Name: extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment
Uploaded: 2016-06-12T14:00:00
Description: Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

Esta investigación fue apoyada por el Departamento de Defensa de mama Cancer Research Program con el número de concesión (ASH, W81XWH-13-1-0010), NIH CA100324, CA100324 PPG, y el Programa Integrado de imagen.

Todos los procedimientos descritos deben realizarse de conformidad con las directrices y regulaciones para el uso de animales vertebrados, incluyendo la aprobación previa del Albert Einstein College of Medicine Institucional Cuidado de Animales y el empleo. 1. Generación de fluorescencia tumores etiquetados y macrófagos asociados a tumores <ol><li> Generar células tumorales marcadas con fluorescencia cruzando el modelo espontáneo, autóctona, ratón diseñado genéticamente mamaria del cáncer, donde la m...

<ol>
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interacciones celulares que se producen de forma espontánea en el microambiente del tumor puede conducir a cambios en la motilidad de las células tumorales y intravasación. -Alta resolución de imagen intravital del tejido tumoral vivo permite la visualización de la dinámica multicelulares que pueden ser altamente 10,13,24 transitoria. Punto final en ensayos in vivo o imágenes con lapso de tiempo adquiridos con puntos de tiempo discretos pueden proporcionar información esencial sobre ...

La difusión de células tumorales de tumor de mama primario se ha demostrado que la participación células tumorales no sólo, pero el anfitrión de las células del estroma incluyendo los macrófagos y células endoteliales. Además, la vasculatura del tumor es anormal con aumento de la permeabilidad 1. Por lo tanto, la determinación de cómo las células tumorales, los macrófagos y las células endoteliales interactúan para mediar en la permeabilidad vascular y intravasación de células tumorales en el microambiente del tumor primario es importante para entender la metástasis. La comprensión de la cinética de la permeabilidad vascular, la intravasación de células tumorales y el mecanismo de señalización subyacente de las interacciones multicelulares en el microambiente tumoral puede proporcionar información crucial en el desarrollo y administración de terapias contra el cáncer. El principal medio de estudio de la permeabilidad vascular de tumores in vivo ha sido la medición de tintes extravasculares como el azul 2, dextranos Evans alto peso molecular (155 kDa)3 y fluoróforo o proteínas de radiotrazadores conjugado (incluyendo albúmina) 4 en puntos fijos de tiempo después de la inyección del colorante. Los avances en microscopía, modelos animales y tintes fluorescentes han permitido la visualización de procesos celulares y la permeabilidad vascular en animales vivos a través de microscopía intravital 5. Imágenes de animales vivos con la adquisición de imágenes estáticas o secuencias corto lapso de tiempo sobre varios puntos de tiempo no permite la comprensión completa de eventos dinámicos en el microambiente tumoral 6,7. De hecho, la adquisición de imagen estática durante el transcurso de 24 horas demostró que la vasculatura del tumor es permeable, sin embargo la dinámica de la permeabilidad vascular no se observó 6. Por lo tanto, extendida continua de imágenes de lapso de tiempo de hasta 12 horas capta la cinética de eventos dinámicos en el microambiente tumoral. Este protocolo describe el uso de multiphot prolongado lapso de tiempoen la microscopía intravital para estudiar eventos multicelulares dinámicos en el microambiente tumoral. Múltiples tipos de células en el microambiente del tumor se marcan con tintes inyectables o mediante el uso de animales transgénicos que expresan proteínas fluorescentes. El uso de un catéter de vena de la cola colorantes vasculares o proteínas pueden ser inyectados después del inicio de formación de imágenes para adquirir datos cinética de eventos multicelulares en el microambiente tumoral. Para imágenes de células vivas del tumor mamario está expuesto a través de la preparación quirúrgica de un colgajo de piel. Las imágenes se adquieren por hasta 12 horas utilizando un microscopio multifotónica equipado con múltiples tubos fotomultiplicadores (PMT) detectores 8. Mediante el uso de filtros adecuados, un algoritmo de sustracción permite 4 detectores PMT para adquirir 5 señales fluorescentes en el microambiente tumoral simultáneamente 9. microscopía de alta resolución multifotónica intravital captura resolución de celda de imagen única de las interacciones tumor-estroma en el microambiente del tumor, lo que lleva a una mejor uOMPRENDER de la permeabilidad vascular y intravasación de células tumorales 10-13. Específicamente, reveladas muy localizadas, eventos de permeabilidad prolongados de formación de imágenes intravital transitorios vasculares que se producen de forma selectiva en los sitios de interacción entre una célula tumoral, un macrófago y una célula endotelial (definido como el microambiente tumoral de metástasis, TMEM 14) 13. Además, la intravasación de células tumorales se produce sólo en TMEM y se espacial y temporalmente correlacionado con la permeabilidad vascular 13. fue posible la resolución de una sola célula de la dinámica de estos eventos a través del uso de prolongado lapso de tiempo múltiple de fotones microscopía de las células marcadas con fluorescencia en el microambiente tumoral.

