Gostaríamos de agradecer a Nikon Imaging Center da Harvard Medical School, Waters especificamente Jennifer, Lara Petrak e Salmon Wendy, à formação e à utilização de microscópios seus timelapse. Gostaríamos também de agradecer a Rosa Nunes e Achim Besser pelas valiosas discussões. Este trabalho foi financiado pelo NIH Grant 5695837 (para M. Iwanicki) e GM064346 a JSB; por uma concessão do Dr. Miriam e G. Sheldon Adelson Medical Research Foundation (para ICC).

1. Ovarian Cancer Formação Spheroid celular <ol><li> RFP-expressando células cancerosas do ovário são cultivadas em meio de base de 10% (um meio de cultura de célula personalizado contendo uma mistura 50:50 de 199 e MCDB105, 10% de soro bovino fetal inactivado e 1% pen-strep). Para expressar RFP em células cancerosas do ovário não marcados, transfectar as células com um plasmídeo contendo RFP e seleccionar para células que expressam RFP. Alternativamente, os vectores virais podem ser usados ​​para expressar as proteínas fluorescentes transitoriamente, ou as células podem ser pré-incubar com um vermelho fluorescente de células rastreador corante (Invitrogen). </li><li> Antes da formação de esferóides de cancro do ovário, é necessário preparar-adesão baixa 96 pratos de cultura de fundo redondo bem. Para produzir as placas de aderência baixa de cultura, 30μl poli-HEMA (6mg polyhydroxyethylmethacrylate em 1 ml de 95% EtOH) solução é adicionada a cada poço de uma bem Corning 96 prato de cultura de células. As placas de 96 poços são incubadas em um 37 ° C não-umidificado incubadora para evaporar o etanol, leaving uma película de poli-HEMA em cada poço. Esta película de poli-HEMA impede que as células se liguem ao fundo do poço, forçando as células a crescer em suspensão 18. [Alternativamente, Ultra-Low placas de fixação de cultura (Corning) pode ser usado em vez de poli-HEMA pratos revestidos.] </li><li> Após os de baixa aderência placas de cultura são preparados, trypsinize uma placa de células de ovário, cancro, sedimentar as células em uma centrífuga de mesa (Heraeus) a 900 RCF durante 3 minutos, aspirar o sobrenadante e re-suspender em meio de base de 10%. </li><li> Contar as células utilizando um hemocitómetro. </li><li> Ajustar a concentração de tais células que existem 100 células por 50 l de meio de base de 10%. </li><li> Adicionar 50 l da suspensão de células em suspensão uniformemente diluído a cada poço da placa de cultura de 96 poços de poli-HEMA revestido. </li><li> Incubar a placa de 96 poços em um 37 ° C incubador de cultura de células durante 16 horas (esta quantidade de tempo deve ser aumentada ou diminuída dependendo da quantidade de tempo que leva parauma linha de células particular para formar esferóides multicelulares ou desejado condições experimentais) para permitir que as células de cancro do ovário a agrupar em conjunto, formando um único esferóide multicelular em cada poço. Algumas células tumorais podem sofrer apoptose durante este período, por isso, é importante escolher um tempo anterior à indução de apoptose. </li></ol> 2. Formação de monocamada celular mesotelial <ol><li> Em um capuz de cultura de células, de pré-revestimento dos poços de uma 6 bem prato MatTek com fundo de vidro com fibronectina por adição de 2 mL de uma fibronectina 5μg / ml de solução PBS a cada poço do prato e incubando à temperatura ambiente durante 30 minutos. A qualidade óptica do vidro de fundo em pratos MatTek permitir a alta resolução de imagem microscópica. </li><li> GFP-expressando células mesoteliais são cultivadas em meio de base de 10%. Trypsinize uma placa de células mesoteliais, girar em uma centrífuga de mesa (Heraeus) a 900RPM por 3 minutos, aspirar o sobrenadante, e re-suspensão em