Name: Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment (Video) | JoVE Journal
Uploaded: 2016-06-12T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Learn intravital multiphoton microscopy for visualizing real-time multicellular dynamics in the tumor microenvironment. This technique enables high-resolution observation of vascular permeability and cellular interactions in vivo.

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5 minutes

This research was supported by the Department of Defense Breast Cancer Research Program under award number (A.S.H, W81XWH-13-1-0010), NIH CA100324, &#160;PPG CA100324,&#160;and the Integrated Imaging Program.

All procedures described must be performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.
1. Generating Fluorescently Labeled Tumors and Tumor-associated Macrophages
<ol>
	<li>Generate fluorescently labeled tumor cells by crossing the spontaneous, autochthonous, genetically engineered mouse mammary cancer model where the mouse mammary tumor virus long terminal repeat drives the polyoma middle T antigen (MMTV-PyMT) with transgenic mice with fluorescent reporters [i.e., enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP) or Dendra2]8,9.</li>
	<li>Fluorescently label macrophages in PyMT animals with fluorescently labeled tumor cells by crossing genetically engineered mouse models with myeloid- and macrophage- specific fluorescent reporters [i.e., MacGreen15 or MacBlue mice Csf1r-GAL4VP16/UAS-ECFP16] .</li>
	<li>Alternatively, generate fluorescently labeled tumors through orthotopic transplantation of fluorescently labeled PyMT tumor cells (syngeneic), human breast cancer cell lines or primary human patient derived tumors (xenografts)11,17.
	<ol>
		<li>Implant fluorescently labeled PyMT tumor cells into syngeneic mice with fluorescent protein-labeled myeloid cells to generate animals with fluorescently labeled tumor cells and myeloid cells13.</li>
	</ol>
	</li>
	<li>Inject 100 &#181;l of 10 mg/kg fluorescent 70 kDa dextran by intravenous (i.v.) tail vein into severe combined immunodeficiency (SCID) mice with xenograft tumors of human cell lines or patient-derived primary tumors to fluorescently label macrophages 2 hr prior to the start of intravital imaging.</li>
</ol>
2. Microscope Setup and Imaging Preparation
Note: This procedure describes the set-up for intravital imaging on a multiphoton microscope8.
<ol>
	<li>Turn on all microscope and laser components including two-photon lasers and the detectors.</li>
	<li>Turn on the heating box to 30 &#176;C to pre-warm the stage. This step is critical for maintaining the physiological temperature of the animal.</li>
	<li>For use on an inverted microscope place the custom-made stage insert on the microscope stage. The custom insert is a sheet of 1/8&#34; thick aluminum machined to fit in the stage insert space and with a 1&#34; diameter through-hole in the center for imaging.
	<ol>
		<li>Wipe the microscope stage and stage insert with 70% ethanol and air dry.</li>
		<li>Place a large drop of water on the 20X, 1.05&#160;NA microscope objective to maintain optical contact with the cover glass.</li>
		<li>Place cover glass (#1.5 thickness) over the imaging port on the microscope stage insert. Secure in place with lab tape.</li>
		<li>Use a hole punch to punch a 2 cm x 2 cm hole in a flexible rubber pad and place the rubber pad on the microscope stage with the hole in the pad aligned with the hole in the custom stage insert.</li>
	</ol>
	</li>
</ol>
3. Preparation of Tail Vein Catheter and Reagents for Injection during Imaging
<ol>
	<li>Cut a 30 cm length of polyethylene (PE) tubing.</li>
	<li>Using forceps, move the metal needle of a 31 G needle back and forth until it breaks off of the plastic fitting.</li>
	<li>Using forceps, insert the blunt end of the detached needle into the PE tubing.</li>
	<li>Insert a 31 G needle into the other end of the PE tubing.</li>
	<li>Fill a 1 cc syringe with sterile phosphate buffered saline (PBS) and insert it into the 31 G needle and flush all air out of the tubing and needle with PBS. PBS is used for maintaining hydration and blood osmolarity9,18, however isotonic saline may also be used.</li>
	<li>Fill a 1 cc syringe with 200 &#181;l of 3 mg/kg 155 kDa dextran-tetramethylrhodamine (TMR) or quantum dots for vascular labeling.</li>
	<li>Prepare any other injectable proteins such as a 165 amino acid isoform of vascular endothelial growth factor A (VEGFA165) in a 1 cc syringe and place on ice. Inject 0.2 mg/kg VEGFA16519 at a concentration of 0.05 mg/ml.</li>
</ol>
4. Insertion of the Indwelling Tail Vein Catheter
<ol>
	<li>Place the animal under anesthesia using an induction chamber with 5% isoflurane with O2 as the carrier gas.</li>
	<li>Transfer the mouse to the surgical platform once it is fully anesthetized and does not respond to a toe pinch.</li>
	<li>Open isoflurane anesthesia line to the surgical platform, close the anesthesia line to the induction chamber and place the nose cone over the animal&#39;s snout. Reduce the isoflurane from 5% to 2.5%.</li>
	<li>Apply ophthalmic ointment to the eyes of the mouse to prevent dryness during anesthesia.</li>
	<li>Heat the animal under the heating lamp for an additional 2 min to increase circulation in the tail as circulation slows with deep anesthesia.</li>
	<li>Sterilize the mouse tail vein with 70% ethanol.</li>
	<li>Insert the tip of the 31 G needle from the catheter constructed in Section 3 into the lateral tail vein of the animal and push the needle in 2-3 mm.</li>
	<li>Pull back on the syringe to see blood, ensuring the catheter is placed in the tail vein.</li>
	<li>Use a 1 cm length of lab tape placed over the needle to hold the needle in place parallel to the vein along the length of the tail.</li>
</ol>
5. Skin Flap Surgical Procedure to Expose the Mammary Tumor
<ol>
