considerar a preparação de um excesso de 15% do volume final mínima necessária : por exemplo, 17,25 mg em 5,75 ml de uma solução 5 ml). <ol><li> Adicionar pré-aquecido, gota a gota HBSS em primeiro lugar para solubilizar fragmentos de fibrinogénio. Quebrar fragmentos maiores com uma espátula no copo. Agitar o recipiente de tempos a tempos para facilitar a mistura. Não use um disco de agitação durante o procedimento. Dissolver o pó restante pipetando a suspensão para cima e para baixo. </li></ol></li><li> Manter a solução de fibrinogénio morna enquanto SteriLIZING a solução por passagem através de um filtro de 0,22 um. Nota: Se o HBSS não está suficientemente quente ou o fibrinogênio não completamente dissolvido, a solução pode obstruir o filtro. Se for o caso, substituir o filtro de uma ou duas vezes, o que pode diminuir a concentração de fibrinogênio e rigidez, assim, coágulo de fibrina. </li><li> Suspender as células de interesse (por exemplo, células endoteliais) em solução de fibrinogénio pronto-para-uso durante o ajuste do seu volume final, tal como um procedimento alternativo. </li></ol></li><li> Preparar as soluções de trombina. <ol><li> Prepara-se uma solução estoque em ddH2O (50 NIH unidades / ml), em seguida, esterilizá-lo utilizando um filtro de 0,22 um. </li><li> Usar uma proporção / trombina do fibrinogénio ≥1: 0,0075 (v / v), a fim de gerar o gel de fibrina. </li></ol></li><li> Gerando o gel de fibrina. <ol><li> Mantenha o fibrinogênio estéril e trombina soluções estoque em gelo durante todas as etapas seguintes. Os geles de fibrina são permitidos para formar em placas de 24 poços. </li><li> th prontamente sobreposiçãoe a superfície de cada cavidade com a solução de fibrinogénio (200 ul / poço), evitando a formação de bolhas de ar. Processo 6 poços a uma hora. </li><li> Uma vez que a solução de fibrinogénio cobre completamente a superfície dos poços, inclinar a placa a um ângulo de 45 ° e adicionar 1,5 ml de solução de trombina para o primeiro poço soltando a trombina para o centro do poço, e em seguida rodar suavemente a placa horizontalmente para 1-2 seg. <ol><li> Deixar a placa numa posição estável sob a câmara de fluxo laminar (5-10 minutos) até que o processo de gelificação / coagulação foi concluída (NB: o processo de polimerização não deve ser perturbado, por exemplo, através do transporte da placa para a incubadora). </li></ol></li><li> Uma vez que os primeiros seis poços polimerizado, repetir a mesma sequência (ou seja, as 3 etapas anteriores) para os próximos seis poços até que todos os poços foram processadas. </li></ol></li></ol> 3. segunda camada de fibrina Gel e imprensado Collagen plug <ol><li> OpçãoA: (A utilização do colágeno Ligue imediatamente). <ol><li> Certifique-se de que a primeira camada de gel de fibrina polimerizada em todos os poços por delicadamente da inclinação da placa. Colocar a placa de 96 poços contendo lado os tampões de gel de colagénio a lado com a placa de 24 cavidades (contendo os geles de fibrina) para facilitar a transferência das fichas de colagénio. </li><li> Adicionar uma gota de HBSS a cada poço da placa contendo as fichas de colagénio. </li><li> Remover cada vela de colagénio do poço com uma agulha fina montado numa seringa (usado como um identificador) ou usando um micro-colher (veja o vídeo). Transfira cada vela de colágeno para a primeira camada de gel de fibrina usando um ou dois micro-colheres, assegurando ao mesmo tempo que a ficha colágeno é bem centrado no poço e que a esterilidade é bem conservado. </li><li> Overlay o gel de fibrina previamente formada com a segunda camada de solução de fibrinogénio (300 ul / poço) e introduzir a trombina como descrito em 2.3, mantendo um mínimo de 1:. Relação de 0,0075 e uma sequência de seis poços de cada vez &lt;/ Li&gt; </ol></li><li> Opção B (Revestimento do Collagen Plug com uma fina camada de Fator de Crescimento reduzido membrana basal (GFRBM)). <ol><li> Frescos todas as soluções preparadas e instrumentos de antemão e mantê-los a 4 ° C ou em gelo (por exemplo, pipetas, pontas, tubos de ensaio) durante o manuseio desde alíquotas congeladas de GFRBM são muito sensíveis à taxa de aquecimento excessivo durante a descongelação (siga as instruções do fabricante) . </li><li> Após a remoção a partir dos poços da placa, mergulhar cada vela de colagénio durante 2 min num tubo de centrífuga de 1,5 ml em gelo contendo 100 uL de uma solução GRFBM puro. </li><li> Transferir cada vela revestida sobre a primeira camada de fibrina, enquanto assegurando que é bem centralizada, tal como descrito anteriormente. Incubar as placas contendo os plug a 37 ° C durante 5 min para permitir a GRFBM para formar um gel. Adicione a segunda camada de fibrina como no passo 3.1.4. </li></ol></li></ol> 4. Cultura Celular condições do meio <ol><li> Encher cada poço com meio de cultura (quatro00 uL). Os meios de cultura e suplementos serão seleccionados com base na linha celular e as condições experimentais. </li><li> Adicionar aprotinina, um agente antifibrinolítica, ao meio de cultura a uma concentração final de 100 unidades de inibidor de calicreína (KIU) / ml. NOTA: Armazenar as placas numa incubadora de cultura de células de acordo com as condições utilizadas para a linha de células testada. </li><li> Reabastecer culturas com meio fresco a cada dois dias ou de acordo com o programa experimental, e adicionar aprotinina. Antes de adicionar meio fresco, inclinar ligeiramente a placa (com um ângulo de 30-35 °) e inclinar a pipeta contra o lado de aspiração do poço, enquanto cuidadosamente o meio condicionado sob observação constante. </li></ol>

