MB é suportado por financiamento da concessão de uma bolsa de estudo do MRC. KP e AHM são suportados por financiamento da concessão de Wessex Medical Research and Cancer Research UK / RCS (Inglaterra) (C28503/A10013). Somos gratos pelo apoio da Universidade de Southampton Unidade de Pesquisa histoquímica.

1. Estabelecimento de culturas primárias de fibroblastos de cólon explantes <ol><li> Obtenção de amostras de tecido de mucosa de cólon normal humano directamente a partir da sala de operações cirúrgicas, e suspender em 7-10 ml de PBS suplementado com 100 unidades / ml de penicilina, 100 ug / ml de estreptomicina, e 0,25 ng / mL de Fungizone. </li><li> No laboratório, colocar a amostra no centro de um 10 cm de prato de cultura de tecidos e lava-se 3 vezes com PBS / Pen-strep / Fungizone. Não aspirar após a lavagem final. </li><li> Com fórceps estéreis e bisturi, cortar o tecido em pequenos pedaços (cerca de 2 mm) e colocar cada peça no centro de uma cruz desenhada com a lâmina de bisturi em uma placa de 12 poços. Balance o bisturi suavemente sobre o tecido para permitir que ele aderir. </li><li> Cultura das amostras em 750 ul de meio de crescimento de fibroblastos básico (DMEM suplementado com 20% de soro fetal de vitelo (FCS), 100 U / ml de penicilina, 100 ug / ml de estreptomicina e 292 ng / ml de L-glutamina), que deve submerge o tecido sem permitir que flutue. Evite perturbar a placa ao longo dos próximos 5 dias, exceto para re-alimentação a cada 2 dias. </li><li> Ao longo dos 5 dias seguintes fibroblastos começam a crescer lentamente para fora a partir da borda do tecido. Em cerca de sete dias, este será mais visível. Após cerca de 3-4 semanas, e se existem suficientes fibroblastos crescem para fora, adicionar 750 ul de tripsina a cada poço. Depois de células têm destacado (o que pode demorar 5-10 min), piscina e transferir para um frasco de cultura de tecidos T-25. </li></ol> 2. Organotípicas Preparação Preparação do fibroblasto impregnado em gel organotípica: <ol><li> Prepara-se uma suspensão de células de fibroblasto, contendo 5 x 10 5 células por organotípicas em DMEM suplementado com FCS a 10% e 292 ug / ml de L-Glutamina. </li><li> Completar os géis organotípicas no gelo, nas seguintes proporções: <ul><li> 7 volumes de Rat-Tail Colágeno: Matrigel mistura (1:1) </li><li> 1 volume de filtrado 10x DMEM </li><li> 1 volume de FCS </li><li> Um volume de suspensão de células de fibroblasto, contendo 5 x 10 5 fibroblastos Para 9 géis (1 ml por gel) preparar 10 ml e misture com cuidado para evitar bolhas. </li></ul></li><li> Adicionar 1 ml a cada poço de uma placa de 24 poços e incubar durante 1 hora a 37 ° C em uma atmosfera húmida para permitir que os geles para definir. Depois de 1 hora adicionar 1 mL de DMEM (FCS a 10%) / Glut no topo dos géis e retornar para a incubadora durante a noite. </li><li> No dia seguinte, o meio aspirado a partir do topo do gel e da placa 1 ml de meio contendo 5 x 10 5 células epiteliais CRC. </li></ol> Preparação de folhas de nylon revestido de gel: <ol start="5"><li> Prepare-se estéril, folhas de nylon autoclavados medindo 1,5 cm x 1,5 cm, com antecedência. </li><li> Utilizando fórceps estéreis, colocar o número requerido de folhas de nylon em uma placa de cultura de 10 centímetros (geralmente quatro folhas de nylon podem ser acomodados por prato). </li><li> Make up gel no gelo na seguinte ordem (250 mL total devolume é necessário por folha de nylon): <ul><li> 7 volumes de Rat-Tail Colágeno </li><li> 1 volume de filtrado 10x DMEM </li><li> 1 volume de FCS </li><li> 1 volume de DMEM 10% de FCS / Glut </li></ul></li><li> Se a solução é amarela, neutralizar por adição de 0,1 M de NaOH em 50 mL alíquotas até a solução torna-se rosa. </li><li> Adicionar 250 ul desta solução para cada uma das folhas de nylon e incubar a 37 ° C numa atmosfera humidificada de 5% de CO 2 durante 30 min para permitir que o gel para definir. </li><li> Perfazer solução de glutaraldeído a 1% em PBS. Adicionam-se 10 ml a cada 10 centímetros placa de cultura, uma vez que o gel se definir e incubar a 4 ° C durante 1 hora. </li><li> Folhas de nylon de lavagem 3x em PBS 1x e em DMEM (FCS a 10%) / Glu e incubar durante a noite em DMEM / Glu a 4 ° C. </li></ol> Levantando géis organotípicas para grades de aço para invasão: <ol start="12"><li> Pré-preparar grades andaimes dobrando chapas de aço inoxidável em uma formação tripé. Autoclave antes do uso.</li><li> Use uma pinça esterilizada em etanol para colocar uma grade de aço em cada poço de uma placa de seis bem. </li><li> Colocar uma folha de nylon revestido com gel de colagénio com o lado superior, em cada grelha. </li><li> Transferir cuidadosamente os géis organotípicas da placa de 24 cavidades para a folha de nylon levantadas usando uma espátula esterilizada em etanol. </li><li> Encher o poço de DMEM com 10% de FCS / Glut suplementado com FCS a 10% até que a folha de nylon está em contacto com o suporte, mas não submersa. Certifique-se que o meio não toca o gel organotípica. </li><li> Incubar durante 14 dias a 37 ° C numa atmosfera humidificada de 5% de CO 2, substituindo mídia a cada 2 dias. </li></ol> 3. Organotípicas Fixation <ol><li> Remover todo organotípica incluindo folha de nylon bem e coloque em película aderente. </li><li> Bisseccione organotípica e folha de nylon com um bisturi descartável limpo e corrigir as duas metades em formol por 24 horas à temperatura ambiente. </li><li> Substitua formaldeído com 70% ethanol depois de 24 horas e deixe durante a noite antes da inclusão em parafina, corte e coloração. </li></ol> 4. Laser Microdissecção da margem invasiva <ol><li> Secção de 10 um de espessura para uma membrana de lâminas montado. </li><li> Retire a parafina seções aplicando xileno por 1 min, em seguida, retire xileno e corrigir em etanol 75% por mais 1 min. </li><li> Remover etanol e mancha com 0,125% solução Cresyl Violeta por 1 min. Cresyl Violeta destaca células epiteliais e permite fácil discriminação do estroma. </li><li> Retirar violeta de cresilo e aplicar etanol a 100% durante mais 1 min antes da lavagem com etanol a 100%. Deixar as lâminas de ar seco por 30 min. </li><li> Conduzir microdissecção a laser usando plataforma escolhida (por exemplo, o sistema Leica AS). </li><li> Coloque a lâmina de vidro com a parte manchada para ser microdissectados a face para baixo no palco microscópio. </li><li> Montar um tubo de microcentrífuga de 0,5 ml na cassete de recolha e adicionar 50 ul detampão de lise celular (por exemplo, DNA, RNA ou proteína de tampão de lise) na tampa na qual o tecido microdissecção irá recolher. </li><li> Sob visão direta, utilize o joystick para identificar tecido de interesse, e usando a interface do software, anotar a seção de produtos orgânicos manchado, com destaque para as células a serem microdissectados na frente de invasão tumoral. </li><li> Instrua o laser para o fogo, que deve tanto cortar a seção de destaque e impulsionar o tecido microdissecção na tampa do tubo de microcentrífuga. </li><li> Uma vez que o material tenha sido recolhida suficiente, ejectar a cassete de recolha, a fechar o tubo de microcentrífuga e girar suavemente para desenhar o tampão de lise para o fundo do tubo. </li><li> Coloque as amostras em gelo até microdissection está completa. Processe as amostras por um método adequado para extrair a substância a analisar de interesse. </li></ol>

