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

Questa ricerca è stata sostenuta dal Dipartimento della Difesa Breast Cancer Research Program con il numero premio (ASH, W81XWH-13-1-0010), NIH CA100324, CA100324 PPG, e il programma di imaging integrato.

Tutte le procedure descritte devono essere eseguite in conformità con le linee guida e regolamenti per l&#39;uso di animali vertebrati, tra cui la preventiva approvazione da parte del Albert Einstein College of Medicine Istituzionale cura degli animali e del Comitato uso. 1. Generazione di fluorescenza Tumori etichettati e macrofagi associati al tumore <ol><li> Generare cellule tumorali fluorescente attraversando il, autoctona, topo geneticamente modificato modello cancro mammario spontaneo in cui la ripetizi...

<ol>
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interazioni cellulari che si verificano spontaneamente nel microambiente tumorale possono portare a cambiamenti nella motilità delle cellule tumorali e intravasation. High-Resolution Imaging intravitale del tessuto tumorale dal vivo permette la visualizzazione delle dinamiche pluricellulari che possono essere 10,13,24 altamente transitorio. End-point in vivo o immagini time-lapse acquisiti con i punti di tempo discreto grado di fornire informazioni essenziali sui meccanismi molecolari dei processi n...

Diffusione di cellule tumorali dal tumore mammario primario ha dimostrato di coinvolgere non solo le cellule tumorali, ma ospitare cellule stromali compresi macrofagi e cellule endoteliali. Inoltre, il tumore vascolare è anormale con aumento della permeabilità 1. Così, determinare come le cellule tumorali, macrofagi e cellule endoteliali interagiscono per mediare la permeabilità vascolare e intravasation delle cellule tumorali nel microambiente tumorale primaria è importante per la comprensione delle metastasi. Comprendere la cinetica della permeabilità vascolare, intravasation delle cellule tumorali e il meccanismo di segnalazione alla base delle interazioni multicellulari nel microambiente tumorale in grado di fornire informazioni cruciali per lo sviluppo e la gestione delle terapie anti-cancro. I mezzi primari di studiare tumore permeabilità vascolare in vivo è stata la misura di coloranti extravascolare come Evans blue 2, destrani ad alto peso molecolare (155 kDa)3 e fluoroforo o proteine ​​radiotracciante coniugato (tra cui l&#39;albumina) 4 in punti fissi di tempo dopo l&#39;iniezione del colorante. I progressi nella microscopia, modelli animali e coloranti fluorescenti hanno permesso la visualizzazione dei processi cellulari e permeabilità vascolare di animali vivi attraverso la microscopia intravitale 5. Immagini di animali vivi con l&#39;acquisizione di immagini statiche, o sequenze di breve time-lapse in diversi punti di tempo non permette la completa comprensione di eventi dinamici nel microambiente tumorale 6,7. Infatti, l&#39;acquisizione di immagini statiche nel corso di 24 ore ha dimostrato che tumore vascolarizzazione è a tenuta, tuttavia la dinamica della permeabilità vascolare non è stata osservata 6. Così, lungo l&#39;imaging continuo time-lapse fino a 12 ore cattura la cinetica di eventi dinamici nel microambiente tumorale. Questo protocollo descrive l&#39;uso di prolungato multiphot time-lapsesulla microscopia intravitale per studiare eventi multicellulari dinamici nel microambiente tumorale. Più tipi di cellule del microambiente tumorale sono etichettati con coloranti iniettabili o utilizzando animali transgenici che esprimono proteine ​​fluorescenti. Utilizzando un catetere venoso coda coloranti vascolari o proteine ​​possono essere iniettati dopo l&#39;inizio di imaging per acquisire dati cinetici di eventi multicellulari nel microambiente tumorale. Per l&#39;imaging cellulare dal vivo il tumore mammario è esposta attraverso la preparazione chirurgica di un lembo cutaneo. Le immagini vengono acquisite fino a 12 ore utilizzando un microscopio multiphoton dotato di molteplici tubi fotomoltiplicatori (PMT) rilevatori 8. Utilizzando opportuni filtri, un algoritmo di sottrazione consente 4 rivelatori PMT per acquisire 5 segnali fluorescenti nel microambiente tumorale contemporaneamente 9. Ad alta risoluzione multiphoton intravitale microscopia cattura di imaging singola risoluzione delle cellule di interazioni tumore-stroma nel microambiente tumorale, portando ad una migliore understanding della permeabilità vascolare e del tumore delle cellule intravasation 10-13. In particolare, rivelato altamente localizzati, transitori eventi permeabilità prolungati di imaging intravitale vascolari che si verificano selettivamente nei siti di interazione tra una cellula tumorale, un macrofago e una cellula endoteliale (definito come il microambiente tumorale delle metastasi, TMEM 14) 13. Inoltre, intravasation cellule tumorali si verifica solo in TMEM ed è spazialmente e temporalmente correlata con la permeabilità vascolare 13. risoluzione singola cella della dinamica di questi eventi è stato reso possibile attraverso l&#39;utilizzo di prolungato time-lapse multifotonica microscopio di cellule fluorescente nel microambiente tumorale.

<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.

Esteso microscopia time-lapse intravitale permette l&#39;imaging singola cella risoluzione dei processi multicellulari nel microambiente tumorale. Da parte delle cellule tumorali fluorescenza etichettatura, i macrofagi, lo spazio vascolare, e visualizzare la rete di fibre di collagene con il secondo segnale di generazione di armoniche, comparti multipli nel microambiente tumorale sono contemporaneamente monitorati durante l&#39;imaging. Le cellule tumorali marcate con proteine ​​fluo...

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

Questo protocollo descrive l&#39;uso della microscopia multiphoton effettuare prolungato time-lapse imaging di interazioni multicellulari in tempo reale, in vivo a risoluzione singola cellula.

Esteso Time-lapse Imaging intravitale del Real-time multicellulare Dynamics nel microambiente tumorale

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

Multispettrali in tempo reale immagini a fluorescenza per il rilevamento intraoperatoria del linfonodo sentinella in Oncologia Ginecologica

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 ...

Ricerca

Medicina

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

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale 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

Modelli per MAME 4D Live-Imaging cellule di tumore: Interazioni microambiente che Impact progressione maligna

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

In tempo reale Imaging digitale di leucociti-endotelio in ischemia-riperfusione (IRI) del muscolo 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

Vivo Imaging di risposte droga nel microambiente tumorale in modelli murini di cancro al seno

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 imaging di primaria preneoplastiche cellule epiteliali mammarie Derivato da modelli murini geneticamente modificati di cancro al seno

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

Trapianto nella camera anteriore dell&#39;occhio per longitudinale, non invasiva<em&gt; In vivo</em&gt; Imaging con Single-cella di risoluzione in tempo reale

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

Chemioterapia-indotta vascolare Tossicità - in tempo reale<em&gt; In vivo</em&gt; Imaging di 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

Confocale Time lapse imaging come un metodo efficiente per la valutazione citocompatibilità di compositi dentali

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

In tempo reale a raggi X Imaging del polmone volumi di fluido in neonatale topo polmone

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