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The authors have no disclosure.

Como uma nota importante técnica, é essencial que nenhum intervalo está presente na interface entre a central e os géis periféricos. Caso contrário, pode reduzir a capacidade das células para migrar / invadem o gel de fibrina. Um espaço entre o colagénio e os geles de fibrina podem formar durante as primeiras 24 h de cultura, se a trombina não foi apropriadamente diluído. É também possível que a linha de células testada pode levar o gel de colagénio para contrair durante a cultura, fazendo assim com que ...

Investigando as características fundamentais e biomédicas de invasão da célula cancerosa / migração e subsequente criação metástase é objecto de um 1,2 intensa pesquisa. A metástase é o último estágio do câncer e seu manejo clínico permanece indefinida. Uma melhor compreensão de metástases nos níveis celulares e moleculares permitirá o desenvolvimento de terapias mais eficazes 3. Várias propriedades de células metastáticas pode ser explorada in vitro, incluindo a sua stemness 4 e potencial para adquirir um estado de transição (por exemplo, a transição epitelióide-mesenquimal) para migrar e invadem dentro e a partir do tumor primário 5. No entanto, a avaliação in vitro de processos / metástase de invasão tem sido um desafio, uma vez que praticamente exclui a contribuição da circulação de sangue / linfático. culturas organotípicas que incorporam fragmentos de tumor em géis de colagénio têm Previously sido utilizada para monitorizar a agressividade do cancro. Embora a complexidade dos tumores é preservada (por exemplo, a presença de células não-cancerosas), fragmentos tumorais são expostas a difusão forma limitada, a variação de amostragem, e a um crescimento excessivo de células estromais 6. Um método alternativo consiste em crescer as células cancerosas no interior de componentes da matriz extracelular (ECM), que imita o ambiente celular tridimensional (3D). A proliferação de linhas celulares de cancro da mama num gel de colagénio e / ou uma matriz de membrana basal é derivada entre os exemplos mais bem caracterizados de cultura de células 3D. Através da utilização de ambientes de cultura de células específicas 3D, o conjunto desorganizado observado para as células de cancro da mama cultivadas sob condições padrão pode ser revertida para a formação espontânea de ácinos mamaria e estruturas tubulares 7-10. Além disso, a formação de esferóides multicelulares de tumores derivados de células de cancro de adenocarcinoma congregados utilizando técnicas diferentes (por exemplo, pendurado gotas, esferóides flutuante, agar embedment) constitui agora o ensaio de cultura de células 3D mais utilizada 11-13. No entanto, este ensaio é limitado pelo conjunto restrito de linhas celulares de cancro que podem formar esferóides e pela curto período disponível para estudar células nestas condições. Nesta técnica visualizada, que aqui se introduzir um ensaio de cultura de células 3D sofisticado, onde as células cancerosas de interesse são incorporados num gel de colagénio para permitir a formação in vitro de um tumor pseudo-primário que pode ser alternativamente revestido com uma matriz derivada de membrana basal. Uma vez formado, o tumor pseudo-primário é, em seguida, colada em uma matriz acelular (gel de fibrina, no presente caso), o que permite que as células cancerosas para atravessar a interface entre os dois compartimentos da matriz (ver Figura 1). Curiosamente, as estruturas semelhantes a tumores, secundárias provenientes do tumor pseudo-primário, juntamente com as células cancerosas agressivas aparecem nagel de fibrina. Um tal sistema de cultura 3D oferece a flexibilidade necessária para investigar, por exemplo, fármacos anti-cancerígenos, de expressão de genes e célula-célula e / ou interacções célula-ECM 14-16. <img alt="figura 1" src="/files/ftp_upload/54322/54322fig1.jpg" /> Figura 1:.. Visão Geral do Método resumo esquemático do método para gerar o sistema de cultura celular 3D como um modelo para estudos de câncer <a href="https://www.jove.com/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Freeze-dried collagen</td>
			<td>Sigma-Aldrich</td>
			<td>C7661</td>
			<td>from rat tail tendon (soluble dispersion) or home-made (see Rajan et al., ref.#14)</td>
		</tr>
		<tr>
			<td>Fibrinogen (freeze-dried)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F8630</td>
			<td>Type I-S, 65-85% protein with &#8805;75% of protein is clottable</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>EMD Chemicals Inc.</td>
			<td>605157</td>
			<td>&#160;Gibbstown, NJ; NIH units/mg dry weight&#160;</td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Previously from BD Biosciences</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma-Aldrich</td>
			<td>A6279&#160;</td>
			<td>&#160;solution at 5-10TIU/ml (Trypsin Inhibitor Unit)&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Micro-spoons</td>
			<td>Fisher Scientific</td>
			<td>2140115</td>
			<td>Fisherbrand Handi-Hold Microspatula</td>
		</tr>
		<tr>
			<td>96 well plate, round base</td>
			<td>Sarstedt</td>
			<td>3925500</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sarstedt</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s Medium</td>
			<td>Sigma Chemical, Co.</td>
			<td>D5546</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>CAA15-701</td>
			<td>FBS, Canadian origin.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Chemical, Co.</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td>Sigma Chemical, Co.</td>
			<td>H8264</td>
			<td>HBSS</td>
		</tr>
	</tbody>
</table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

