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

Diese Arbeit wurde durch das Department of Defense Breast Cancer Research Program unter Verleihungsnummer (ASH, W81XWH-13-1-0010), NIH CA100324, PPG CA100324 und dem Integrated Imaging-Programm unterstützt.

Alle beschriebenen Verfahren müssen mit den Richtlinien und Vorschriften für den Einsatz von Wirbeltieren, einschließlich der vorherigen Zustimmung des Albert Einstein College of Medicine Institutional Animal Care und Use Committee entsprechend durchgeführt werden. 1. Erstellen von fluoreszenzmarkierten Tumoren und Tumor-assoziierten Makrophagen <ol><li> Generieren Sie fluoreszenzmarkierten Tumorzellen durch die spontane, autochthonen, gentechnisch veränderte Maus Brustkrebsmodell Kreuzung , wo die Maus -...

<ol>
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Zelluläre Interaktionen, die sich spontan in der Tumor-Mikroumgebung auftreten können, auf Veränderungen in der Tumorzellbeweglichkeit und intravasation führen. Hochauflösende intravital Bildgebung von lebenden Tumorgewebe ermöglicht die Visualisierung von mehrzelligen Dynamik , die hochtransienten 10,13,24 sein kann. End-Punkt in - vivo - Tests oder Zeitraffer-Bilder mit diskreten Zeitpunkten erworben wesentlichen Informationen über die molekularen Mechanismen von Prozessen in der Tu...

Verbreitung von Tumorzellen von der primären Mammatumor wurde nicht nur Tumorzellen gezeigt einzubeziehen, aber Host stromalen Zellen, einschließlich Makrophagen und Endothelzellen. Darüber hinaus ist Tumorgefäße mit einer erhöhten Permeabilität 1 abnormal. Somit bestimmen, wie Tumorzellen, Makrophagen und endothelialen Zellen in Wechselwirkung treten Gefäßpermeabilität und Tumorzell intravasation in dem primären Tumor-Mikroumgebung zu vermitteln, ist wichtig für das Verständnis der Metastasierung. die Kinetik der vaskulären Permeabilität, Tumorzelle intravasation und dem darunterliegenden Signalisierungsmechanismus von mehrzelligen Interaktionen im Tumormikroumgebung zu verstehen, können wichtige Informationen bei der Entwicklung und Verabreichung von Antikrebstherapien bereitzustellen. Die primäre Mittel der Tumor vaskuläre Permeabilität in vivo zu studieren die Messung von extravaskulärem Farbstoffe wie Evans - Blau 2, hochmolekulare Dextrane (155 kDa) wurde3 und Fluorophor oder Radiotracer-konjugierte Proteine ​​(einschließlich Albumin) 4 in festen Zeitpunkten nach der Injektion des Farbstoffes. Fortschritte in der Mikroskopie, Tiermodelle und Fluoreszenzfarbstoffe haben die Visualisierung von zellulären Prozessen und vaskuläre Permeabilität in lebenden Tieren durch Intravitalmikroskopie 5 aktiviert. Live - Tierbildgebung mit dem Erwerb von statischen Bildern oder kurzen Zeitraffer-Sequenzen über mehrere Zeitpunkte nicht für das Verständnis von dynamischen Ereignissen im Tumormikromilieu 6,7 erlauben. Tatsächlich statische Bildaufnahme über den Verlauf von 24 Stunden gezeigt , dass Tumorgefäße undicht jedoch die Dynamik der vaskulären Permeabilität wurde 6 nicht beobachtet. So fängt längere kontinuierliche Zeitraffer-Bildgebung bis zu 12 Stunden, um die Kinetik der dynamischen Ereignissen im Tumormikromilieu. Dieses Protokoll beschreibt die Verwendung von erweiterten Zeitraffer multiphotauf Intravitalmikroskopie dynamische vielzelligen Ereignisse in der Tumor-Mikroumgebung zu studieren. Mehrere Zelltypen in der Tumor-Mikroumgebung mit injizierbaren Farbstoffen markiert oder mit transgenen Tieren exprimieren fluoreszierende Proteine ​​verwenden. einen Schwanzvenenkatheter Gefäß Farbstoffe oder Proteine ​​verwendet, kann nach dem Start der Bildgebung injiziert werden kinetischen Daten von mehrzelligen Ereignisse im Tumormikromilieu zu erwerben. Für Live-Cell-Imaging wird die Brusttumor durch die chirurgische Vorbereitung einer Hautlappen ausgesetzt. Bilder werden für bis zu 12 Stunden erworben 8 eine Multiphotonen - Mikroskop ausgestattet mit mehreren Photovervielfacherröhren (PMT) Detektoren. Durch die Verwendung von geeigneten Filtern ermöglicht eine Subtraktion Algorithmus 4 PMT Detektoren 5 Fluoreszenzsignale im Tumormikromilieu zu erwerben gleichzeitig 9. Hochauflösende Multiintravitalmikroskopie erfasst einzelne Zelle auflösende Bildgebung von Tumor-Stroma-Interaktionen im Tumormikromilieu, zu einem besseren führenden uERSTÄNDIGUNG der vaskulären Permeabilität und Tumorzell intravasation 10-13. Insbesondere offenbart verlängerte intravitalAbbildungs ​​stark lokalisierten, transient Gefäßpermeabilität Ereignisse , die selektiv an den Stellen der Interaktion zwischen einer Tumorzelle, einem Makrophagen und einer Endothelzelle (definiert als der Tumor - Mikroumgebung der Metastasierung, TMEM 14) 13 auftreten. Weiterhin tritt Tumorzelle intravasation nur an TMEM und räumlich und zeitlich mit Gefäßpermeabilität 13 korreliert. Einzelne Zell Auflösung der Dynamik dieser Ereignisse wurde möglich durch die Verwendung erweiterter Zeitraffer-Multiphotonen-Mikroskopie von fluoreszenzmarkierten Zellen im Tumormikromilieu.

