This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

Este estudo foi aprovado pelo Institutional Review Board do Hospital Chonnam National University Hwasun e University of South Carolina. amostras TMA compostas de câncer de mama e tecidos mamários normais adjacentes combinados foram fornecidos pelo Biobank de Chonnam National Hwasun Hospital Universitário, um membro da Coreia do Biobank rede. 1. Preparação de Soluções <ol><li> Prepare 1 litro de DNase e RNase água livre. Faça todas as soluções químicas usando dietilpirocarbonato (DEPC) água tratada. Tratar 1 L de água com 500 ul de DEPC e incubar durante 3 horas a temperatura ambiente. Autoclave a água para inactivar DEPC. Cuidado: Não inalar DEPC, uma vez que é conhecido agente cancerígeno. </li><li> Prepare 1x PBS a partir de 10x PBS usando DEPC água tratada. </li><li> Prepare 20x SSC (dissolver 175,3 g de NaCl e 88,2 g de citrato de sódio em 800 ml de água, ajustar o pH a 7,0 com algumas gota de 14 N solução de HCl e ajustar o volume a 1 litro com water), paraformaldeído a 4%, etanol a 95%, etanol a 80% usando água tratada com DEPC. </li><li> Adicionar 8 mL de trietanolamina e 1,05 mL de HCl em 590 ml de água tratada com DEPC e adicionar 1,5 ml de anidrido acético para acetilação. </li><li> Preparar 50 ug / mL de proteinase K em tampão (Tris-HCl a 50, pH 8,0 em água tratada com DEPC). </li><li> Preparar tampão de hibridação constituída por 500 mL de formamida, 250 pL de 20x SSC, 100 ul de solução 50x de Denhardt, 12,5 ul de t-RNA, 2,5 ul de ADN de esperma de arenque, 30 ul de RVC, 0,02 g de reagente de bloqueio e 50 ul de água DEPC. Prepare fresco. </li></ol> 2. Os pontos tecido mamário <ol><li> Corte de 5 mm secções embebidas em parafina fixadas em formalina com micrótomo e aplicar a secção de cobrar slide 14. Nota: O tecido da mama usada aqui foi previamente preparado a partir da Coreia do Biobank rede. </li></ol> 3. Preparação do Tecido <ol><li> Remova a parafina from o tecido por imersão em lâminas 2x frasco Coplin em xileno fresco durante 10 minutos cada e re-hidratar a secção de tecido por 5 min em incubações concentrações decrescentes de etanol (100%, 95% e 80%) em ddH 2 O. Execute esta etapa em um exaustor. </li><li> Mergulha-se o tecido em água tratada com DEPC, durante 5 min. Nesta altura, levar limites em torno secção de tecido com a pena barreira hidrofóbica. </li><li> Corrigir o tecido com PFA a 4% durante 15 min, seguido por uma lavagem com PBS. </li><li> Mergulha-se o tecido em 0,3% de Triton X-100 / PBS durante 10 minutos seguido por uma lavagem com PBS. </li><li> Incubar amostra com uma solução de / ul de proteinase K 50 ^ g durante 15 min a 37 ° C. Em seguida, incubar as secções de tecido com trietanolamina e anidrido acético durante 5 min à temperatura ambiente, seguido por uma lavagem com PBS. Sem lavagem com PBS é necessária entre o tratamento com proteinase K e / tratamento anidrido acético trietanolamina. </li></ol> 4. Hibridização <ol><li> Lavar o tecido completamente e incuBATE a secção de tecido com tampão de hibridação durante 2 a 3 horas à temperatura ambiente. Tipicamente 120 ul de tampão de hibridação é suficiente para cobrir a secção de tecido. </li><li> Diluir 5&#39;-DIG marcado com sonda LNA a concentração final de 3:00 em 120 ul de solução de hibridação. À medida que a concentração de estoque geralmente é de 40 ng / ul, adicionar 3 ul de estoque de tampão de hibridação de 120 ul e incubar mistura a 95 ° C durante 5 min. </li><li> Incubar secção de tecido com pernoite sonda a 54 ° C (Tm) em câmara úmida. Normalmente 120 mL é suficiente para cobrir uma seção. Criar umidade usando duas toalhas de papel molhado. O tamanho ideal da câmara é de 10 polegadas por 8 polegadas. </li><li> Nota: Tm pode ser diferente para diferentes miRNA. 54 ° C é de mencionar para miR-489. </li></ol> 5. lavagem rigorosa <ol><li> Lave o tecido com 5x SSC para 7 min (após o passo de hibridação, RNase condições livres não são mais necessários). </li><li> Lave a sagacidade secção de tecidoh 1x SSC durante 7 min, duas vezes a 57 ° C. </li><li> Lava-se a secção de tecido com SSC 0,2x durante 7 minutos a 57 ° C. </li><li> Lava-se a secção de tecido com SSC 0,2x durante 7 min à temperatura ambiente. </li><li> Incubar secção de tecido em PBS durante 10 min. </li></ol> 6. Bloqueio <ol><li> Incubar a secção de tecido com tampão de bloqueio à temperatura ambiente durante 1 hora numa câmara húmida (Para fazer um tampão de bloqueio e juntar 2 ml de FBS, 10 ul de Tween-20 e 3 pi de BSA em 18 m de PBS. Loja tampão de bloqueio a 4 ° C). </li></ol> 7. Anticorpo primário Incubação e Desenvolvimento <ol><li> Diluir 1: anticorpo anti-DIG-AP 300 em tampão de bloqueio e incubar a secção de tecido com o anticorpo, a 4 ° C durante a noite. </li><li> Lavar as seções com tampão de lavagem 3 vezes, durante 5 minutos cada (tampão fornecido pelo kit de desenvolvimento). </li><li> Desenvolver a secção de tecido com um kit comercial para fosfatase alcalina que produz CY-3 luz fluorescente. </li> &lt;li&gt; Lava-se a secção com tampão de lavagem durante 5 minutos. secção mancha com 100 ul de 2,5 ng / mL de corante Hoechst, durante 5 minutos, seguido de uma lavagem de 5 min com tampão de lavagem. </li><li> Montar o tampão com secção de montagem de acordo com o protocolo do fabricante. Use 15 ul por secção de tecido e limpe solução de montagem excessiva com lenço de papel. Selar o slide com unhas polonês claro para evitar vazamentos. </li><li> Observe o microscópio fluorescente seção usando com ampliação de 10x lente de modo total é de 100X. </li></ol>

