Financiado pelo NIH CA051008 subvenções P30 CA009686 e T32 (ATR).

1. Geração de esferóides <ol><li> Prepare a suspensão de células individuais para pendurar culturas gota a separar culturas aderentes de células de cancro de ~ 70% de confluência utilizando uma lavagem com PBS, seguido por exposição a uma solução tripsina-EDTA 0,05%. <ol><li> Neutraliza-se a solução de tripsina com meio de cultura de células e contagem das células, utilizando-se uma alíquota da suspensão de células. Nota: O meio de cultura de células específico dependerá da linha celular a ser testada. Seguir as recomendações da ATCC media, onde o meio de cultura celular é normalmente de DMEM + 10% de FBS. Mais informação pode ser encontrada na Tabela Materiais. </li><li> Efectuar uma diluição para permitir a sementeira de 500-1000 células por 20 ul gota de meio de cultura celular. </li></ol></li><li> Adquirir 10 cm de placa e adicionam-se 5 ml de PBS estéril para o fundo. Nota: Esta etapa protege o enforcamento cai de evaporação. Baixo custo &quot;bacteriológicos&quot; pratos grau pode ser usado em lugar de pratos de grau de cultura de células, como as células não irá contactuar a superfície de cultura. </li><li> Usar uma pipeta multi-canal para transferir gotas de 20 ul da suspensão de células diluída com a superfície interna da tampa. <ol><li> Pipetar 40 gotas (5 linhas de 8 gotas) sobre a tampa da placa de 10 cm. </li></ol></li><li> Inverter a tampa e coloque sobre o prato de cultura. Incubam-se as culturas de gota em suspensão a 37 ° C durante 72 h, para gerar esferóides. <ol><li> Vire a tampa de uma forma confiante, ainda controlada. Nota: Inverter a tampa muito rápido ou muito lento pode causar as gotículas de mudar. Algumas linhas de células podem formar esferóides em 48 horas ou menos, enquanto outros podem exigir mais do que 72 horas para produzir agregados compactos. </li></ol></li></ol> 2. Incorporação de Spheroids em Matrix 3D <ol><li> Descongelar uma aliquota de crescimento reduzida de factor porão materiais de membrana, a 4 ° C durante a noite antes de incorporação dos esferóides. <ol><li> Opcional: poços camada com ECM de antecedência para evitar a interação potencial esferóide com o tecido culture superfície. <ol><li> Pipetar 200 l de ECM por cavidade em uma placa de 24 cavidades. </li><li> Inclinar a placa para assegurar que a ECM cobre toda a superfície dos poços. </li><li> Retirar cuidadosamente o excesso ECM com uma pipeta. </li><li> Incubar a placa até que os poços estão secas: 3 horas à temperatura ambiente ou durante a noite a 4 ° C. </li></ol></li></ol></li><li> Recolher os esferóides inclinando a tampa da placa de cultura de tecidos e de partilha dos meios de comunicação. Transferir os meios de comunicação com os esferóides em um tubo de microcentrífuga de 1,5 ml. </li><li> Permitir que 10 min durante os esferóides para assentar no fundo do tubo de microcentrífuga. Esferóides deve ser visível a olho nu. </li><li> Misturar 100 mL de materiais de membrana basal com 100 ul de frio (4 ° C) colagénio do tipo I, em um tubo pré-refrigerada separada. Manter-se a mistura a 4 ° C para evitar qualquer gel solidifique prematuramente. <ol><li> Utilize pontas de pipetas pré-refrigerados se transferir pequenos volumes. </li><li> Dê cuidado aomisturando o material de membrana basal e colágeno tipo I para evitar a formação de bolhas de ar. Nota: As bolhas de ar podem dificultar a imagiologia mais tarde no