<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.

Erweiterte Zeitraffer-Mikroskopie ermöglicht einzelne intravital Zellauflösende Bildgebung von mehrzelligen Prozesse im Tumormikromilieu. Durch fluoreszenz Markierung von Tumorzellen, Makrophagen, den vaskulären Raum und Visualisieren des Kollagenfasernetzwerk der zweiten Harmonischen Erzeugungssignal, mehrere Kompartimente in der Tumor-Mikroumgebung verwendet werden gleichzeitig während der Bildgebung verfolgt. Tumorzellen mit fluoreszierenden Proteinen markiert können in transgene...

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

Dieses Protokoll beschreibt die Verwendung von Multiphotonen - Mikroskopie ausgedehnte Zeitraffer-Bildgebung von mehrzelligen Interaktionen in Echtzeit durchzuführen, in vivo auf Einzelzellauflösung.

Erweiterte Zeitraffer-Intravital Imaging von Echtzeit-Vielzellige Dynamics im Tumormikromilieu

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

Multispectral Real-time Fluoreszenz-Imaging für die intraoperative Detektion des Sentinel Lymph Node in Gynäkologische Onkologie

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 ...

Forschung

Medizin

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

Evaluation von Nanopartikel-Aufnahme in Tumoren in Echtzeit über 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 ...

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

MAME Modelle für die 4D-Bildgebung lebender Zellen von Tumor: Mikroumgebung Wechselwirkungen, die Einfluss auf maligne Progression

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

Real-time Digital Imaging von Leukozyten-Endothel-Interaktion in der Ischämie-Reperfusionsschaden (IRI) der Ratte Cremastermuskel

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

Live-Imaging of Drug Responses in der Tumor-Mikroumgebung in Mausmodellen von Brustkrebs

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

Zeitraffer-Imaging of Primary Präkanzeröse Brustepithelzellen aus gentechnisch veränderten Mausmodellen der Breast Cancer Abgeleitet

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 in die Vorderkammer des Auges für Längs-, Non-invasive<em&gt; In vivo</em&gt; Imaging mit Single-cell Auflösung in Echtzeit

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

Chemotherapie-induzierte vaskuläre Toxizität - Echtzeit-<em&gt; In-vivo-</em&gt; Imaging von Vessel Impairment

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

Konfokale Zeitraffer Imaging als eine effiziente Methode für die Bewertung der Cytocompatibility Dental Composites

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

Echtzeit-Röntgenbildgebung von Lungenflüssigkeitsvolumen in Neonatal Mauslunge

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