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 estudio fue aprobado por el Comité de Ética del Hospital de la Universidad Nacional de Chonnam Hwasun y la Universidad de Carolina del Sur. TMA muestras compuestas de cáncer de mama y tejidos de mama normales adyacentes emparejados fueron proporcionados por el Biobanco del Hospital Hwasun Chonnam Universidad Nacional, un miembro de la Red de Biobancos Corea. 1. Preparación de la solución <ol><li> Preparar 1 l de DNasa y RNasa libre de agua. Realiza todas las soluciones químicas mediante el uso de dietilo (DEPC) de agua tratada. Tratar 1 L de agua con 500 l de DEPC y se incuba durante 3 horas a temperatura ambiente. Autoclave el agua para inactivar DEPC. Precaución: No se requieren medidas DEPC ya que es conocido carcinógeno. </li><li> Preparar 1x PBS 10x de PBS utilizando agua tratada con DEPC. </li><li> Preparar 20x SSC (disolver 175,3 g de NaCl y 88,2 g de citrato de sodio en 800 ml de agua, ajustar el pH a 7,0 con unas pocas gota de 14 N solución de HCl y ajustar el volumen a 1 L con water), 4% de paraformaldehído, 95% de etanol, 80% de etanol con agua tratada con DEPC. </li><li> Añadir 8 ml de trietanolamina y 1,05 ml de HCl en 590 ml de agua tratada con DEPC y añadir 1,5 ml de anhídrido acético para la acetilación. </li><li> Preparar 50 g / l de proteinasa K en tampón (50 mM Tris-HCl, pH 8,0 en DEPC agua tratada). </li><li> Preparar tampón de hibridación compuesto por 500 l de formamida, 250 l de 20x SSC, 100 l de 50x solución de Denhardt, 12,5 l de t-RNA, 2.5 l de ADN de esperma de arenque, 30 l de RVC, 0,02 g de reactivo de bloqueo y 50 l de agua DEPC. Preparar fresco. </li></ol> 2. Las secciones de tejido de mama <ol><li> Cortar 5 micras secciones incluidas en parafina fijadas con formalina con microtomo y aplicar la sección 14 para cargar diapositivas. Nota: El tejido mamario se utiliza aquí se preparó previamente de la Red de Biobancos Corea. </li></ol> 3. Preparación del tejido <ol><li> Retire la parafina lado a otrom el tejido mediante la inmersión de diapositivas en 2x jarra Coplin en xileno fresco durante 10 minutos cada uno y rehidratar la sección de tejido por incubaciones de 5 min en concentraciones decrecientes de etanol (100%, 95% y 80%) en ddH 2 O. Realice este paso en una campana de humos. </li><li> Sumergir el tejido en DEPC agua tratada durante 5 min. En este punto completar límites en torno sección de tejido con la pluma barrera hidrofóbica. </li><li> Fijar el tejido con 4% PFA durante 15 min seguido de un lavado con PBS. </li><li> Sumergir el tejido en 0,3% de Triton X-100 / PBS durante 10 min seguido de un lavado con PBS. </li><li> Incubar la muestra con una solución / l de proteinasa K 50 mg durante 15 minutos a 37 ° C. A continuación incubar las secciones de tejido con trietanolamina y anhídrido acético durante 5 min a temperatura ambiente, seguido de un lavado con PBS. No se requiere un lavado con PBS entre el tratamiento con proteinasa K y / anhídrido acético tratamiento trietanolamina. </li></ol> 4. La hibridación <ol><li> Lavar el tejido a fondo y incubate la sección de tejido con tampón de hibridación para 2-3 horas a temperatura ambiente. Típicamente 120 l de tampón de hibridación es suficiente para cubrir la sección de tejido. </li><li> Diluir 5&#39;-DIG sonda marcada LNA a la concentración final de 3 horas, en la solución de hibridación 120 l. A medida que la concentración de reserva por lo general es de 40 ng / l, añadir 3 l de tampón de hibridación de valores para 120 l e incubar la mezcla a 95 ° C durante 5 min. </li><li> Incubar sección de tejido durante la noche con la sonda a 54 ° C (Tm) en una cámara húmeda. Típicamente 120 l es suficiente para cubrir una sección. Crear humedad mediante el uso de dos toallas de papel húmedo. El tamaño ideal de la cámara es de 10 pulgadas por 8 pulgadas. </li><li> Nota: Tm puede ser diferente para diferentes miARN. 54 ° C es la mención de miR-489. </li></ol> 5. rigurosidad de lavado <ol><li> Lavar el tejido con 5 x SSC durante 7 minutos (después de la etapa de hibridación, RNasa condiciones libres ya no son necesarios). </li><li> Lavar la sección de tejido ingenioh 1x SSC durante 7 minutos dos veces a 57 ° C. </li><li> Lavar la sección de tejido con 0,2 x SSC durante 7 minutos a 57 ° C. </li><li> Lavar la sección de tejido con 0,2 x SSC durante 7 minutos a temperatura ambiente. </li><li> sección de tejido en PBS en incubación durante 10 min. </li></ol> 6. Bloqueo <ol><li> Incubar la sección de tejido con tampón de bloqueo a temperatura ambiente durante 1 hr en una cámara húmeda (Para hacer el amortiguador de bloqueo añadir 2 ml de FBS, 10 l de Tween-20 y 3 l de BSA en 18 m de PBS. Tienda tampón de bloqueo a 4 DO). </li></ol> 7. Anticuerpo primario Incubación y Desarrollo <ol><li> Diluir 1: 300 de anticuerpo anti-DIG-AP en tampón de bloqueo y se incuba la sección de tejido con el anticuerpo a 4 ° C durante la noche. </li><li> Se lavan las secciones con tampón de lavado 3 veces, durante 5 minutos cada uno (tampón proporcionado por el kit de desarrollo). </li><li> Desarrollar la sección de tejido con un kit comercial para la fosfatasa alcalina que produce CY-3 luz fluorescente. </li> &lt;li&gt; Lavar la sección con tampón de lavado durante 5 min. sección de la mancha con 100 l de 2,5 ng l de colorante / Hoechst durante 5 min seguido de un lavado de 5 min con tampón de lavado. </li><li> Montar la sección con tampón de montaje de acuerdo con el protocolo del fabricante. Utilice 15 l por sección de tejido y limpie solución de montaje con el tejido excesivo limpie. Sellar la diapositiva con esmalte de uñas transparente para evitar fugas. </li><li> Observar al microscopio fluorescente usando la sección con un aumento de 10X lente de modo total es de 100X. </li></ol>