meio base de 10%. Océlulas mesoteliais aqui utilizados foram já expressando GFP quando eles foram obtidos, mas não marcados células mesoteliais podem ser produzidos por transfecção com um plasmídeo contendo ADNc de GFP, ou pré-incubação das células em um verde fluorescente de células rastreador corante (Invitrogen). </li><li> Após a incubação a fibronectina de 30 minutos (no passo 2.1), lavar os poços do prato MatTek com 2mL de PBS. </li><li> Aspirar o PBS e placa de 6 x10 5 de células mesoteliais por poço em cada poço do prato MatTek 6 bem. Incubar o prato MatTek em um 37 ° C incubador de cultura de células durante a noite para permitir que as células mesoteliais para anexar o prato e formam uma monocamada. </li></ol> 3. Ensaio Apuramento mesotelial celular <ol><li> Use uma pipeta para coletar os esferóides de câncer de ovário a partir da placa de 96 poli-HEMA bem revestido. </li><li> Aspirar o meio a partir de um poço do prato MatTek 6 poços contendo uma monocamada de células mesoteliais. Lavar uma vez com 2 ml de PBS. Adicionar todos os esferóides a partir do 96 bem ptarde para um poço de o prato MatTek (~ o ​​número 3x de esferóides que vão a ser trabalhada a conta para a aterragem esferóides por parte do prato que não pode ser trabalhada). </li><li> Coloque o prato MatTek com a fase de um microscópio de fluorescência invertido Widefield capaz de realizar lapso de tempo de imagem para a duração de pelo menos 8 horas. Use um palco motorizado para posições de imagens múltiplas no prato, com múltiplos eventos de intercalação esferóides, em um único experimento. Usamos uma Nikon Ti-E Invertido Motorizado Widefield Microscópio de fluorescência time-lapse com Sistema de Foco integrado perfeita e baixo [abertura 20x 0,75-numérica (NA)] ampliação / diferencial NA interferência de contraste (DIC) óptica, um transiluminador de halogênio Nikon com 0,52 NA distância de trabalho longa condensador (DPM), Nikon rápido (&lt;100 milissegundos de comutação tempo) de excitação e emissão filtros (GFP Ex 480/40, Em 525/50, RFP-mCherry Ex 575/50 Em 640/50), Sutter rápido transmissíveis Desligar e caminho de epifluorescência luz inteligenteters, uma Nikon fase linear codificado motorizado, um Hamamatsu ORCA-AG resfriado charge-coupled device câmera (CCD), uma custom-built câmara de incubação microscópio com a temperatura e CO controle 2, Nikon NIS Elements AR software versão 3, e um TMC mesa de isolamento de vibração. </li><li> As células de câncer de ovário esferóides vai assentar no fundo do prato e coloque a monocamada de células mesoteliais. Colete GFP, RFP e imagens de fase de 20 + esferóides / monocamada interações, a cada 10 minutos, por 8 horas. </li><li> Os RFP-expressam ovarianos esferóides de células cancerosas em invadir a monocamada de células GFP-expressando mesotelial criação de um buraco na monocamada. Após 8 horas, medir os tamanhos dos furos traçando os buracos negros nas imagens GFP utilizando elementos de software (ou outro software adequado, tal como J imagem). Normalizar o tamanho do furo para o tamanho inicial esferóide dividindo o tamanho do orifício de 8 horas com o tamanho do esferóide na imagem RFP correspondente no tempo zero. Neste examplo, o tamanho do buraco só foi medida uma vez, mas pode ser medido várias vezes ao longo da experiência de oito horas para entender melhor a dinâmica da intercalação. </li></ol> 4. Os resultados representativos Neste exemplo, comparou a capacidade de apuramento de mesotelial OVCA433 ovarianos esferóides de células cancerosas que têm expressão atenuada do talina-1 para controlar OVCA433 esferóides. OVCA433 esferóides de cada grupo foram adicionados a um prato MatTek contendo monocamadas de células mesoteliais