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 forskningen ble støttet av det amerikanske forsvarsdepartementet Breast Cancer Research Program under award nummer (ASH, W81XWH-13-1-0010), NIH CA100324, PPG CA100324 og Integrated Imaging Program.

Alle prosedyrer beskrevet må utføres i samsvar med retningslinjer og regler for bruk av virveldyr, inkludert godkjennelse fra Albert Einstein College of Medicine Institutional Animal Care og bruk komité. 1. Generering fluorescensmerkede Svulster og tumor-assosiert Makrofager <ol><li> Generer fluorescensmerkede tumorceller ved å krysse den spontane, innfødt, genmodifisert mus mammary kreft modell der musebrysttumorvirus lang terminal gjenta driver polyoma midtre T-antigen (MMTV-PyMT) med transgene mus med ...

<ol>
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Cellular interaksjoner som oppstår spontant i svulsten mikromiljøet kan føre til endringer i tumor cellemotilitet og intravasation. Høy oppløsning intra avbildning av levende svulstvev tillater visualisering av multi-mobilnettet dynamikk som kan være svært forbigående 10,13,24. End-point in vivo eller time-lapse bilder ervervet med diskrete tidspunkter kan gi viktig informasjon om molekylære mekanismer av prosesser i svulsten mikromiljøet. Intrabilde studier har blitt brukt til å studere p...

Spredning av tumorceller fra den primære brysttumor har vist seg å medføre ikke bare tumorceller, men vert stromale celler innbefattende makrofager og endotelceller. Videre er tumorvaskulatur unormal med økt permeabilitet 1. Således bestemmer hvor tumorceller, makrofager og endotelceller interagere for å mediere vaskulær permeabilitet og tumorcelle intravasation i den primære svulsten mikromiljøet er viktig for forståelse metastasering. Forstå kinetikk vaskulær permeabilitet, tumorcelle intravasation og den underliggende signal mekanismen av flercellede interaksjoner i svulsten mikromiljøet kan gi viktig informasjon i utvikling og forvaltning av anti-kreft terapi. Det primære middel for å studere tumor vaskulær permeabilitet in vivo har vært måling av ekstravaskulære fargestoffer så som Evans blå 2, høye vekt dekstraner (155 kDa) molekyl3 og fluorofor eller radiotracer-konjugerte proteiner (inkludert albumin) 4 ved faste tidspunkter etter injeksjon av fargestoffet. Fremskritt i mikroskopi, har dyremodeller og fluorescerende fargestoffer aktivert visualisering av cellulære prosesser og vaskulær permeabilitet i levende dyr gjennom intramikros 5. Levende dyr bildebehandling med oppkjøpet av statiske bilder eller kort time-lapse sekvenser over flere tidspunkter tillater ikke for fullstendig forståelse av dynamiske hendelser i svulsten mikromiljøet 6,7. Faktisk statiske bildeoverføring i løpet av 24 timer viste at tumor vaskulaturen er utett, men dynamikken i vaskulær permeabilitet ble ikke observert 6. Dermed utvidet kontinuerlig time-lapse imaging opptil 12 timer fanger kinetikken av dynamiske hendelser i svulsten mikromiljøet. Denne protokollen beskriver bruk av utvidede tidsforsinkelse multiphotpå intramikroskopi for å studere dynamiske flercellede hendelser i svulsten mikromiljøet. Flere celletyper i svulsten mikromiljøet er merket med fargestoffer injiserbare eller ved anvendelse av transgene dyr som uttrykker fluorescerende proteiner. Ved hjelp av en halevenekateter vaskulære fargestoffer eller proteiner kan injiseres etter starten av avbildning for å erverve kinetiske data fra flercellede hendelser i svulsten mikromiljøet. For levende celle bildebehandling melke svulsten er eksponert gjennom kirurgisk utarbeidelse av en hud klaff. Bildene er kjøpt i opptil 12 timer med en multiphoton mikroskop utstyrt med flere photomultiplier rør (PMT) detektorer 8. Ved å bruke egnede filtre, gjør en subtraksjonsalgoritmen 4 PMT detektorer for å skaffe 5 fluorescerende signaler i svulsten mikromiljøet samtidig ni. Høy oppløsning multiphoton intramikros fanger enkelt celle oppløsning avbildning av tumor-stroma interaksjoner i svulsten mikromiljøet, fører til en bedre understanding av vaskulær permeabilitet og tumorcelle intravasation 10-13. Spesielt utvidede intrabilde avdekket svært lokaliserte, forbigående vaskulære permeabilitet hendelser som oppstår selektivt på områder for interaksjon mellom en svulst celle, en makrofag og en endotelceller (definert som svulstens mikrometastaser, TMEM 14) 13. Videre oppstår tumorcelle intravasation bare ved TMEM og er romlig og tidsmessig korrelert med vaskulær permeabilitet 13. Enkelt celle oppløsning av dynamikken i disse hendelsene ble gjort mulig ved bruk av forlenget time-lapse multiphoton mikroskopi av fluorescerende merkede celler i tumoren mikromiljøet.

<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.

Utvidet time-lapse intravital mikroskopi muliggjør enkelt celle oppløsning avbildning av flercellede prosesser i svulsten mikromiljøet. Ved fluorescently merking kreftceller, makrofager, det vaskulære plass, og visualisere kollagen fiber nettverk ved hjelp av den andre harmoniske generasjon signal, er flere avdelinger i svulsten mikromiljøet samtidig spores under bildebehandling. Tumorceller merket med fluorescerende proteiner kan frembringes i transgene mus som har blitt gjort i MM...

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 protokollen beskriver bruken av multiphoton mikroskopi for å utføre forlenget time-lapse avbildning av flercellede vekselvirkninger i sanntid, in vivo ved enkelt celle oppløsning.

Utvidet Time-lapse intra Imaging av Real-time Flercellede Dynamics i svulstens 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-celle Imaging av tumor: mikromiljøet interaksjoner som Impact Ondartet Progresjon

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 av leukocyttantigener-endotelial Interaksjon i iskemi-reperfusjon Injury (IRI) av Rat 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

Lever Imaging of Drug Responses i svulsten mikromiljøet i musemodeller av 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 av Primary Preneoplastic mammary epitelceller Avledet fra genmodifisert mus modeller av Breast Cancer

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

Transplantasjon i fremre kammer av øyet for Longitudinal, Ikke-invasive<em&gt; In vivo</em&gt; Imaging med encellede oppløsning i sanntid

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

Cytostatikaindusert Vaskulær Toxicity - Real-time<em&gt; In vivo</em&gt; Imaging av Vessel Nedskrivning

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

Konfokalt Time Lapse Imaging som en effektiv metode for Cytocompatibility Evaluering av 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 av Lung Fluid Volumene 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 ...