a cultura de células de mamífero em monocamadas é amplamente utilizado para estudar vários processos fisiológicos e moleculares. No entanto, esta abordagem para estudar as células que crescem frequentemente gera artefactos indesejados. Portanto, a cultura de células em um ambiente tridimensional (3D), muitas vezes o uso de componentes da matriz extracelular, surgiu como uma alternativa interessante devido à sua estreita semelhança com o nativo no tecido vivo ou órgão. Foi desenvolvido um sistema de cultura celular 3D utilizando dois compartimentos, a saber, (i) um compartimento central contendo células cancerosas embebidas numa actuação gel de colagénio como um tumor macrospherical pseudo-primário e (ii) um compartimento livre de células periférica feita de um gel de fibrina, isto é, um componente de matriz extracelular diferente do utilizado no centro, no qual as células cancerígenas podem migrar (invasão frente) e / ou formar microparticulas tumores representam tumores secundários ou satélite. A formação de tumores satélite no compartimento periférico énotavelmente correlacionados com a agressividade conhecido ou origem metastático das células tumorais nativas, que faz com que este sistema de cultura 3D original. Esta abordagem de cultura de células pode ser considerada para avaliar a capacidade de invasão de células do cancro e a motilidade, as interacções célula-matriz extracelular e como um método para avaliar as propriedades de drogas anti-cancro.

Como mencionado anteriormente, uma característica interessante deste ensaio de cultura de células 3D é que as células de cancro não só podem migrar a partir do bujão de colagénio para o gel de fibrina adjacente, mas também estabelecer tumores secundários (por exemplo, estruturas, por satélite tumoral). Isto pode ser directamente observado com um microscópio de contraste de fase invertida com ampliações de baixa e alta, através da espessura do gel, especialmente co...

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Tridimensional Cultura Ensaio para Explorar Cancer Invasividade celular e formação de tumor Satélite

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

Research

JoVE Journal

Medicine

10.3K visualizações.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Vídeo: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

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

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

Sistema de cultura de células 3D para estudar a invasão e avaliação terapêutica em câncer de bexiga

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Pesquisa do câncer

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 ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Monitoramento da invasão celular cancerígena e citotoxicidade de células T na cultura 3D

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

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 ...

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

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

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

Tridimensional modelo de co-cultura para Interaction Tumor estromal-

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

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

Tridimensional (3D) Tumor Esferóide Invasion Assay

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

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

Pintainho Coração Invasion Ensaio para testar a invasão das células neoplásicas e da Actividade de compostos potencialmente anti-invasivos

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

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 ...

3D Culture Basement Membrane Assay: Culturing Cancer Cells on 3D Basement Membrane Protein Matrix to Study Cell Invasion

Source: Cvetkovi&#263;, D., et al. <a href="https://www.jove.com/v/51341/quantification-breast-cancer-cell-invasiveness-using-three">Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model.</a> J. Vis. Exp. (2014).
This video 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.

Ensaio de Membrana Basal de Cultura 3D: Cultura de Células Cancerígenas em Matriz de Proteína de Membrana Basal 3D para Estudar a Invasão Celular

Source: Cvetkovi&#263;, D., et al. Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model. J. Vis. Exp. (2014).
This video ...