<ol>
	<li>Nystrom, M. L., Thomas, G. J., Stone, M., Mackenzie, I. C., Hart, I. R., Marshall, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J Pathol. 205 (4), 468-475 (2005).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-153+supports+colorectal+cancer+progression+via+pleiotropic+effects+that+enhance+invasion+and+chemotherapeutic+resistance.">miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance.</a> Cancer Res. , (2013).</li><li>Bullock, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+actions+of+miR-21+highlight+the+critical+role+of+deregulated+stromal+microRNAs+during+colorectal+cancer+progression.">Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.</a> Cell Death Dis. 20 (4), (2013).</li><li>Jenei, V., Nystrom, M. L., Thomas, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+invasion+in+an+organotypic+model.">Measuring invasion in an organotypic model.</a> Methods Mol Biol. 769, 223-232 (2011).</li><li>De Wever, O., Demetter, P., Mareel, M., Bracke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+myofibroblasts+are+drivers+of+invasive+cancer+growth.">Stromal myofibroblasts are drivers of invasive cancer growth.</a> Int J Cancer. 123 (10), 2229-2238 (2008).</li><li>Fries, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+fibroblast+heterogeneity+and+the+role+of+fibroblast+subpopulations+in+fibrosis.">Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.</a> Clin Immunol Immunopathol. 72 (3), 283-292 (1994).</li><li>Moutasim, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betel-derived+alkaloid+up-regulates+keratinocyte+alphavbeta6+integrin+expression+and+promotes+oral+submucous+fibrosis.">Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.</a> J Pathol. 223 (3), 366-377 (2011).</li><li>Winter, J., Jung, S., Keller, S., Gregory, R. I., Diederichs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+roads+to+maturity:+microRNA+biogenesis+pathways+and+their+regulation.">Many roads to maturity: microRNA biogenesis pathways and their regulation.</a> Nat Cell Biol. 11 (3), 228-234 (2009).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+miRNA+expression+patterns+using+total+RNA+extracted+from+matched+samples+of+formalin-fixed+paraffin-embedded+(FFPE)+cells+and+snap+frozen+cells.">Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.</a> BMC Biotechnol. 7 (36), (2007).</li><li>Szafranska, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+molecular+characterization+of+formalin-fixed,+paraffin-embedded+tissues+by+microRNA+expression+profiling.">Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.</a> J Mol Diagn. 10 (5), 415-423 (2008).</li></ol>