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There is nothing to disclose.

O objetivo deste estudo foi determinar o nível de expressão miRNA em tecidos de câncer de mama utilizando miRNA hibridização in situ. Deve ser notado que nesta experiência, todas as condições foram optimizadas para o tecido do cancro da mama. Optimização adicional pode ser necessário para outros tipos de tecidos. Todas as soluções deve ser feita com água com DEPC e todos os recipientes a ser usado deve ser lavado com água tratada com DEPC e subsequentemente autoclavadas. Mesmo fraca contaminação RNase pode interferir com o resultado final do experimento. Como miARN é uma sequência muito curto, é necessário o uso de uma sonda de LNA para facilitar uma melhor hibridação com o alvo. Além disso, após a fixação do tecido, o tratamento com RNase A proteinase K é absolutamente necessário caso contrário as proteínas podem interferir com a hibridação da sonda com o miARN endógeno. Além disso, antes da incubação da secção de tecido com a sonda, esta última deve ser fervida para 95 ° C para se desdobrar qualquerestruturas secundárias. Após incubação durante a noite, é importante lavar com concentrações variáveis ​​de SSC à temperatura acima mencionada no protocolo para reduzir a ligação não específica. Para o desenvolvimento de fluorescência, incubar secção de tecido com substrato durante pelo menos 1 h. É aconselhável para observar a secção ao longo da incubação desenvolvimento excessivo de modo a que possa ser evitada. Certifique-se de montar a seção com os meios de comunicação de montagem fornecidos com o kit. Há um fator limitante que deve-se levar em consideração. Se a expressão miRNA-alvo é menor no tecido que vai ser difícil de detectar a sua expressão usando esta técnica. É sempre aconselhável realizar este protocolo com um controlo positivo, como U6 assim que a técnica pode ser confirmada e todos os reagentes são assegurados por estar trabalhando e eles são RNase livre. É possível a utilização de uma sonda marcada, em vez de biotina DIG sonda marcada, em caso de sinal fracoal com DIG sonda marcada. amplificação do sinal é possível usando o sistema biotina-avidina. Avidina tem forte afinidade para a biotina. Ao aproveitar essa afinidade, é possível marcar sonda de biotina com duplex avidina-biotina que é anexar com fosfatase alcalina. Ao utilizar esta estratégia, é possível amplificar o sinal para baixo miRNA abundante também. O campo de miRNA está emergindo rapidamente, uma vez que miRNAs desempenham papéis críticos em processos celulares importantes e estão fortemente ligados ao desenvolvimento do câncer. Além disso, estes miARNs também pode ser utilizado como biomarcador no diagnóstico do cancro. Vários estudos têm indicado uma forte correlação entre a expressão de miRNA e parâmetros clínicos tais como o estágio do tumor sugerindo a possibilidade de aplicação de expressão miRNA no prognóstico do câncer. Esta técnica também pode ser utilizada para análise de expressão de miARN em outros tipos de cancro. Por exemplo, a expressão de miR-182 e miR-503 foram analisadas em tecidos de pacientes com cancro do cólon usando em Sihibridação tu 15. análise de expressão gênica tradicional pode revelar nível de expressão miRNA em tecidos mas não pode revelar a localização a partir do qual a estrutura miRNA está sendo expresso. Usando esta técnica, pode-se facilmente encontrar o nível de expressão de miRNA, bem como localização.