ZT. Seis esferóides de cada grupo foram fotografadas a cada 10 minutos durante oito horas (Figura 4, Filme 3, Filme 4). Os furos produzidos na monocamada pelos esferóides de espalhamento foram medidos e seis posições de cada grupo foram em média. A Figura 4 mostra que a área média de apuramento criado por talina 1 esferóides knockdown era significativamente menor do que a área média criado por esferóides controle, sugerindo que é necessária para talina depuração por O mesotelialVCA433 câncer de ovário esferóides. <img alt="A Figura 1" src="/files/ftp_upload/3888/3888fig1.jpg" /> Figura 1. Metástase do câncer de ovário. Tumores ovarianos desenvolver a partir do epitélio de superfície do ovário ou trompas de falópio. As células tumorais / clusters romper com o tumor primário e recolher na cavidade peritoneal. As células tumorais podem então agregam para formar esferóides multicelulares. Esferóides, em seguida, anexar as monocamadas de células mesoteliais que revestem a cavidade peritoneal. As células mesoteliais são excluídos por baixo do esferóide anexa do cancro do ovário, permitindo que os esferóides para obter acesso à membrana basal subjacente. Filme 1. Metástase do câncer de ovário. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">Clique aqui para ver filme</a> . <img alt="A Figura 2" src="/files/ftp_upload/3888/3888fig2.jpg" /> Figura 2. </strOng&gt; linha células mesoteliais a superfície do tecido peritoneal humana e são excluídos por baixo implantes de células de cancro do ovário. <img alt="A Figura 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> Figura 3. Ensaio Apuramento mesoteliais. Esferóides de cancro do ovário são formadas por incubação 100 RFP-expressando células do ovário do cancro por poço em um poli-HEMA revestido prato de cultura de 96 poços de fundo redondo, a 37 ° C durante 16 horas. Poli-HEMA impede que as células se liguem ao prato de cultura, permitindo que as células permaneçam em suspensão e aderirem umas às outras para formar um único agrupamento por poço. Monocamadas de células mesoteliais são preparados por plaqueamento 6x10 5 células mesoteliais por poço em um fibronectina revestido 6 prato MatTek bem e incubando a placa a 37 ° C durante 16 horas. Os esferóides são então transferidos para o prato MatTek com a monocamada mesotelial e as duas populações de células são gravadas a cada 10 minutos durante 8 horas, utilizando um Nikon Ti-E Inverted Motorizado Widefield Microscópio de fluorescência time-lapse e software Elements. Filme 2. Ensaio Apuramento mesoteliais. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">Clique aqui para ver filme</a> . <img alt="A Figura 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> Figura 4. Atenuação de talina expressão 1 em OVCA433 esferóides diminui a capacidade de apuramento mesotelial. OVCA433 esferóides (vermelho) com e sem expressão atenuada do talina 1 foram autorizados a juntar-se e invadir em uma monocamada ZT mesotelial (verde). As duas populações de células foram fotografadas a cada 10 minutos durante 8 horas, utilizando um Nikon Ti-E invertido motorizado Widefield Fluorescência lapso de tempo microscópio e software elementos. O gráfico mostra que talina 1 atenuaçãodiminui significativamente a depuração célula mesotelial (parcela Quantile com barras verdes no meio). Filme 3. Controle OVCA433 esferóides (vermelho) invasores em uma monocamada mesotelial (verde). <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">Clique aqui para ver filme</a> . Movie 4. Atenuação de talina expressão 1 em OVCA433 esferóides (vermelho) diminui mesotelial (verde) a capacidade de apuramento. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">Clique aqui para ver filme</a> .