Os autores não divulgar os potenciais conflitos de interesse.

Aqui nós descrevemos um método para isolar e caracterizar especificamente as células tumorais que tenham adquirido a capacidade de invadir o estroma associado câncer durante os primeiros estágios da progressão metastática em um 3-dimensional construção de co-cultura de células epiteliais e estromais. Modelos de co-cultura composto por células epiteliais CRC justapostos com um estroma sintético contendo ex vivo fibroblastos cólon humanos foram usados ​​para estudar a...

Organotípicas co-culturas são modelos que ajudam na reconstrução do microambiente do tumor in vivo em 3-dimensões justapondo células epiteliais malignas e as células do estroma em um gel de colágeno contendo essenciais componentes da matriz extracelular 1-3 engenharia de tecidos. Principalmente concebido como um método de medir a invasão do tumor, organotypics reduzir a dependência em modelos animais in vivo, e evitar os problemas de outras técnicas in vitro tais como ensaios de Transwell, em que as células invasoras são forçados artificialmente em um estado de dispersão mono-2-4. A inclusão de células estromais nestes modelos, tais como fibroblastos, reflecte o papel fundamental do microambiente do tumor na regulação da invasão maligna e metástase 3-4, no entanto, a heterogeneidade fenotipica do estroma é de tal modo que, a fim de maximizar a relevância fisiológica de organotípicas modelos, específica do órgão, anatomicamente precisos ex vivofibroblastos devem ser incluídos na medida do possível 3,6. Organotypics são uma plataforma versátil para estudar as interações entre células tumorais e estromais e são cada vez mais utilizados em novas maneiras de examinar o impacto sobre a invasão do tumor de inibidores químicos e alterações no gene alvo 7,8. Estudar a margem de tumor invasivo é uma perspectiva particularmente emocionante. Em modelos organotípicas, cancro colorectal estabelecido (CRC) linhas celulares produzem tipicamente uma camada epitelial bem estratificada, e em secção transversal, as células que adquiriram a capacidade de invasão da matriz extracelular (ECM) são facilmente discerníveis. À luz da heterogeneidade de micro-topográfico estabelecido de tumor e do tumor associado células estromais in vivo, extraindo estas células utilizando microdissecção laser e estudá-los de forma isolada, pode revelar percepções biológicas importantes sobre a origem da metástase do cancro colo-rectal e de como ela pode ser mais eficiente segmentado. Eun a seguinte metodologia, todos os pacientes que doaram amostras de tecido prestados consentimento informado, eo estudo foi aprovado pelo comitê de ética das instituições regionais de pesquisa.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colorectal cancer cell lines - example shown SW480</td>
			<td style="width:132px;">ATCC</td>
			<td style="width:119px;">ATCC CCL-228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matrigel</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon membrane</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">VVLP01300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Metal grid</td>
			<td style="width:132px;">The Mesh Company</td>
			<td>WSS20-A4</td>
			<td>themeshcompany.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser microdissection platform</td>
			<td style="width:132px;">Leica Microsystems</td>
			<td style="width:119px;">Leica AS LMD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Membrane mounted slides</td>
			<td style="width:132px;">Molecular devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cresyl Violet</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">1052350025</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