O cancro da mama é um dos tumores malignos mais comuns que afetam a população feminina em todo o mundo. Mais de 1,3 milhões de casos de câncer de mama são notificados a cada ano no mundo 1,2. Embora as células tumorais têm sido tradicionalmente considerados como biologicamente homogênea e altamente proliferativa, tornou-se evidente que o câncer de mama é geneticamente e clinicamente heterogênea. A resistência aos fármacos anticancerígenos prevalentes é uma característica de cancros da mama avançado, conduzindo à mortalidade na maioria dos pacientes através da sua promoção da progressão do cancro e metástases à distância 3. MicroRNAs (miRNAs) são uma classe de pequenas moléculas de RNA de aproximadamente 22 nucleótidos de comprimento que regulam a expressão do gene através do bloqueio da tradução do mRNA ou mediar a degradação de ARNm 4. Mais de 2.000 miRNAs diferentes foram até agora identificados em humanos, que estão ligadas a doenças humanas 5. Uma crescente variedade de estudos have demonstraram que miARN pode funcionar como oncogenes e supressores tumorais e são frequentemente desreguladas em tumores 6. miARNs impacto fortemente toda a tumorigénese principal, controlando as principais reguladores da progressão do ciclo celular, senescência, apoptose e autofagia, juntamente com os da motilidade de células tumorais, invasão e metástase 7. Expressão aberrante miRNA foi também associada a implicações clínicas, tais como estágio, diferenciação, prognóstico e resposta ao adjuvante terapia 8. Em particular, o aumento da expressão de miR-146 está correlacionada com mau prognóstico em pacientes com câncer de pulmão 9. Outro estudo demonstrou que a expressão perdida de miRNA-let7b está ligada a fenótipos malignas e, consequentemente, pior sobrevida 6. Compreender os tipos de células e estruturas que expressam miRNAs é uma parte importante de compreender os mecanismos através dos quais alterações nas células de expressão influência miRNA etecido fenótipos 10. Certos miARNs encontrado para ser segregado em células do estroma são tomadas por células epiteliais e, especificamente, agir sobre os seus objectivos. Na mesma linha, o miR-212 e miR-132 que são segregadas por estroma e regular as interacções epiteliais-estromal requeridas para crescimento ductal durante 11 desenvolvimento da glândula mamária. O objetivo geral deste trabalho é explicar um protocolo detalhado para detectar a expressão de miRNA em tecidos de cancro da mama através de miRNA hibridização in situ. Temos todas as condições optimizadas para ensaiar os níveis de expressão de miARN-489 em amostras de pacientes de cancro da mama. Para este fim, foi utilizado um DIG marcado bloqueado ácido sonda (LNA) nucleico em nosso experimento. Alguns dos seus nucleótidos tinha uma modificação LNA, que é, uma ponte metileno que liga &#39;átomo de oxigénio e 4&#39; a 2 átomos de carbono do esqueleto ribose que fixa as sequências de nucleótidos de tal modo que ele fica &quot;bloqueado no lugar&quot; após a hibridização. Como resultado, é muito mais difícilpara o complexo hibridado para desnaturar 12,13.

