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

Denne forskning blev støttet af det amerikanske forsvarsministerium Breast Cancer Research Program under award nummer (ASH, W81XWH-13-1-0010), NIH CA100324, PPG CA100324, og den integrerede Imaging Program.

Alle beskrevne procedurer skal udføres i overensstemmelse med retningslinjer og regler for brug af hvirveldyr, herunder forudgående godkendelse fra Albert Einstein College of Medicine Institutional Animal Care og brug Udvalg. 1. Generering fluorescensmærkede Tumorer og tumor-associerede makrofager <ol><li> Generere fluorescensmærkede tumorceller ved at krydse den spontane, autokton, gensplejset mus brystcancer model, hvor musebrysttumorvirus lange terminale gentagelse driver polyoma middle T-antigen (MMTV-...

<ol>
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Cellulære interaktioner, der forekommer spontant i tumormikromiljøet kan føre til ændringer i tumorcellemotilitet og intravasation. Høj opløsning intravital billeddannelse af levende tumorvæv tillader visualisering af multi-cellulære dynamik, der kan være meget forbigående 10,13,24. End-point in vivo assays eller time-lapse billeder erhvervet med diskrete tidspunkter kan give væsentlige oplysninger om molekylære mekanismer i processer i tumor mikromiljø. Intravital billeddannende unders?...

Udbredelse af tumorceller fra primære brysttumor har vist sig at inddrage ikke kun tumorceller, men vært stromale celler, herunder makrofager og endotelceller. Endvidere tumorvaskulatur er unormal med forøget permeabilitet 1. Således bestemme, hvor tumorceller, makrofager og endotelceller interagerer at mediere vaskulær permeabilitet og tumorcelle intravasation i den primære tumor mikromiljø er vigtig for forståelsen metastase. Forståelse kinetikken af ​​vaskulær permeabilitet, tumor celle intravasation og den underliggende signalering mekanisme flercellede interaktioner i tumoren mikromiljø kan give afgørende oplysninger i udvikling og administration af anti-behandlinger mod kræft. Det primære middel til at studere tumor vaskulær permeabilitet in vivo har været målingen af ekstravaskulære farvestoffer, såsom Evans blue 2, højmolekylære dextraner (155 kDa)3 og fluorophor eller radiotracer-konjugerede proteiner (herunder albumin) 4 ved faste tidspunkter efter injektion af farvestoffet. Forbedringer i mikroskopi, har dyremodeller og fluorescerende farvestoffer aktiveret visualisering af cellulære processer og vaskulær permeabilitet i levende dyr ved intravital mikroskopi 5. Levende dyr billeddannelse med købet af statiske billeder eller kort tid-lapse-sekvenser over flere tidspunkter ikke giver mulighed for det fuldstændig forståelse af dynamiske begivenheder i tumoren mikromiljø 6,7. Faktisk statisk billede erhvervelse i løbet af 24 timer viste, at tumorvaskulatur er utæt, men dynamikken i vaskulær permeabilitet blev ikke observeret 6. Således extended kontinuert tid-lapse billeddannelse op til 12 timer indfanger kinetikken for dynamiske begivenheder i tumormikromiljøet. Denne protokol beskriver anvendelsen af ​​extended time-lapse multiphotpå intravital mikroskopi for at studere dynamiske flercellede begivenheder i tumormikromiljøet. Flere celletyper i tumormikromiljøet er mærket med injicerbare farvestoffer eller ved at anvende transgene dyr, der udtrykker fluorescerende proteiner. Ved hjælp af en halevene kateter kan injiceres vaskulære farvestoffer eller proteiner efter begyndelsen af ​​billeddannelse til at erhverve kinetiske data af multicellulære begivenheder i tumormikromiljøet. For levende celler den brysttumor er eksponeret gennem den kirurgiske forberedelse af en hudlap. Billeder er erhvervet i op til 12 timer ved hjælp af en multifoton mikroskop udstyret med flere fotomultiplikatorrør (PMT) detektorer 8. Ved hjælp af passende filtre, en subtraktion algoritme giver 4 PMT-detektorer til at erhverve 5 fluorescerende signaler i tumor mikromiljø samtidigt 9. Høj opløsning multifoton intravital mikroskopi indfanger enkelt celle opløsning billeddannelse af tumor-stroma interaktioner i tumormikromiljøet, hvilket fører til en bedre understanding af vaskulær permeabilitet og tumorcelle intravasation 10-13. Specifikt udvidede intravital imaging afslørede stærkt lokaliseret, forbigående vaskulær permeabilitet hændelser, der forekommer selektivt ved steder for interaktion mellem en tumorcelle, en makrofag og en endotelcelle (defineret som tumormikromiljøet af metastase, TMEM 14) 13. Endvidere tumorcelle intravasation forekommer kun på TMEM og er rumligt og tidsligt korreleret med vaskulær permeabilitet 13. Enkelt celle opløsning af dynamikken i disse begivenheder blev muliggjort gennem anvendelse af længere tid-lapse multifoton mikroskopi af fluorescens-mærkede celler i tumor mikromiljø.

<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.

Udvidet tid-lapse intravital mikroskopi muliggør enkelt celle opløsning billeddannelse af flercellede processer i tumor mikromiljø. Ved fluorescens mærkning tumorceller, makrofager, det vaskulære rum, og visualisere netværket kollagen fiber ved hjælp af den anden harmoniske generation signal, er flere rum i tumoren mikromiljø samtidig spores under billedbehandling. Tumorceller mærket med fluorescerende proteiner kan genereres i transgene mus som det er sket i MMTV-PyMT-Dendra2 m...

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

Denne protokol beskriver anvendelsen af multifoton mikroskopi til at udføre længere tid-lapse billeddannelse af multicellulære interaktioner i realtid, in vivo ved enkelt celle opløsning.

Udvidet Time-lapse Intravital Imaging af Real-time flercellede Dynamics i Tumor mikromiljø

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

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 ...

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 ...

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

Mame Modeller for 4D live-cell imaging af tumor: mikromiljø interaktioner, Impact malignt 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

Realtid Digital Imaging af leukocyt-endotel Interaktion med iskæmi-reperfusionsskade (IRI) af rotte Cremaster Muscle

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

Levende Imaging af Drug respons i den tumormikromiljøet i musemodeller af Breast Cancer

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 af primære præneoplastiske brystepitelceller celler afledt fra gensplejsede musemodeller for brystkræft

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 ind i det forreste kammer i øjet til langsgående, ikke-invasiv<em&gt; In vivo</em&gt; Imaging med Single-celle Opløsning i realtid

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

Kemoterapi-induceret vaskulær toksicitet - Real-time<em&gt; In vivo</em&gt; Imaging af Vessel Nedskrivninger

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

Konfokal Time Lapse Imaging som en effektiv metode til at Cytocompatibility Evaluering af 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

Real-time X-ray Imaging af Lung Fluid Mængder i Neonatal Mouse Lung

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