Invadir o câncer colorretal células (CRC) adquiriram a capacidade de se libertar de suas células-irmãs, infiltrar o estroma, e remodelar a matriz extracelular (ECM). Caracterizando a biologia desse grupo fenotipicamente distintos de células pode melhorar substancialmente a nossa compreensão dos eventos iniciais durante a cascata metastática. A invasão tumoral é um processo dinâmico, facilitado pela interacção bidireccional entre epitélio maligno e o estroma cancro associado. A fim de analisar as respostas específicas de células no interface estroma do tumor que se combinaram organotípicas de co-cultura e as técnicas de micro-dissecção de laser. Modelos organotípicas, em que constituintes do estroma-chave, tais como fibroblastos são 3-dimentioanally co-cultivados com células epiteliais cancerosas, são ferramentas experimentais altamente manipuláveis ​​que permitem invasão e interações câncer-estroma a serem estudados em quase phcondições ysiological. Laser microdissection (LMD) é uma técnica que implica a dissecção cirúrgica e extração de vários estratos dentro do tecido do tumor, com nível de precisão mícron. Ao combinar estas técnicas com perfil genômico, transcriptomic e epigenética pretendemos desenvolver uma compreensão mais profunda das características moleculares de invadir as células do tumor e tecido circundante do estroma, e ao fazê-lo, potencialmente revelar novos biomarcadores e oportunidades para o desenvolvimento de drogas no CRC.

Temos aplicado o método de cima para várias combinações de linhas celulares de CRC e células estromais. Um exemplo apresentado aqui é linhagens de células SW480 epitelial CRC com ex vivo fibroblastos humanos primários do cólon. 3-dimensional co-culturas são construídas (Figura 1), e submetido a microscopia de captura laser de microdissecção (Figura 2), e analisados ​​por microRNA (miRNA) perfis (Figura 3) para a comparação destes pequenos RNA...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Perfil molecular de células de laser e microdissecados do estroma, modelos de cancro colorectal sintéticos engenharia de tecidos representa uma abordagem nova e manipulável para caracterizar e dissecar a biologia distinta na interface entre as células tumorais na frente invasiva e células estromais de cancro associado.

Profiling Molecular do microambiente do tumor invasivo em um modelo 3-Dimensional de células de câncer colorretal e<em&gt; Ex vivo</em&gt; Os fibroblastos

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

molecular-profiling-invasive-tumor-microenvironment-3-dimensional

Research

JoVE Journal

Medicine

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

11.0K visualizações.  University of Southampton School of Medicine.  Perfil molecular de células de laser e microdissecados do estroma, modelos de cancro colorectal sintéticos engenharia de tecidos representa uma abordagem nova e manipulável para caracterizar e dissecar a biologia distinta na interface entre as células tumorais na frente invasiva e células estromais de cancro associado. 

Vídeo: Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Na imagem de bioluminescência vivo Tumor de mama Dynamics Hipóxia metástase do cancro cerebral em um modelo de rato

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Medicina

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modelagem e imagem 3-Dimensional Invasion cela coletiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Triagem para Modificadores Melanoma usando um modelo de tumor Zebrafish autóctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Viver de imagem de Respostas de drogas no microambiente tumoral em modelos do rato do cancro da mama

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Início do Carcinoma de mama metastático por Segmentação do ductal Epithelium com Adenovírus-Cre: Um Modelo Novel de camundongo transgênico do Câncer de Mama

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Um<em&gt; Ex vivo</em&gt; Modelo para estudar a ação do hormônio no peito humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Isolamento e propagação de células tumorais circulantes de um cancro do modelo do rato

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Um tumor cerebral / Slice organotípicas Co-cultura Sistema de Estudos de Tumor Microenvironment e alvejado droga Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...