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There is nothing to disclose.

El objetivo de este estudio fue determinar el nivel de expresión de los genes miARN en los tejidos de cáncer de mama utilizando miARN hibridación in situ. Hay que señalar que en este experimento, todas las condiciones se optimizaron para el tejido de cáncer de mama. Una optimización adicional puede ser necesario para otros tipos de tejidos. Todas las soluciones deben hacerse con agua DEPC y todos los contenedores que se utilicen deben ser enjuagados con agua tratada con DEPC y posteriormente en autoclave. Incluso una ligera contaminación RNasa puede interferir con el resultado final del experimento. Como miARN es una secuencia muy corta, es necesario el uso de una sonda de LNA para facilitar mejor la hibridación con la diana. Además, después de la fijación del tejido, el tratamiento con RNasa libre de la proteinasa K es absolutamente necesario de otro modo las proteínas pueden interferir con la hibridación de la sonda con la endógeno miRNA. Además, antes de la incubación de la sección de tejido con la sonda, esta última debe ser hervida a 95 ° C a desplegarse cualquierestructuras secundarias. Después de incubar durante la noche, es importante lavar con diferentes concentraciones de SSC a la temperatura mencionada anteriormente en el protocolo para reducir la unión no específica. Para el desarrollo de la fluorescencia, incubar sección de tejido con el sustrato durante al menos 1 hr. Es conveniente observar la sección a lo largo de la incubación de manera que el desarrollo excesivo se puede evitar. Asegúrese de montar la sección con los medios de montaje suministrados con el kit. Hay un factor limitante que uno debe tomar en consideración. Si el objetivo miARN expresión es menor en el tejido que va a ser difícil de detectar su expresión usando esta técnica. Siempre es recomendable realizar este protocolo con un control positivo como U6 lo que la técnica puede ser confirmada y todos los reactivos se aseguran estar trabajando y que son libres de RNasa. Es posible utilizar una sonda marcada con biotina en lugar de sonda marcada con DIG en caso de señal débilal con DIG sonda marcada. la amplificación de la señal es posible mediante el uso de sistema de biotina-avidina. La avidina tiene una fuerte afinidad por la biotina. Al tomar ventaja de esta afinidad, es posible etiquetar sonda de biotina con avidina-biotina duplex que es adjuntar con fosfatasa alcalina. Mediante el uso de esta estrategia, es posible amplificar la señal de bajo miRNA abundante también. El campo de miARN está emergiendo rápidamente, ya que los miRNAs juegan un papel crítico en los procesos celulares fundamentales y están fuertemente vinculados con el desarrollo del cáncer. Además, estos miRNAs también se pueden utilizar como biomarcadores en el diagnóstico de cáncer. Varios estudios han indicado una fuerte correlación entre la expresión de los genes miARN y los parámetros clínicos tales como el estadio del tumor sugiere la posible aplicación de la miARN expresión en el pronóstico del cáncer. Esta técnica también se puede utilizar para el análisis de expresión de los genes miARN en otros tipos de cáncer. Por ejemplo, la expresión de miR-182 y miR-503 se analizó en los tejidos de pacientes con cáncer de colon usando en SIma hibridación de 15. análisis de expresión génica tradicional puede revelar el nivel de expresión de los genes miARN en el tejido, pero no puede revelar la ubicación desde la que la estructura está siendo expresada miARN. Mediante el uso de esta técnica, se puede encontrar fácilmente el nivel de expresión de los genes miARN, así como la ubicación.

