Name: mirna expression analyses in prostate cancer clinical tissues
Uploaded: 2015-09-08T14:00:00
Description: Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

Agradecemos al Dr. Roger Erickson por su apoyo y asistencia en la preparación del manuscrito. Este trabajo fue apoyado por el Instituto Nacional del Cáncer, de los Institutos Nacionales de Salud (Número de Grant RO1CA177984; RO1CA138642), el proyecto de programa de VA sobre el cáncer de próstata (BX001604).

Se obtuvieron fijado en formol, embebido en parafina (FFPE) o muestras de cáncer de próstata congelados frescos del SFVAMC. Las muestras procedían de pacientes con cáncer de próstata sometidos a prostatectomía radical en SFVAMC. Escrito el consentimiento informado se obtuvo de todos los pacientes y el estudio fue aprobado por el Comité de la UCSF en Investigación en Seres Humanos. Alternativamente, los tejidos de cáncer de próstata microarrays se adquirieron de fuentes comerciales y se utilizan para miARN anal...

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En este artículo se describe un flujo de trabajo simplificado para la expresión de los genes miARN perfiles de FFPE archivado o tejidos clínicos sobre el cáncer de próstata congelados frescos. En el cáncer de próstata, varios estudios sugieren un papel importante de microRNAs en cáncer de próstata iniciación, progresión y metástasis. Sin embargo, los resultados en conflicto a menudo se obtuvieron en un miRNA específico 22 desde la extracción y análisis de miRNA métodos difieren ampliamente. En...

