Somos gratos a Ken Iwata (OSI Pharmaceuticals, New York, EUA) para reagentes e Miller Fred (Barbara Ann Karmanos Cancer Intitute, Detroit, EUA) para as linhas de celular.

1. Preparação da solução Methocel (500 mL) <ol><li> Autoclave g 6 de metilcelulose em uma garrafa de 500 ml contendo um agitador magnético. </li><li> Dissolver a metilcelulose em 250 ml de DMEM/F12 pré-aquecido sem soro (60 ° C) por 20 min. </li><li> Adicione 250 ml de DMEM/F12 (RT), contendo soro de cavalo 10%. Misturassem durante a noite (4 ° C). </li><li> Clara a solução por centrifugação (5000g, 2h, 4 ° C). </li><li> Tome a solução clara e altamente viscoso para um novo tubo (cerca de 90-95% da solução-mãe). </li><li> Armazenar a 4 ° C até o uso. </li></ol> 2. Cultura esferóide <ol><li> Prepare uma solução de 20% Methocel (10 ml e 40 Methocel meio de crescimento ml). </li><li> Lave as células duas vezes com PBS, adicionar 0,1% de tripsina e células incubar a 37 ° C </li><li> Ressuspender as células em meio de crescimento e contagem das células. </li><li> Prepare uma suspensão de 10 000 células por ml em Methocel 20%. </li><li> Adicione a suspensão de células para o fundo s 96 bem rodadaplacas USPENSÃO (100 l por poço). </li><li> No dia seguinte, verificar a formação de esferóides nas placas de suspensão. Esferóides estará pronto para uso após 1-3 dias. </li></ol> 3. Preparação de colágeno <ol><li> Prepare uma solução sobre o gelo, de 8 de colágeno ml, 1 ml PBS 10x, 1 ml NaOH 0,1 N e 3 gotas 0,1 N HCl. Verifique se o pH é de 7,4 por manchar um pouco da solução em tiras indicador de pH. Ajustar o pH se fora do intervalo. </li><li> Adicionar 50 ml de solução de colágeno neutralizado nos poços de uma placa de 96 poços </li><li> Incubar a 37 ° C até que o colágeno é solidificada (90 min). </li><li> Prepare-Methocel colágeno ligante soluções no gelo: para cada ligante parte 1 de pipeta de solução de colágeno. Adicionar 20 ml por ng de colágeno TGF-β. Após a diluição com Methocel e difusão sobre as diferentes camadas, a concentração final de TGF-β será de 5 ng / ml Mix em vortex. Adicionar 1 parte de Methocel solução. Misturar em vortex. </li></ol> 4. Esferóide incorporarding <ol><li> Coloque uma ponta de 200 mL, que tem cerca de 5 mm da ponta cortada, em uma pipeta de 200 mL. Sugar um esferóide com a pipeta e cuidadosamente dispensar o meio em um prato vazio. Observe se o esferóide fica dentro da ponta. O esferóide é visível como um ponto branco. Sem soltar o êmbolo, sugar até 100 mistura de colágeno Methocel-L e dispensar o esferóide na mistura de colágeno em colágeno revestido-96 também. Faça isso por 12 esferóides para cada condição. </li><li> Deixe colágeno solidificar pelo menos 30 minutos a 37 ° C </li><li> Remover bolhas de ar com uma ponta de 200 mL. </li><li> Adicionar 50 ul de meio com soro 1,6% e inibidores dissolvido em DMSO. Para SB-431542 adicione 4 ml de solução estoque (10 mM) a 1 ml de meio. Depois de difusão, a concentração final do inibidor será de 10 mM. TGF-β e inibidores vai manter o curso do experimento. Tirar fotos dos esferóides sob o microscópio usando aumento de 40 vezes. </li><li> Depois de 48h de estimulação com TGF-β e / ou inibidores, tirar fotos dos esferóides sob o microscópio usando aumento de 40 vezes. </li><li> Medir a área dos esferóides invadindo após 2 dias e subtrair a área inicial. Medir a área de um esferóide: imagem do Aberto do esferóide em Adobe Photoshop Extended (figura 2A) e selecione a ferramenta de seleção rápida (figura 2B). O ponteiro do mouse se transforma em um círculo com um &#39;plus&#39; (+) sinal (figura 2C). Enquanto arrasta o cursor sobre o esferóide, Photoshop irá reconhecer as fronteiras do esferóide. Se uma área fora do esferóide é selecionada, esta região pode ser excluído da seleção pressionando a tecla Alt ou usando a ferramenta de seleção negativa que é indicado por um sinal - na barra de ferramentas (). O cursor muda para um círculo com um &quot;menos&quot; (-) sinal e arrastando o cursor sobre a área selecionada erroneamente, esta área é removida da seleção (figura 