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O &quot;Ensaio Apuramento mesotelial&quot; aqui apresentado usa de lapso de tempo de microscopia para monitorar as interações de esferóides de câncer de ovário multicelulares e monocamadas de células mesoteliais, em detalhe espacial e temporal. Anteriormente, vários grupos 8-14 tinha usado ensaios de ponto de extremidade para mostrar que as células de câncer de ovário juntar-se e invadir em monocamadas de células mesoteliais. Este ensaio é único na medida em que utiliza células marcadas por fluo...

<table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> OVCA433 células cancerígenas de ovário </td><td></td><td></td><td> Presente do Dr. Dennis Slamon </td></tr><tr><td> Zt células mesoteliais </td><td></td><td></td><td> Presente do Dr. Tan Ince </td></tr><tr><td> Meio 199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> Célula Applications Inc. </td><td> 117-500 </td><td></td></tr><tr><td> FBS, inativado pelo calor </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> Pen-Strep </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Placas de 96 poços </td><td> Corning Costar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polyhydroxyethylmethacrylate (poli-HEMA) </td><td> Sigma Aldrich </td><td> 192.066-25G </td><td> Para poli-HEMA solução dissolver o pó poli-HEMA 6mg em 1 ml de EtOH a 95% </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> Diluir a 95% em 2 0 dH </td></tr><tr><td> Capuz de cultura de células </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> De cultura de tecidos incubadora </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> incubadora para poli-HEMA placas </td><td> Instrumentos Labline </td><td> Imperial III 305 </td><td></td></tr><tr><td> Centrífuga de mesa </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 prato fundo de vidro bem </td><td> MatTek corp. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> A fibronectina </td><td> Sigma </td><td> F1141-1mg </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td> Microscópio Timelapse: </td><td></td><td></td><td></td></tr><tr><td> Microscópio </td><td> Nikon </td><td></td><td> Ti-E Invertido Motorizado microscópio lapso de tempo de fluorescência com sistema integrado Foco Perfeito </td></tr><tr><td> Lente </td><td> Nikon </td><td></td><td> 20X-0,75 apeture numérica </td></tr><tr><td> Transiluminador de halogéneo </td><td> Nikon </td><td></td><td> 0,52 NA condensador de longa distância de trabalho </td></tr><tr><td> Os filtros de excitação e emissão </td><td> Chroma simples filtros passa em Nikon habitação </td><td></td><td> GFP Ex 480/40, Em 525/50 RFP-mCherry Ex 575/50 Em 640/50 </td></tr><tr><td> Transmissíveis e Epifluoresce caminho da luz </td><td> Sutter </td&gt; <td></td><td> Persianas inteligentes </td></tr><tr><td> Linear-codificado estágio motorizado </td><td> Nikon </td><td></td><td></td></tr><tr><td> Câmera do dispositivo resfriado Charged-Coupled </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Câmara de incubação com microscópio de temperatura e de CO 2 controlo </td><td> custom-built </td><td></td><td></td></tr><tr><td> Mesa de isolamento de vibração </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS Elements software </td><td> Nikon </td><td></td><td> Versão 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