El cáncer de próstata es un tumor maligno masculina comúnmente diagnosticado que es una de las principales causas de la mortalidad relacionada con el cáncer entre los hombres. En Estados Unidos, se estima que 220.800 nuevos casos y 27.540 muertes se reportaron en 2015 1. El cáncer de próstata es una enfermedad heterogénea con enfermedad muy variable supuesto- tumores pueden ser indolente o muy agresivo. Un reto fundamental en el manejo clínico del cáncer de próstata es el que plantea la insuficiencia de los que actualmente se utilizan métodos / biomarcadores para la detección de enfermedades, el diagnóstico, el pronóstico y el tratamiento 2. Métodos de detección actuales incluyen antígeno (PSA) prostático específico y un examen rectal digital (DRE), seguido de las biopsias de próstata 3. Antígeno específico de próstata (PSA) es el biomarcador de cáncer de próstata más ampliamente utilizado que ha revolucionado significativamente la gestión clínica y la mejora de las tasas de supervivencia 4. Sin embargo, debido a las limitaciones inherentes de PSA incluyendo lack de especificidad, cribado basado en PSA ha llevado a más de diagnóstico y sobre el tratamiento de la enfermedad. En vista de esto, los intensos esfuerzos se dirigen hacia la búsqueda de alternativas biomarcadores de cáncer de próstata, en particular los que puede predecir la agresividad de la enfermedad y conducir mejores decisiones de tratamiento 4,5. En los últimos años, los microRNAs (miRNAs) han surgido como prometedores biomarcadores de cáncer de próstata alternos. Los microARNs (miRNAs) constituyen una clase conservado evolutivamente de los pequeños RNAs no codificantes que suprimen la expresión de genes post-transcripcional a través de interacciones específicas de secuencia con los 3&#39;-regiones no traducidas (UTRs) de mRNA objetivos afines. Se estima que&gt; 60% de los mRNAs se conservan objetivos de miRNAs 6. genes miARN se encuentran en regiones intergénicas o dentro de intrones o exones de proteína / no proteico 7 genes de codificación. Estos genes son preferentemente transcritos por la ARN polimerasa II en primamiRNAs ry (pri-miRNAs, varias kilobases de largo) que bucle tallo en forma de estructuras secundarias forma de horquilla. Estos pri-miRNAs se procesan en miRNAs precursores (pre-miRNAs, 60-75 nucleótidos de largo) que se exportan al citoplasma y tratados posteriormente en miRNAs maduros (18-25 nucleótidos de largo) 08.10. miRNAs regulan procesos celulares clave, incluyendo la proliferación, el desarrollo, la diferenciación y la apoptosis 11. Los estudios sugieren una desregulación generalizada de los perfiles de expresión de miRNA en diversos tumores malignos humanos incluyendo el cáncer de próstata 12-15. los perfiles de expresión de genes miARN se han notificado a ser ampliamente desregulado en el cáncer de próstata primario y metastásico. Alterada la expresión de miRNA se han relacionado con la progresión del cáncer de próstata, la agresividad y la recurrencia destacando el potencial pronóstico de miRNAs 12,14,16-19. Un creciente cuerpo de evidencia indica que los miRNAs juegan importantes papeles mecanicistas en la iniciación del cáncer de próstata, el desarrollo, el progresoion y la metástasis. En general, los miRNAs están surgiendo alternativas como biomarcadores prometedores para el diagnóstico del cáncer de próstata y el pronóstico que pueden distinguir entre los tejidos y ayuda normales y cancerosas en la estratificación de los tumores de próstata 12. También, miRNAs son objetivos importantes para el desarrollo de terapias eficaces contra el cáncer de próstata 20. Debido a su pequeño tamaño y resistencia a la actividad de RNasa endógena, miRNAs son biomarcadores estables que se pueden detectar fácilmente en tejidos fijados con formalina y 21 en las biopsias de próstata 22. Por otra parte, los perfiles de expresión de miRNAs se han comparado en los tejidos congelados y formol fija y se han encontrado para ser fuertemente correlacionada 21. Sin embargo, la expresión de los genes miARN perfiles en los tejidos clínicos sobre el cáncer de próstata a menudo es un reto debido a la heterogeneidad del tumor, los errores de muestreo, la contaminación del estroma etc. El desarrollo de miRNAs como biomarcadores eficaces para el cáncer de próstata en gran medida relies en su detección precisa en tejidos clínicos. Aquí se describe un flujo de trabajo simplificado utilizado en nuestro laboratorio para la expresión de los genes miARN perfiles de FFPE archivado o especímenes clínicos sobre el cáncer de próstata congelados frescos. Empleamos una combinación de PCR cuantitativa en tiempo real e hibridación in situ para miRNA los análisis de muestras clínicas, con el primero produciendo más información cuantitativa y el segundo para la visualización de la expresión diferencial de los genes miARN potenciales biomarcadores en una variedad de tejidos. Dentro de este flujo de trabajo, optimizamos las metodologías existentes para la extracción de miARN de FFPE y tejidos de la próstata congelados, la expresión análisis por Taqman-sonda basada miARN RT-PCR y miARN en la técnica de hibridación in situ utilizando ácido nucleico bloqueado (LNA) a base de sondas 23. Sondas basados ​​en LNA ofrecen mayor sensibilidad y especificidad en comparación con el ADN o ARN-sondas basadas y permite la detección robusta de todas las secuencias de genes miARN, independientemente de su contenido de GC y también permiten la discriminaciónción de las familias miARN. Nuestro protocolo miRNA ISH optimizado se puede aplicar a las diapositivas de cáncer de próstata de tejido o microarrays de tejidos de cáncer de próstata (TMA), con este último ofrece el potencial para acelerar el descubrimiento de biomarcadores miRNA.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems&#160;</td>
			<td>RM2255</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus Autopix for LCM</td>
			<td>Arcturus/ Life Technologies</td>
			<td>LCM1621/LCM1110</td>
			<td>Alternatively, Arcutus Xt system from Life Technolgies can be used.&#160;</td>
		</tr>
		<tr>
			<td>CapSure Macro LCM Caps</td>
			<td>Life Technologies</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy FFPE Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right">217504</td>
			<td></td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4351106</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman MicroRNA Reverse Transcription kit&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4366596</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman Fast Universal PCR master mix&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG labeled LNA probe for U6</td>
			<td>Exiqon</td>
			<td>99002-01</td>
			<td></td>
		</tr>
		<tr>
			<td>BM Purple AP substrate</td>
			<td>Roche</td>
			<td align="right">11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-1</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-2</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-8</a></td>
			<td></td>
		</tr>
		<tr>
			<td>AP-conjugated anti-digoxigenin antibody</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-7</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting media&#160;</td>
			<td>Vector Laboratories&#160;</td>
			<td>H-5501&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (guanidine isothiocyanate-phenol reagent)&#160;</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/15596018">15596-018</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin</td>
			<td>Statlab</td>
			<td>SL200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Statlab</td>
			<td>SL201&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