protocolo de se tornar incorporado no gel. </li></ol></li><li> Aspirar os esferóides a partir da porção inferior 40 ul do tubo de microcentrífuga, e combinar com o material da membrana basal / mistura de colagénio do tipo I. Tenha cuidado para evitar a formação de bolhas de ar quando a mistura. Nota: A solução contendo 100 ul de materiais de membrana basal, 100 ul de colagénio do tipo I, e 40 ul de esferóides criará material suficiente para 4 3D culturas independentes. Isto pode ser escalado para cima ou para baixo. </li><li> Pipetar 40 ul de gotas da mistura viscosa para os centros das cavidades de uma placa de 24 poços. Manter o nível de placa para evitar que a mistura de correr para o lado do poço. Nota: Uma coluna única na placa de 24 cavidades - 4 poços - é recomendado para cada condição a ser testado. </li><li> Colocar a placa intoa 37 ° C incubadora e deixar em repouso por 30 min. A cultura 3D irá polimerizar durante este período. </li><li> Lentamente submergir as culturas 3D em 1 mL de meios de cultura celular. <ol><li> Certifique-se de que a mídia está quente ao adicioná-lo nos poços para promover ainda mais a polimerização gel. </li><li> Passo crítico: Delicadamente adicionar a mídia para as culturas 3D. Pipetando os meios muito rapidamente nas cavidades pode levar ao descolamento das culturas em 3D a partir da superfície da cultura de tecidos. </li></ol></li></ol> 3. Monitoramento e Análise Spheroid Invasion <ol><li> Invasão imagem dos esferóides na matriz 3D circundante em pontos de tempo decidido pelo investigador. Adquirir fotografias usando um microscópio invertido com a lente objetiva de 20x. Nota: Ideal pontos de tempo irá variar de acordo com a linha de células que está sendo testado. Linhas de células mais invasivos começará sua saída dos esferóides logo após o plaqueamento e, portanto, levar fotografia inicials dentro de um par de horas após o plaqueamento. Geralmente, as fotografias são tiradas a 0 h (após o plaqueamento), 24 horas e 48 horas. Invasão conclui tipicamente de 24 a 48 h após o plaqueamento. </li><li> Quantificar a capacidade invasiva utilizando software de análise de imagem como Imagem J. <ol><li> Analise invasão celular como a distância a partir da borda do esferóide. <ol><li> Use a ferramenta straight draw para marcar o raio e / ou a distância máxima invasiva. </li><li> Clique em &quot;Analisar&quot; no menu superior e clique em &quot;Measure&quot; para exibir a medição do comprimento. </li></ol></li><li> Analisar invasão como a área total invasiva fora do esferóide. <ol><li> Use a ferramenta de desenhar à mão livre para traçar a fronteira do esferóide e / ou total área invasiva. </li><li> Clique em &quot;Analisar&quot; no menu superior e clique em &quot;Measure&quot; para exibir a medição da área. </li></ol></li><li> Use um plugin como ROI Ferramentas do Gerenciador de criar medidas para uma pilha de imagens. </li></ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Este estudo foi avaliado o desempenho de um painel de linhas celulares de cancro em um ensaio de invasão esferóide (Tabela 1). De um modo geral, descobrimos que a formação do esferóide é aumentada em mais as linhas de células epiteliais de aspecto, em que a presença de junções célula-célula promove a formação de uma arquitectura do tipo esferóide. Linhas celulares conhecidas que foram submetidos a uma transição epitelial-mesenquimal, tais como MDA-MB-231 não formar esferóides na cultu...