El cáncer de mama es una de las neoplasias más comunes que afectan a la población femenina en todo el mundo. Más de 1,3 millones de casos de cáncer de mama cada año se registran en todo el mundo 1,2. Aunque las células tumorales han sido tradicionalmente considerado como biológicamente homogénea y altamente proliferativas, se ha hecho evidente que el cáncer de mama es genéticamente y clínicamente heterogéneos. La resistencia a medicamentos contra el cáncer prevalentes es una característica de los cánceres de mama avanzados, que conduce a la mortalidad en la mayoría de los pacientes a través de su facilitación de la progresión del cáncer y la metástasis a distancia 3. MicroARNs (miRNAs) son una clase de moléculas de ARN pequeñas de aproximadamente 22 nucleótidos de longitud que regulan la expresión génica mediante el bloqueo de la traducción del ARNm o la mediación de la degradación del ARNm 4. Más de 2.000 diferentes miRNAs Hasta ahora se han identificado en humanos, que están vinculados a enfermedades humanas 5. Un creciente gama de estudios VHAe demostró que los miRNAs pueden funcionar como oncogenes y supresores tumorales y son a menudo mal regulada en tumores 6. miRNAs afecta fuertemente la totalidad de la tumorigénesis primaria mediante el control de los principales reguladores de la progresión del ciclo celular, la senescencia, la apoptosis y la autofagia, junto con los de la motilidad de células tumorales, invasión y metástasis 7. La expresión aberrante de los genes miARN se ha asociado también con implicaciones clínicas como la etapa, la diferenciación, el pronóstico y respuesta al tratamiento adyuvante 8. En particular, el aumento de expresión de miR-146 se correlaciona con mal pronóstico en pacientes con cáncer de pulmón 9. Otro estudio ha demostrado que la expresión perdida de miARN-let7b está vinculada a fenotipos malignos y, en consecuencia, una baja supervivencia 6. La comprensión de los tipos de células y estructuras que expresan miRNAs es una parte importante de la comprensión de los mecanismos a través del cual las alteraciones en la célula de influir en la expresión de los genes miARN ytejido fenotipos 10. Ciertos miRNAs encontrado para ser secretada en las células del estroma se toman en por las células epiteliales y actúan específicamente sobre sus objetivos. En la misma línea, el miR-212 y miR-132 son secretadas por estroma y regulan las interacciones epitelio-estroma necesarios para derivación ductal durante el desarrollo de la glándula mamaria 11. El objetivo general de este trabajo es explicar un protocolo detallado para detectar la expresión de los genes miARN en tejidos de cáncer de mama a través de miRNA hibridación in situ. Hemos optimizado todas las condiciones para someter a ensayo los niveles de expresión de los genes miARN-489 en muestras de pacientes con cáncer de mama. Con este fin, se utilizó una DIG marcado bloqueado sonda de ácido nucleico (LNA) en nuestro experimento. Algunos de sus nucleótidos tenían una modificación LNA, es decir, un puente de metileno que conecta &#39;átomo de oxígeno y 4&#39; de la 2 átomo de carbono del esqueleto de ribosa que fija el nucleótido de tal manera que permanece &quot;bloqueado en su lugar&quot; después de la hibridación. Como resultado, es mucho más difícilpara el complejo hibridado para desnaturalizar 12,13.