<table border="0" cellpadding="0" cellspacing="0" width="637">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">155 kDa dextran-tetramethylrhodamine isothiocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">T1287</td>
			<td style="width:167px;">reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">70 kDa dextran-Texas Red</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">D-1830</td>
			<td>reconstitute at 10 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">10 kDa dextran-fluorescein isothyocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">FD10S</td>
			<td>reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Qdot 705 ITK Amino (PEG) Quantum Dots</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">Q21561MP</td>
			<td>Dilute 25 uL in 175 uL of 1 X PBS for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MMTV-PyMT mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">2374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">26051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-EGFP mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">1 x PBS</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Isoethesia (isoflurane)</td>
			<td style="width:95px;">Henry Schein Animal Health</td>
			<td align="right" style="width:128px;">50033</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen</td>
			<td style="width:95px;">AirTech</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">1 mL syringe, tuberculin slip tip</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">30G x 1 (0.3 mm X 25 mm) needle</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">305128</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Polyethylene micro medical tubing&#160;</td>
			<td style="width:95px;">Scientific Commodities Inc</td>
			<td style="width:128px;">BB31695-PE/1</td>
			<td>0.28 mm I.D. X 0.64 mm O.D.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope coverglass</td>
			<td style="width:95px;">Corning</td>
			<td style="width:128px;">2980-225</td>
			<td>thickness 1.5, 22 X 50 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MouseOx oximeter, software and sensors</td>
			<td style="width:95px;">STARR Life Sciences</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Laboratory tape</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:128px;">159015R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">soft rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8514K62</td>
			<td>Ultra-Soft Polyurethane Film, 3/16&#8221; Thick, 12&#34; x 12&#34;, 40 Oo Durometer, Plain Back</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">hard rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8568K615</td>
			<td>High-Strength Neoprene Rubber Sheet 1/4&#34; Thick, 12&#34; X 12&#34;, 50A Durometer,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope</td>
			<td style="width:95px;">Olympus</td>
			<td style="width:128px;"></td>
			<td>The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">7-Punch set</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td style="width:128px;">3429A12</td>
			<td>, 1/4&#34; to 1&#34; Hole Diameter, for Hammer-Driven Hole Punch,&#160;</td>
		</tr>
	</tbody>
</table>

extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

Extendido microscopía intravital de lapso de tiempo permite la resolución de la célula de imagen única de los procesos multicelulares en el microambiente tumoral. Por las células tumorales con fluorescencia etiquetado, macrófagos, el espacio vascular, y la visualización de la red de fibras de colágeno mediante la segunda señal de generación de armónicos, múltiples compartimentos en el microambiente del tumor se realiza un seguimiento de forma simultánea durante la exploraci?...

Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Este protocolo describe el uso de la microscopía multifotónica para realizar extendida time-lapse de las interacciones multicelulares en tiempo real, en vivo a una resolución de una sola célula.

Prolongado lapso de tiempo intravital de imágenes de tiempo real multicelular Dinámica en el microambiente tumoral

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Research

JoVE Journal

Medicine

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

Multiespectrales en tiempo real imágenes de fluorescencia para la detección intraoperatoria del ganglio centinela en Oncología Ginecológica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Investigación

Medicina

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

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 ...

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

MAME Modelos de 4D Live de células de imagen de un tumor: Interacciones microambiente que la progresión maligna del impacto

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

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

En tiempo real de imágenes digitales de los leucocitos del endotelio interacción en el daño por isquemia-reperfusión (IRI) del músculo cremáster Rata

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

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

Imágenes en directo de las respuestas de drogas en el microambiente tumoral en modelos de ratón de cáncer de mama

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

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse de imágenes de Primaria preneoplásicas células epiteliales mamarias derivadas de los modelos de ratones genéticamente modificados del cáncer de mama

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Trasplante en la cámara anterior del ojo para Longitudinal, no invasiva<em&gt; En vivo</em&gt; Imagen con una sola célula de resolución en tiempo real

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Inducida por la quimioterapia Vascular Toxicidad - En tiempo real<em&gt; En vivo</em&gt; Imagen de Deterioro del buque

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

Confocal Lapso de tiempo de imágenes como un método eficaz para la Evaluación Cytocompatibility de composites dentales

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

En tiempo real de imágenes de rayos X de los pulmones de volúmenes de líquido en pulmón de ratón neonatal

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...