2D). Se necessário, várias regiões podem ser selecionados pressionando o Shift botão. Para trabalhar com mais precisão, o tamanho do ponteiro do mouse pode ser ajustada alterando o diâmetro da escova na barra de ferramentas (Figura 2E). Quando o esferóide todo é selecionada, a área da seleção é calculada através da análise MEDIDA na barra de menu ou pressionando Ctrl + Shift + M (Figura 2F). A janela de gravação de medição aparecerá mostrando o nome do arquivo e da área da seleção em pixels, assim como outros dados (figura 2G). Se várias seleções são medidos, a primeira linha mostra a soma de todas as áreas. As medições das seleções individuais são apresentados nas linhas abaixo. Os dados de todas as medições podem ser exportados para um arquivo de texto e aberto em Excel. </li></ol> 5. Resultados representativos: Um exemplo do ensaio esferóide com MCF10A1 (M1), as células epiteliais mamárias normais, H-RAS transformou MCF10AneoT (M2) e M2 células derivadas MCF10CA1a (M4) é dada na Figura 3. Células M1 apresentaram apenas invasão fracos semestimulação, mas invadiu significativamente melhor após estimulação por TGF-β. Em contraste, a RAS-transformado M1-M2 derivados já invadiu de forma eficiente, sem adição de estímulos, e isso aumentou ainda mais vezes 4-5 sobre TGF-β tratamento. Células M4 apresentou o maior invasão, com e sem TGF-β disso. Em seguida, examinamos se poderíamos interferir com TGF-β induzida por invasão neste modelo esferóide uso de inibidores químicos. Para isso, usou SB-431542, um análogo de ATP e inibidor seletivo da atividade da quinase de TβRI, bem como ativina tipo IB receptor (ActRIB) e activina receptor-like kinase (ALK) 7 12. Como esperado, SB-431542 inibiu TGF-β induzida por invasão de M1, M2 e M4 células (Fig. 4), indicando que a invasão TGF-β-induzida é TβRI kinase-dependente. Além disso, a invasão dos esferóides basal também foi fortemente inibida, sugerindo que a invasão basal também é deindependentes em TGF-β. <img alt="Figura 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> Figura Fluxograma 1. Do ensaio 3D esferóide. Primeiro, as células são banhados sob condições não-aderentes em placas em forma de u suspensões. Células aggregrate em esferóides multcellular e pode ser incorporado em uma matriz de colágeno. Para esta matriz de colágeno, que são capazes de invadir. <img alt="Figura 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> Figura 2. Quantificação da área do esferóides usando o Adobe Photoshop Extended. (A) A imagem do esferóide é aberto no Adobe Photoshop Extendend Selection (B) da ferramenta de selecção rápida (C) Arrastar o cursor sobre o esferóide para selecionar a área da remoção (D) esferóide de uma área wongly incluído usando a negativos de selecção rápida ferramenta de ajuste (E) do tamanho do pincel da ferramenta de seleção rápida de forma mais precisa selecionar a área (F) Selecção domedição de comando para gravar (G) Exemplo de um registro de medição. <img alt="Figura 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> Figura 3. TGF-β induzida por invasão de esferóides de MCF10A1 espontaneamente imortalizado (M1) (A), RAS-transformado MCF10AneoT (M2 (B) e seus derivados MCF10CA1a metastático (M4) (C). M1, M2 e M4 esferóides embutidas em colágeno foram tratados com 5ng/ml de TGF-β por 48h, conforme indicado. AC Representante imagens tomadas no dia da incorporação e dois dias depois. D invasão relativa diferença foi quantificada como área no dia 2 de menos dia 0. Os resultados são expressos como média ± sd Adaptado de 13. <img alt="Figura 4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="Figura 4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> Figura 4. Invasão esferóide Basal e TGF-β induzida é inibida by SB-431542. Esferóides M1, M2 e M4 foram incorporados em colágeno e tratados com TGF-β 5ng/ml e / ou 10 mM SB-431542, conforme indicado. Imagens representativas tomada após dois dias (A). Invasão relativa diferença foi quantificada como área no dia 2 de menos dia 0 (B). Os resultados são expressos como média ± sd Adaptado de 13.