	<li>With the animal on the surgical platform swab the tumor and the ventral surface with 70% ethanol.&#160;Note: As described, this procedure is not performed completely aseptically.&#160; For long imaging sessions where infection can become a confounding factor, it is recommended to use proper aseptic technique.</li>
	<li>Using sterile forceps, lift the ventral skin and with sterile scissors make a subcutaneous incision along the ventral midline approximately 1 cm in length. Avoid puncturing the peritoneum during the incision.</li>
	<li>Gently cut the connective tissue attaching the mammary gland and tumor away from the peritoneum to expose the mammary tumor.</li>
	<li>Using scissors and forceps, gently remove and cut away the fascia and fat from the exposed surface of the tumor while maintaining the integrity of the tumor vasculature and minimizing bleeding. This step is critical in maintaining the tumor architecture and vasculature supply of the tumor.</li>
</ol>
6. Animal Preparation for Microscopy
<ol>
	<li>Transfer the animal to the pre-warmed imaging stage with the exposed tumor placed on the coverslip on the stage insert over the microscope objective for imaging. Take care to transfer the tail vein catheter gently with the animal so as not to dislodge the needle.</li>
	<li>Place the anesthesia nose cone over the animal&#39;s snout to maintain anesthesia set to 3% isoflurane.</li>
	<li>Position the animal so that the tumor rests in the hole of the rubber pad and makes contact with the glass coverslip.</li>
	<li>Use two rubber pads to gently hold the tumor in place and fix them to the microscope stage with lab tape to reduce movement during image acquisition.</li>
	<li>Fill the chamber from the rubber pad with PBS to keep the tissue hydrated and maintain optical contact with the coverslip.</li>
	<li>Start monitoring the animal&#39;s vital signs with a pulse oximeter probe. Attach a clip sensor to the thigh of the animal. Alternatively a collar that is configured to fit around the neck can be used.</li>
	<li>Place the heating box over the animal to maintain 30 &#176;C 20.</li>
	<li>Slowly reduce the level of isoflurane to 0.5-1% to maintain anesthesia and maintain blood flow.</li>
</ol>
7. Image Acquisition and Injection of Fluorescent Dyes and Injectable Proteins
<ol>
	<li>Using the microscope eyepiece focus on the fluorescent tumor cells on the surface of the tumor.</li>
	<li>Select an area of interest by finding areas with flowing blood vessels. The selection of an area of interest with flowing blood vessels is critical for assessing tumor vasculature.</li>
	<li>Once a region of interest has been selected switch the microscope into multiphoton imaging mode.</li>
	<li>Set the upper and lower limits of a z-series. Set the upper limit of the z-series by using the focus adjuster to move the objective to the desired start location and clicking on the Z position &#34;Top&#34; button. Determine the upper limit of the z-series by visualizing the collagen fiber network at the surface of the tumor in the second harmonic generation (SHG) channel and observing when no cells but only collagen fibers are visible.
	<ol>
		<li>Set the lower limit of the z-series by moving the objective to the desired imaging depth (typically 50 - 150 &#181;m) and clicking the Z position &#34;Bottom&#34; button.</li>
		<li>Set the step size by typing the desired value into the step size field. Determine step size based on considerations for resolution and acquisition time (typically 2 &#181;m is used for high-resolution 3D reconstruction and 5 &#181;m otherwise).</li>
	</ol>
	</li>
	<li>Set the time-lapse interval by switching to the Time-Lapse panel and entering the desired lapse time into the Time-Lapse field. For optimal temporal resolution set the time interval to 0 sec for continuous imaging. Note: For long time-lapse imaging it is recommended to set the time interval to 10 sec between acquisitions to replenish the objective immersion liquid.</li>
	<li>Once the parameters for imaging have been determined, slowly inject 155 kDa dextran-TMR.
	<ol>
		<li>Remove the syringe with PBS in the tail vein catheter and replace with the syringe containing 155 kDa dextran-TMR taking care not to introduce any bubbles into the line.</li>
		<li>Slowly inject 155 kDa dextran-TMR into the mouse, with a maximum volume of 200 &#181;l, and replace the syringe with syringe containing PBS. CRITICAL STEP: Perform the injection slowly and take precautions to avoid getting solution outside of the vein.</li>
	</ol>
	</li>
	<li>Start the acquisition of the Z-stack time-lapse imaging by clicking on the Z-Stack and Time-Lapse buttons to depress them and then clicking on the record button.</li>
	<li>If other fluorescent dextrans, i.e., 10 kDa dextran-fluorescein isothiocyanate (FITC), or proteins, i.e., VEGFA165, have been prepared in advance, inject them after the start of image acquisition for a t = 0 min start.
	<ol>
		<li>Remove the syringe with PBS in the tail vein catheter and replace with the syringe containing 10 kDa dextran-FITC or VEGFA165 taking care not to introduce any bubbles into the line.</li>
		<li>Slowly inject injectables into the mouse through the tail vein catheter and replace the syringe with a syringe containing PBS.</li>
	</ol>
	</li>
	<li>Every 30-45 min, slowly inject 50 &#181;l of PBS or saline to maintain hydration of the animal.</li>
</ol>
8. Euthanasia
<ol>
	<li>At the termination of image acquisition, euthanize the animal.
	<ol>
		<li>Increase the isoflurane to 5%.</li>
		<li>Keep the animal under 5% isoflurane until 30 sec after it ceases to breathe and remove the animal from the stage.</li>
		<li>Perform cervical dislocation to ensure complete euthanasia.</li>
	</ol>
	</li>
</ol>
9. Image Processing
<ol>
	<li>Acquire images at 16-bit TIFF files for each individual channel at each time point and save sequentially.</li>
	<li>Perform separation of spectral overlap (i.e., GFP and CFP), elimination of x-y drift and the generation of movies from time-lapse sequences using established methods in ImageJ9. Perform 3D surface reconstructions of high-resolution images13.</li>
</ol>

