Name: extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment
Uploaded: 2016-06-12T14:00:00
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Denna forskning stöddes av försvarsdepartementet Breast Cancer Research programmet under tilldelning nummer (ASH, W81XWH-13-1-0010), NIH CA100324, PPG CA100324, och den integrerade bildprogrammet.

Alla förfaranden som beskrivs måste utföras i enlighet med de riktlinjer och regler för användning av ryggradsdjur, inklusive förhandsgodkännande av Albert Einstein College of Medicine Institutional Animal Care och användning kommittén. 1. Generera fluorescerande Tumörer och tumörassocierade makrofager <ol><li> Generera fluorescensmärkta tumörceller genom att korsa den spontana, autoktona, gentekniskt mus bröstcancer modell där musbrösttumörvirus långa terminala upprepningen driver polyoma mi...

<ol>
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Cellulära interaktioner som förekommer spontant i tumören mikro kan leda till förändringar i tumörcellrörlighet och intravasering. Högupplöst intravital avbildning av levande tumörvävnad medger visualisering av flercelliga dynamik som kan vara mycket övergående 10,13,24. Slutpunkt in vivo eller time-lapse bilder som förvärvats med diskreta tidpunkter kan ge viktig information om molekylära mekanismer för processer i tumören mikromiljö. Intravital imaging studier har använts för a...

Spridning av tumörceller från den primära brösttumör har visats involvera inte bara tumörceller, men värd stromala celler inkluderande makrofager och endotelceller. Dessutom är tumörvaskulatur onormalt med ökad permeabilitet en. Således är viktig för att förstå metastaser bestämma hur tumörceller, makrofager och endotelceller samverkar för att förmedla vaskulär permeabilitet och tumörcell intravasering i den primära tumören mikromiljö. Att förstå kinetiken för vaskulär permeabilitet, tumörcell intravasering och mekanismen för flercelliga interaktioner i tumören mikro underliggande signalering kan ge viktig information för att utveckla och administration av anti-cancerterapier. Det primära sättet att studera tumör vaskulär permeabilitet in vivo har varit mätning av extravaskulära färgämnen som Evans blå 2, högmolekylära dextraner (155 kDa)3 och fluorofor eller radiotracer-konjugerad proteiner (inkluderande albumin) 4 vid fasta tidpunkter efter injektion av färgämnet. Framsteg inom mikroskopi, har djurmodeller och fluorescerande färgämnen möjlig visualisering av cellulära processer och vaskulär permeabilitet i levande djur genom intravital mikroskopi 5. Levande djur avbildning med förvärvet av statiska bilder eller kort time-lapse-sekvenser under flera tidpunkter inte möjliggöra fullständig förståelse av dynamiska händelser i tumören mikro 6,7. Faktum är att statisk bildtagning under loppet av 24 timmar visade att tumörkärl är läckande, men dynamiken i vaskulär permeabilitet observerades inte sex. Således, förlängd kontinuerlig tidsförlopp avbildning upp till 12 timmar fångar kinetiken av dynamiska händelser i tumören mikromiljö. Detta protokoll beskriver användningen av förlängda tidsförlopp multiphotpå intravital mikroskopi för att studera dynamiska flercelliga händelser i tumören mikromiljö. Flera celltyper i tumören mikro är märkta med injicerbara färgämnen eller genom att använda transgena djur som uttrycker fluorescerande proteiner. Med användning av en svansven kateter vaskulära färgämnen eller proteiner kan injiceras efter starten av bildbehandling för att förvärva kinetiska data av multicellulära händelser i tumörmikromiljön. För levande cell imaging brösttumör är exponerad genom den kirurgiska beredningen av en hud klaff. Bilder förvärvas för upp till 12 timmar med en multifotonmikroskop utrustad med flera fotomultiplikatorrör (PMT) detektorer 8. Genom att använda lämpliga filter, en subtraktion algoritm möjliggör 4 PMT detektorer för att förvärva 5 fluorescerande signaler i tumörens mikromiljö samtidigt 9. Högupplöst multifoton intravital mikroskopi fångar enda cell upplösning avbildning av tumör stroma interaktioner i tumören mikro, vilket leder till en bättre understanding av vaskulär permeabilitet och tumörcell intravasering 10-13. Specifikt förlängda intravital imaging avslöjade mycket lokal, övergående vaskulära permeabilitets-händelser som inträffar selektivt vid ställen för interaktion mellan en tumörcell, en makrofag och en endotelcell (definierad som tumörens mikro av Metastas, TMEM 14) 13. Dessutom sker tumörcell intravasering endast på TMEM och rumsligt och tidsmässigt korrelerade med vaskulär permeabilitet 13. Enda cell upplösning av dynamiken i dessa händelser möjliggjordes genom användning av förlängda tidsförlopp multifoton mikroskopi av fluorescerande celler i tumören mikromiljö.

<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.

Förlängd tidsförlopp intravital mikroskopi möjliggör enda cell upplösning avbildning av flercelliga processer i tumören mikromiljö. Genom fluorescerande märkning tumörceller, makrofager, det vaskulära utrymmet och visualisera kollagenfibernätet med hjälp av andra harmoniska generationen signal, är flera fack i tumören mikro samtidigt spåras under avbildning. Tumörceller märkta med fluorescerande proteiner kan genereras i transgena möss, såsom har skett i MMTV-pymt-Den...

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

Detta protokoll beskriver användningen av multifotonmikroskop för att utföra förlängda tidsförlopp avbildning av flercelliga interaktioner i realtid, in vivo på en enda cell upplösning.

Utökad Time-lapse Intravital avbildning av realtid flercelliga Dynamics i tumörens mikro

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

Multispektrala realtid Fluorescens Imaging för intraoperativ Identifiering av Sentinel Lymph nod i gynekologisk onkologi

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

Utvärdering av nanopartiklar Upptag i tumörer i realtid med hjälp 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 för 4D Live-cell imaging av tumör: mikromiljö Interaktioner som påverkar malign 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 bildbehandling av leukocyt-endotel interaktion i ischemi-reperfusionsskada (IRI) i råtta kremastermuskeln

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

Realtidsundersökningen av Drug svar i tumören mikromiljön i musmodeller av bröstcancer

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 avbildning av primära preneoplastiska bröstepitelialceller celler från genetiskt modifierade musmodeller av bröstcancer

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 i den främre ögonkammaren för längsgående, icke-invasiv<em&gt; In vivo</em&gt; Imaging med encelliga Upplösning 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-inducerad vaskulär toxicitet - Realtids<em&gt; In vivo</em&gt; Avbildning av Vessel Nedskrivningar

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

Confocal Time Lapse Imaging som en effektiv metod för Cytocompatibility utvärdering 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

Realtidsröntgen avbildning av lungvätska Volymerna i neonatal mus Lung

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