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			<td height="21" style="height:21px;width:216px;">50X Denhard&#39;t&#160;</td>
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			<td height="21" style="height:21px;width:216px;">t-RNA</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">109541</td>
			<td>Reconstitute in DEPC water</td>
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			<td height="21" style="height:21px;width:216px;">Herring Sperm DNA</td>
			<td style="width:159px;">Promega&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Roche Blocking&#160;</td>
			<td style="width:159px;">Roche</td>
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			<td height="21" style="height:21px;width:216px;">RVC</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:119px;">50-812-650</td>
			<td>Before use spin down it at 16.1 RCF and take supernatant&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miR-489 probe&#160;</td>
			<td style="width:159px;">Exiqon</td>
			<td style="width:119px;">38599-01</td>
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			<td height="21" style="height:21px;width:216px;">Nuclease free BSA</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">711454</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Primary antibody-anti DIG</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">11093274910</td>
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			<td height="21" style="height:21px;width:216px;">Diethyl Pyrocarbonate&#160;</td>
			<td style="width:159px;">Sigma</td>
			<td align="right" style="width:119px;">1609478</td>
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clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

tecido do cancro da mama humano foi utilizado para determinar a expressão de miARN-489. Ducto da glândula mamaria e células epiteliais foram encontrados para expressar significativamente mais elevados de miARN-489 níveis que o tecido adjacente do tumor em dois pacientes (Figura 1A e 1B). Isto demonstra claramente que a expressão de miARN-489 foi perdido no tecido tumoral, sugerindo que a actividade supressora do tumor de miARN-489. Para confirmar ainda mais estes resultados, o tecido tumoral e tecido normal adjacente a partir do mesmo paciente foram comparados. Como pode ser observado na Figura 2A e 2B, o tecido normal foi encontrado para expressar mais de miARN-489 do que o seu tecido tumoral adjacente. 10 mm nas imagens indica barra de escala. As imagens foram tiradas usando lente de 10X. <img alt="figura 1" src="/files/ftp_upload/53928/53928fig1.jpg" /> Figura 1: Expressão de miR-489 no tecido mamário normal. Tanto as células tumorais estrutura adesiva e normais são exibidos na mesma secção. Os tecidos são de pacientes com câncer de mama diferentes. ducto mamário normal e glândula mamária expressar miRNA-489 estando ausente no tecido tumoral. Barra de escala = 10 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53928/53928fig2.jpg" /> Figura 2:. Expressão de miR-489 em paciente par o cancro da mama Uma micro matriz de tecido foi usado para estudar a expressão de miR-489 em um paciente com cancro da mama. Um paciente par representativo é mostrado aqui. Barra de escala = 10 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Análise clínico-patológico de miRNA Expressão em cancro da mama tecidos usando miRNA<em&gt; In Situ</em&gt; Hibridização

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

clinicopathological-analysis-mirna-expression-breast-cancer-tissues

Nanyang Technological University

Research

JoVE Journal

Medicine

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

6.7K visualizações.  University of South Carolina. Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Vídeo: Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Análise de biofilme oral de Palatal Expansores por fluorescência de hibridização in situ e microscopia confocal a laser Scanning

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Medicina

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 ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling de MicroRNAs estrogênio regulados em células do cancro da mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificação de cancro da mama invasiva das células usando um modelo (3D) tridimensional

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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

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

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

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

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

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Formação Mammosphere Ensaio de cancro da mama humano tecidos e linhas celulares

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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 ...

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 ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

As análises de expressão de miRNA no câncer de próstata tecidos clínicos

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

Biologia de sistemas de regulação metabólica pelo receptor de estrogênio sinalização no cancro da mama

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...