O câncer de ovário é a quinta causa principal de mortes relacionadas ao câncer nos Estados Unidos 1. Apesar de uma resposta positiva inicial para terapias, 70 a 90 por cento das mulheres com câncer de ovário desenvolver com novas metástases, ea recorrência é muitas vezes fatal 2. É, portanto, necessário compreender como metástases secundárias surgir, a fim de desenvolver melhores tratamentos para o cancro intermediária e fase tardia do ovário. Metástase do cancro do ovário ocorre quando as células malignas destacar a partir do local do tumor primário e disseminar por toda a cavidade peritoneal. As células disseminadas pode formar aglomerados multicelulares, ou esferóides, que irá permanecer quer acopladas, ou implantes para órgãos dentro da cavidade peritoneal 3 (Figura 1, Filme 1). Todos os órgãos dentro da cavidade peritoneal são revestidas com um único, a camada contínua de células mesoteliais 4-6 (Figura 2). No entanto, as células mesoteliais estão ausentes da parte de baixoperitoneais massas tumorais, como revelado por estudos de micrografia electrónica excisadas humanos secções de tecido de tumor 3,5-7 (Figura 2). Isto sugere que as células mesoteliais são excluídos por baixo da massa do tumor por um processo desconhecido. Anterior, em experiências in vitro demonstraram que as células cancerosas do ovário primárias anexar mais eficientemente para a matriz extracelular do que para células mesoteliais 8, e os estudos mais recentes mostraram que os primários peritoneais células mesoteliais realmente proporcionar uma barreira para a adesão celular de ovário cancro e invasão (em comparação com adesão e invasão em substratos que não foram cobertos com células mesoteliais) 9,10. Isto sugere que as células mesoteliais actuar como uma barreira contra a metástase do cancro do ovário. Os mecanismos celulares e moleculares pelos quais as células de câncer de ovário violam esta barreira, e excluir o mesotélio, até recentemente, permaneceu desconhecido. Aqui descrevemos ªmetodologia e para um ensaio in vitro que os modelos de interacção entre esferóides de células cancerosas do ovário e células mesoteliais in vivo (Figura 3, Filme 2). O protocolo foi adaptado de métodos descritos anteriormente para analisar as interações ovarianos de células tumorais com monocamadas mesoteliais 8-16, e foi descrita pela primeira vez em um relatório mostrando que as células tumorais de ovário utilizar uma ativação dependente de integrinas de miosina e força de tração para promover a exclusão do células mesoteliais de 17 sob um tumor esferóide. Este modelo leva vantagem de lapso de tempo a microscopia de fluorescência para monitorar as populações de células de dois em tempo real, fornecendo informação espacial e temporal na interação. As células de cancro do ovário expressar vermelho proteína fluorescente (SDP), enquanto as células mesoteliais expressar a proteína verde fluorescente (GFP). RFP expressando esferóides de células cancerígenas de ovário anexar à monocamada expressando GFP-mesotelial. A propagação esferóides, invadir, eforçar as células mesoteliais de lado a criação de um buraco na monocamada. Este buraco é visualizado como o espaço negativo (preto) na imagem GFP. A área do orifício pode ser então medido quantitativamente analisar as diferenças na actividade folga entre o controlo e as populações experimentais de cancro do ovário e / ou células mesoteliais. Este ensaio requer apenas um pequeno número de células de ovário cancerosas (100 células por esferóides X 20-30 esferóides por condição), de modo que é possível realizar este ensaio usando preciosos primárias amostras de células tumorais. Além disso, este ensaio pode ser facilmente adaptado para rastreio de alto rendimento.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

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

O ensaio de depuração mesotelial descrito aqui leva vantagem das células marcadas com fluorescência e de microscopia de lapso de tempo de vídeo para visualizar e medir quantitativamente as interações de esferóides de câncer de ovário multicelulares e monocamadas de células mesoteliais. Este ensaio modela os passos iniciais da metástase do câncer de ovário.

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 ...

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

14.4K visualizações.  Harvard Medical School. O ensaio de depuração mesotelial descrito aqui leva vantagem das células marcadas com fluorescência e de microscopia de lapso de tempo de vídeo para visualizar e medir quantitativamente as interações de esferóides de câncer de ovário multicelulares e monocamadas de células mesoteliais. Este ensaio modela os passos iniciais da metástase do câncer de ovário.

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Ensaio Metástase Experimental

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medicina

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Modelos MAME para formação de imagens 4D Live-célula do tumor: Interações microambiente que a progressão de Impacto maligno

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal de imagem de angiogênese em um modelo de rato nu de Câncer de mama utilizando a ressonância magnética, tomografia computadorizada volumétrica e ultra-som

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Heterotípica tridimensional<em&gt; In Vitro</em&gt; Modelagem de Estroma-epitelial de ovário Interações durante o início e progressão do câncer

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Modelos de metástase óssea

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Em Enriquecimento vitro de câncer de ovário de iniciação Células Tumorais

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

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 ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In Vivo e Ex Vivo Abordagens para o Estudo do cancro do ovário metastático Colonização de Estruturas Láctea natural em Peritoneal adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

A quantificação da Intra-peritoneal Ovarian Cancer Metástase

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...