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Nós estabelecemos um modelo esferóide em que MCF10A1 células e seus derivados malignos invadem em uma matriz de colágeno de uma forma TGF-β-dependente. Primeiro, esferóides são formados em placas redondas de 96 poços de fundo de suspensão na presença de metilcelulose. Naturalmente, também em forma de U poli-HEMA placas revestidas podem ser usados ​​para esta finalidade. Metilcelulose é absolutamente necessário nesta etapa, já que as células morrerão na ausência de metilcelulose. TGF-β é adicionado...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> Metil celulose </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avançados Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> indicador de pH tiras </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> De 96 poços de suspensão placas </td><td> Greiner Bio-one </td><td> 650 185 </td><td></td></tr><tr><td> De 96 poços de adesão TC placas </td><td> Greiner Bio-one </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-β tem papéis opostos na progressão do câncer de mama, agindo como um supressor de tumor na fase inicial, mas estimulando a invasão e metástase na tarde 1,2 palco. Além disso, o TGF-β é freqüentemente overexpressed no câncer de mama e sua expressão se correlaciona com o prognóstico pobre e metástases 3,4. Os mecanismos pelos quais TGF-β induz a invasão não são bem compreendidos. TGF-β provoca suas reações celulares via TGF-β tipo II (TβRII) e eu tipo (TβRI) receptores. TGF-β sobre induzida por formação heteromeric complexo, TβRII fosforila o TβRI. O TβRI ativada inicia a sua via de sinalização intracelular canônica pela Smads receptor fosforilar (R-Smads), ou seja Smad2 e Smad3. Estes ativados R-Smads formam complexos com heteromeric Smad4, que se acumulam no núcleo e regulam a transcrição de genes-alvo 5. Além do anteriormente dvia Smad escribed, os resultados receptor de ativação na ativação de diversas outras organizações não-Smad vias de sinalização, por exemplo Mitógeno proteína quinase ativada (MAPK) Vias 6. Para estudar o papel da TGF-β em diferentes estágios do câncer de mama, fizemos uso do sistema de célula MCF10A. Este sistema é composto de células espontaneamente imortalizado MCF10A1 (M1) de mama epiteliais 7, o H-RAS transformou M1 derivados MCF10AneoT (M2), que produz lesões pré-malignas em camundongos 8, eo MCF10CA1a M2 derivados (M4), que foi estabelecida a partir de xenoenxertos M2 e os formulários de alto grau de carcinomas com a capacidade de metástase para o pulmão 9. Esta série MCF10A oferece a possibilidade de estudar as respostas de células com diferentes graus de malignidade que não são influenciados por uma base genética diferente. Para a análise de TGF-β induzida por invasão, geramos homotypic MCF10A esferóide celular embe culturasdded em uma matriz de colágeno 3D in vitro (Fig. 1). Tais modelos se assemelham tumores humanos in vivo, estabelecendo um gradiente de oxigênio e nutrientes, resultando em células ativas e invasiva sobre as células fora e quiescente ou mesmo necrose no interior dos 10 esferóide. Esferóide ensaios com base também foram mostrados para melhor recapitular resistência às drogas do que as culturas em monocamada 11. Este sistema MCF10 modelo 3D permite estudar o impacto do TGF-β sinalização nas propriedades invasivas das células de mama em estágios diferentes de malignidade.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

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

Um ensaio para medir quantitativamente Transforming Growth Factor (TGF)-β induzida por invasão em gel 3-dimensional colágeno é descrito. Este ensaio se aproveita da série MCF10A de linhas de células, que representam diferentes fases de desenvolvimento de câncer de mama. Este método pode ser adotado para ser usado com outras linhagens de células e pode ser usado para investigar outras possíveis ativadores ou inibidores de invasão.

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 ...

spheroid-assay-to-measure-tgf-induced-invasion

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JoVE Journal

Medicine

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

21.7K visualizações.  Leiden University Medical Centre. Um ensaio para medir quantitativamente Transforming Growth Factor (TGF)-β induzida por invasão em gel 3-dimensional colágeno é descrito. Este ensaio se aproveita da série MCF10A de linhas de células, que representam diferentes fases de desenvolvimento de câncer de mama. Este método pode ser adotado para ser usado com outras linhagens de células e pode ser usado para investigar outras possíveis ativadores ou inibidores de invasão.

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

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

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

Método para medir tom de músculo axial e proximal

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Precisão em Medicina Dentária, uma maneira nova de medir justeza e precisão

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Telemetria Intrauterine medir Rato contrátil Pressão<em&gt; In Vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

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 ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

A membrana Chorioalantoica Chick in vivo modelo para avaliar invasão perineural em câncer de cabeça e pescoço

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...