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

Cette recherche a été soutenue par le programme d&#39;imagerie intégrée Programme de recherche sur le cancer du sein Département de la Défense sous le numéro d&#39;attribution (ASH, W81XWH-13-1-0010), NIH CA100324, CA100324 PPG, et.

Toutes les procédures décrites doivent être effectuées conformément aux directives et réglementations relatives à l&#39;utilisation des animaux vertébrés, y compris l&#39;approbation préalable de l&#39;Albert Einstein College of Medicine Institutional Animal Care et utilisation Comité. 1. Génération de fluorescence Tumeurs Labellisées et Macrophages associés aux tumeurs <ol><li> Générer des cellules tumorales marquées par fluorescence en traversant le autochthonous modèle spontané, une sour...

<ol>
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les interactions cellulaires qui se produisent spontanément dans le microenvironnement tumoral peut conduire à des modifications de la motilité des cellules tumorales et intravasculaire. Haute résolution intravitale imagerie du tissu tumoral en temps réel permet de visualiser la dynamique multicellulaire qui peut être très 10,13,24 transitoire. Point final dans des essais in vivo ou images time-lapse acquises avec des points de temps discrets peuvent fournir des informations essentiel...

Dissémination des cellules tumorales de la tumeur mammaire primaire a été montré que des cellules tumorales implique non seulement, mais les cellules hôtes stromales, y compris les macrophages et les cellules endothéliales. En outre, le système vasculaire tumoral est anormale avec une augmentation de la perméabilité. Ainsi, la détermination de la façon dont les cellules tumorales, les macrophages et les cellules endotheliales interagissent pour médier la perméabilité vasculaire et intravasculaire de cellules tumorales dans le microenvironnement de la tumeur primaire est importante pour la compréhension des métastases. La compréhension de la cinétique de la perméabilité vasculaire, intravasculaire de cellules tumorales et le mécanisme de signalisation sous-jacente des interactions multicellulaires dans le microenvironnement tumoral peut fournir des informations cruciales dans le développement et l&#39;administration des traitements anti-cancéreux. Le moyen principal de l&#39; étude de la perméabilité vasculaire tumorale in vivo a été la mesure des colorants extravasculaires tels que le bleu d&#39; Evans 2, les dextrans de haut poids moléculaire (155 kDa)3 et fluorophore ou des protéines de radiotraceurs conjugués (y compris l&#39; albumine) 4 en des points fixes de temps après l&#39; injection du colorant. Les progrès de la microscopie, des modèles animaux et des colorants fluorescents ont permis la visualisation des processus cellulaires et la perméabilité vasculaire chez les animaux vivants par microscopie intravitale 5. Imagerie animale en direct avec l&#39;acquisition d&#39;images statiques ou de courtes séquences time-lapse sur plusieurs points de temps ne permet pas à la compréhension complète des événements dynamiques dans le microenvironnement de la tumeur 6,7. En effet, l&#39; acquisition d&#39;image statique au cours de 24 heures a montré que le système vasculaire tumoral est non étanche, mais la dynamique de la perméabilité vasculaire n&#39;a pas été observée 6. Ainsi, l&#39;imagerie en continu prolongé le temps écoulé jusqu&#39;à 12 heures capture la cinétique d&#39;événements dynamiques dans le microenvironnement tumoral. Ce protocole décrit l&#39;utilisation de longue time-lapse multiphotla microscopie intravitale pour étudier les événements dynamiques multicellulaires dans le microenvironnement tumoral. plusieurs types de cellules dans le microenvironnement tumoral sont marquées avec des colorants injectables ou en utilisant des animaux transgéniques exprimant des protéines fluorescentes. Au moyen d&#39;un cathéter veine de la queue des colorants ou des protéines vasculaires peuvent être injectées après le début de formation d&#39;image pour acquérir des données d&#39;événements cinétique multicellulaires dans le microenvironnement tumoral. Pour l&#39;imagerie des cellules vivantes de la tumeur mammaire est exposée à travers la préparation chirurgicale d&#39;un lambeau de peau. Les images sont acquises jusqu&#39;à 12 heures en utilisant un microscope multiphoton équipé de tubes photomultiplicateurs multiples (PMT) détecteurs 8. À l&#39;aide de filtres appropriés, un algorithme de soustraction permet de 4 détecteurs pour acquérir PMT 5 signaux fluorescents dans le microenvironnement tumoral simultanément 9. La microscopie à haute résolution multiphotonique intravitale capture seule résolution cellulaire imagerie des interactions tumeur-stroma dans le microenvironnement de la tumeur, ce qui conduit à une meilleure uCOMPRENDRE de la perméabilité vasculaire et des cellules tumorales intravasation 10-13. Plus précisément, l&#39; imagerie intravitale révélés très localisés, les accidents vasculaires transitoires étendues de perméabilité sélective qui se produisent au niveau des sites d&#39;interaction entre une cellule tumorale, un macrophage et une cellule endothéliale (défini comme le microenvironnement tumoral des métastases, TMEM 14) 13. En outre, intravasculaire de cellules tumorales se produit uniquement à TMEM et est spatialement et temporellement corrélée avec la perméabilité vasculaire 13. résolution cellulaire unique de la dynamique de ces événements a été rendue possible grâce à l&#39;utilisation de longue time-lapse multiphoton microscopique des cellules marquées par fluorescence dans le microenvironnement de la tumeur.

<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.

La microscopie Extended time-lapse intravitale permet seule résolution cellulaire imagerie des processus multicellulaires dans le microenvironnement de la tumeur. Par les cellules tumorales marquage par fluorescence, les macrophages, l&#39;espace vasculaire et de visualiser le réseau de fibres de collagène en utilisant le deuxième signal de génération d&#39;harmoniques, de multiples compartiments dans le microenvironnement tumoral sont simultanément suivis au cours de l&#39;imager...

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

Ce protocole décrit l&#39;utilisation de la microscopie multiphotonique pour effectuer prolongée imagerie time-lapse d&#39;interactions multicellulaires en temps réel, in vivo à la résolution d&#39;une seule cellule.

Time-lapse Extended Imagerie intravitale du temps réel multicellulaire Dynamics dans le microenvironnement de la tumeur

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

Multispectrale en temps réel d&#39;imagerie par fluorescence pour la détection peropératoire du ganglion sentinelle en oncologie gynécologique

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 ...

Recherche

Médecine

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

Evaluation de la fixation des nanoparticules dans les tumeurs en temps réel par imagerie intravitale

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

Modèles de MAME pour 4D imagerie des cellules vivantes de la tumeur: Interactions microenvironnement Progression impact malin

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 temps réel d&#39;imagerie numérique de leucocytes-endothélial interaction dans l&#39;ischémie-reperfusion (IRI) du muscle Cremaster Rat

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

Vivez l&#39;imagerie des réponses de drogues dans le microenvironnement des tumeurs dans les modèles murins de cancer du sein

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

Imagerie time-lapse de primaires prénéoplasiques cellules épithéliales mammaires issues de modèles de souris génétiquement modifiées de cancer du sein

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

Transplantation dans la chambre antérieure de l&#39;oeil pour longitudinale, non-invasive<em&gt; In vivo</em&gt; Imagerie avec une seule cellule de résolution en temps réel

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

Induits par la chimiothérapie vasculaire Toxicité - en temps réel<em&gt; In vivo</em&gt; Imagerie de déficience navire

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

Confocale Accéléré Imaging comme une méthode efficace pour l&#39;évaluation cytocompatibilité de composites dentaires

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 temps réel d&#39;imagerie à rayons X du poumon Volumes fluide dans néonatale Souris Lung

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