mirna expression analyses in prostate cancer clinical tissues

Un reto fundamental en el cáncer de próstata (CaP) manejo clínico se plantea por la insuficiencia de los biomarcadores que actualmente se utilizan para la detección de la enfermedad, diagnóstico, pronóstico y tratamiento. En los últimos años, microRNAs (miRNAs) han surgido como biomarcadores alternativas prometedoras para el diagnóstico y el pronóstico del cáncer de próstata. Sin embargo, el desarrollo de miRNAs como biomarcadores eficaces para el cáncer de próstata depende en gran medida su detección precisa en tejidos clínicos. miARN análisis en muestras clínicas de cáncer de próstata a menudo es un reto debido a la heterogeneidad del tumor, los errores de muestreo, la contaminación del estroma etc. El objetivo de este artículo es describir un flujo de trabajo simplificado para miARN analiza en FFPE archivado o especímenes clínicos sobre el cáncer de próstata fresco congelado usando una combinación de cuantitativa en tiempo real PCR (RT-PCR) e hibridación in situ (ISH). Dentro de este flujo de trabajo, optimizamos las metodologías existentes para la extracción de miRNA de FFPE y tissu próstata congeladoES y la expresión por análisis Taqman-sonda basada miARN RT-PCR. Además, se describe un método optimizado para ISH análisis de detección formiRNA en los tejidos de la próstata utilizando ácido nucleico cerrado (LNA) - sondas basadas. Nuestro protocolo miRNA ISH optimizado se puede aplicar a las diapositivas de cáncer de próstata de tejido o microarrays de tejidos de cáncer de próstata (TMA).

Perfiles de expresión de miR-203 en muestras clínicas de cáncer de próstata primario LCM por RT-PCR análisis (Figura 1) Análisis de RT-PCR de la expresión relativa de miR-203 en LCM tejidos de cáncer de próstata primario y las regiones normales adyacentes emparejados se llevó a cabo como se describe en Saini et al. 15 RNU48 se utilizó como control. La siguiente tabla resume la relativa expresión de miR-203 en los tejidos tumorales de cáncer d...

Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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, ...

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Medicine

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluación de la absorción de nanopartículas en los tumores en tiempo real utilizando intravital imagen

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Investigación

Medicina

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER+ and R26-SmoM2YFP+ mice in this protocol); 2) inducing SmoM2YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

Los análisis de población celular durante la carcinogénesis de piel

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

MR Imagen Molecular del cáncer de próstata con un agente de contraste dirigidos Pequeño Molecular CLT1 Péptido

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

La identificación basada en microarrays de expresión individual HERV Loci: Aplicación al descubrimiento de biomarcadores en el cáncer de próstata

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un modelo murino de cáncer de próstata humano Metástasis ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

El aislamiento de las células madre del cáncer de muestras de cáncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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 ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generación del paciente con cáncer de próstata Derivado modelos de xenoinjertos de células tumorales circulantes

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Trabajar con Tejidos Humanos para la Investigación del Cáncer Traslacional

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients&#039; Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

Análisis miARN exosomal en células no pequeñas del cáncer de pulmón (NSCLC) de plasma de los pacientes a través de qPCR: una herramienta de biopsia líquida factible

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...