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			<td height="20" style="height:20px;width:216px;">ELF-97</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td style="width:119px;">E6604</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Denhard&#39;t&#160;</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td align="right" style="width:119px;">750018</td>
			<td></td>
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		<tr height="21">
			<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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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Herring Sperm DNA</td>
			<td style="width:159px;">Promega&#160;</td>
			<td style="width:119px;">D1815</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Roche Blocking&#160;</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">11096176001</td>
			<td></td>
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		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
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		<tr height="21">
			<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>
			<td></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.

Se utilizó el tejido de cáncer de mama humano para determinar la expresión de miRNA-489. Se encontraron conducto de la glándula mamaria y las células epiteliales de expresar significativamente más altos miARN-489 niveles que el tejido adyacente tumor en dos pacientes (Figura 1A y 1B). Esto demuestra claramente que la expresión de miRNA-489 se ha perdido en el tejido tumoral, lo que sugiere una actividad supresora de tumores de miARN-489. Para confirmar aún más estos resultados, el tejido tumoral y el tejido normal adyacente del mismo paciente se compararon. Como se observa en la Figura 2A y 2B, se encontró que el tejido normal de expresar más miRNA-489 que su tejido tumoral adyacente. 10 micras de las imágenes indica la barra de escala. Las imágenes fueron tomadas mediante el uso de lentes de 10X. <img alt="Figura 1" src="/files/ftp_upload/53928/53928fig1.jpg" /> Figura 1: La expresión de miR-489 en el tejido mamario normal. Tanto las células tumorales y la estructura de conducción normales se muestran en la misma sección. Los tejidos son de diferentes pacientes de cáncer de mama. Conductos mamarios normales y glándulas mamarias expresan los genes miARN-489, mientras ausente en el tejido tumoral. Barra de escala = 10 micras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53928/53928fig2.jpg" /> Figura 2:. La expresión de miR-489 en el par de Pacientes con Cáncer de mama Un micro matriz de tejido se utilizó para estudiar la expresión de miR-489 en un paciente con cáncer de mama. Un par paciente representativo se muestra aquí. Barra de escala = 10 micras. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53928/53928fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta 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.

Análisis clínico-patológica de los genes miARN expresión en el cáncer de mama mediante el uso de tejidos miARN<em&gt; In Situ</em&gt; La hibridación

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

Video: 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.

Análisis de biofilm oral del paladar Expansores por fluorescencia de hibridación in situ y microscopía de escaneo láser confocal

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

Investigación

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.

Multimodal de imágenes de la angiogénesis en un modelo de rata desnuda de las metástasis óseas del cáncer de pecho utilizando imágenes de resonancia magnética, tomografía computarizada y la ecografía volumétrica

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.

Perfiles de microARN regulados por estrógeno en las células del cáncer de 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.

La cuantificación de la invasividad de las células del cáncer de mama El uso de un modelo (3D) Tres Dimensiones

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.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

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.

Mammosphere Ensayo Formación de cáncer de mama humano tejidos y líneas 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.

En vivo y ex vivo para estudiar Cáncer de ovario metastásico colonización de Estructuras Vía lugar en 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.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

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).

Los análisis de expresión de miRNA en tejidos clínicos sobre el cáncer de próstata

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.

Biología de Sistemas de regulación metabólica por el estrógeno receptor de señalización en cáncer de mama

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