Está provado que a motilidade célula cancerosa é preditiva do potencial metastático, uma vez que tal comportamento facilita a invasão através da membrana basal e da entrada no sistema circulatório 1. Mais investigação sobre a motilidade tem-se centrado na forma como as células se comportam de uma configuração bidimensional (2D), embora se tornando-se amplamente reconhecido que o movimento das células em uma matriz tridimensional (3D) é mais representativo de como estas mesmas células vão realmente 2 comportar in vivo. Sistemas de cultura 3D são cada vez mais usados ​​para estudar comportamentos celulares que vão desde a morfologia celular, a cinética de crescimento e de sensibilidade às drogas 3. O desejo de controlar a invasão de células do cancro no contexto de um meio 3D levou à síntese de técnicas anteriormente estabelecidas, envolvendo a geração de agregados de células de 3D ​​(esferóides) através do método de cultura de gota em suspensão 4, seguido por incorporação destes esferóides numa extracelular 3D matriz (ECM) composta de colágeno e da membrana basal materiais 5. Este método visa aperfeiçoar estas técnicas anteriormente estabelecidas, fornecendo uma abordagem simplificada que pode ser facilmente utilizado para comparar invasão sob uma variedade de condições experimentais. Mais formas tradicionais para avaliar a motilidade celular in vitro são o ensaio da ferida-zero e o ensaio transwell 6. O primeiro ensaio de motilidade celular descreve uma configuração em 2D, e é, por conseguinte, independente de uma variedade de características críticas para a invasão in vivo, por exemplo, actividade de protease de 7. O ensaio de invasão de células Transwell pode melhor modelo, quando os insertos de cavidades são revestidas com substratos ECM, mas apenas um único parâmetro, isto é, o aparecimento de células sobre a superfície oposta da membrana é medida, e muitas nuances de invasão de células não são, portanto, facilmente observáveis. Em contraste com estas técnicas, o ensaio de invasão de células de cancro esferóide (figura 1 &lt;/ forte&gt;) permite a monitorização em tempo real da invasão celular, num ambiente que não só é fisiologicamente relevante, mas também permite características importantes específica da linha de células a serem visualizadas, tais como migração de células indivíduo vs colectiva 8. Este método também proporciona vantagens em relação aos ensaios padrão de crescimento de cultura 3D. A geração de agregados celulares através do método de gota em suspensão inicialmente restringe o movimento celular, de modo a que as células serão incentivados a invadir após esta restrição será levantada. Além disso, uma vez que a restrição é levantada, a saída de células irá prosseguir numa direcção uniforme, que pode então ser convenientemente quantificado. Os materiais de ECM mais populares usados ​​para esferóides ensaios de células de câncer são Matrigel e colágeno tipo I, em que cada um destes componentes tem papéis importantes e distintas em influenciar o comportamento metastático. Matrigel é uma mistura de proteínas segregadas por células de sarcoma produzidos Engelbreth Holm Swarm-rato, e é enriquecido em basemeproteínas da membrana nt, tais como laminina, entactina, e colágeno tipo IV 9. Por esta razão Matrigel é doravante referido como &quot;materiais de membrana basal.&quot; Estes materiais de membrana basal proporcionar ligandos essenciais necessários para adesão integrina durante a invasão de células de cancro 10, além de muitas outras proteínas que exercem uma variedade de efeitos no comportamento das células 11. Em comparação, o colagénio do tipo I, geralmente preparado a partir de digestões de ácido dos tendões e outras estruturas de colagénio densas 12, é um material de matriz muito mais simples que serve como um elemento estrutural do tecido do estroma e tecidos conjuntivos de suporte e órgãos do corpo. Foi demonstrado que as características físicas do colagénio pode regular um número de características de motilidade celular; Por exemplo, o alinhamento de fibras de colagénio na interface tumor-estromal permite que as células cancerosas migram subsequentemente ao longo dessas fibrilas ao invadir o estroma 13 em. No ensaio aqui apresentado, tanto o colágeno tipo I e materiais da membrana basal são utilizados como ferramentas para estudar as interações célula de câncer 3D-estroma. O efeito de inibição ou estimulação de caminhos que controlam invasão pode ser monitorizada após as células terem sido incorporados na matriz 3D. As células podem ser pré-tratados durante o crescimento nas gotas de suspensão ou aquando da transferência para a cultura 3D, dependendo se um tratamento prolongado será necessário para modular a invasão. Para tratamentos mais curtos, recomenda-se que a droga ser misturada com a suspensão esferóide após a recolha, assim como os meios de comunicação que vai rodear as culturas em 3D, para facilitar a exposição adequada da droga para as células. Em seguida, as células normais ou associados a tumores do estroma podem ser misturados com o material de matriz para avaliar o seu papel na modulação da invasão de células tumorais, ou para determinar o comportamento de células influências parácrino e autócrino de sinalização. Esta ideia foi demonstrado em um estudo onde a co-cultura de colsobre câncer e células endoteliais em gotas de suspensão levou a uma rede vascular dentro dos esferóides 14. Finalmente, os componentes ECM também pode ser alterado, como a invasão da célula cancerosa é afetado por diferentes substratos 15. O método apresentado abaixo, assim, fornecer um quadro para a avaliação da invasão da célula cancerosa sob uma variedade de condições. De um modo geral verificou-se que nem todas as linhas de células irá criar esferóides nas gotas de suspensão e linhas de células epiteliais de aspecto tipicamente formar esferas regulares.

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

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.

Usando o ensaio de invasão esferóide (figura 1), um painel de linhas celulares de cancro foi testado quanto à sua capacidade para formar esferóides, bem como para a quantidade de egresso de células expostas após o implante numa matriz 3D que consiste em materiais de membrana basal e colagénio tipo I (Tabela 1). Estes resultados demonstram que nem toda linha celular de cancro criará esferóides bem formadas, onde as linhas de células que possuem uma morfologia epitelial in v...

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

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 ...

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Medicine

32.3K visualizações.  Georgetown University. 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. 

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

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

A técnica de impedância elétrica em tempo real baseado medir Invasão de monocamada de células endoteliais por células cancerosas

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Medicina

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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.

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

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 ...

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 ...

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

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

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

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

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

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

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 ...

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 ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Concentre Formação: Um Ensaio Celular para Determinar o Potencial oncogénica de um Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusões artéria carótida para farmacocinéticas e farmacodinâmicas Análise de taxanos em Ratos

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...