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<li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc Natl Acad Sci U S A. 107 (42), 18115-18120 (2010).</li>
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</ol>

The authors have nothing to disclose.

Cellular interactions that occur spontaneously in the tumor microenvironment can lead to changes in tumor cell motility and intravasation. High-resolution intravital imaging of live tumor tissue permits the visualization of multi-cellular dynamics that can be highly transient10,13,24. End-point in vivo assays or time-lapse images acquired with discrete time points can provide essential information on molecular mechanisms of processes in the tumor microenvironment. Intravital imaging studies have been ...

Dissemination of tumor cells from the primary mammary tumor has been shown to involve not only tumor cells, but host stromal cells including macrophages and endothelial cells. Furthermore, tumor vasculature is abnormal with increased permeability1. Thus, determining how tumor cells, macrophages and endothelial cells interact to mediate vascular permeability and tumor cell intravasation in the primary tumor microenvironment is important for understanding metastasis. Understanding the kinetics of vascular permeability, tumor cell intravasation and the underlying signaling mechanism of multicellular interactions in the tumor microenvironment can provide crucial information in the development and administration of anti-cancer therapies.
The primary means of studying tumor vascular permeability in vivo has been the measurement of extravascular dyes such as Evans blue2, high molecular weight dextrans (155 kDa)3 and fluorophore or radiotracer-conjugated proteins (including albumin)4 at fixed time points after injection of the dye. Advancements in microscopy, animal models and fluorescent dyes have enabled the visualization of cellular processes and vascular permeability in live animals through intravital microscopy5.
Live animal imaging with the acquisition of static images, or short time-lapse sequences over several time points does not allow for the complete understanding of dynamic events in the tumor microenvironment6,7. Indeed, static image acquisition over the course of 24 hr demonstrated that tumor vasculature is leaky, however the dynamics of vascular permeability was not observed6. Thus, extended continuous time-lapse imaging up to 12 hours captures the kinetics of dynamic events in the tumor microenvironment.
This protocol describes the use of extended time-lapse multiphoton intravital microscopy to study dynamic multicellular events in the tumor microenvironment. Multiple cell types in the tumor microenvironment are labeled with injectable dyes or by using transgenic animals expressing fluorescent proteins. Using a tail vein catheter vascular dyes or proteins can be injected after the start of imaging to acquire kinetic data of multicellular events in the tumor microenvironment. For live cell imaging the mammary tumor is exposed through the surgical preparation of a skin flap. Images are acquired for up to 12 hours using a multiphoton microscope equipped with multiple photomultiplier tubes (PMT) detectors8. By using appropriate filters, a subtraction algorithm enables 4 PMT detectors to acquire 5 fluorescent signals in the tumor microenvironment simultaneously9. High-resolution multiphoton intravital microscopy captures single cell resolution imaging of tumor-stroma interactions in the tumor microenvironment, leading to a better understanding of vascular permeability and tumor cell intravasation10-13. Specifically, extended intravital imaging revealed highly localized, transient vascular permeability events that occur selectively at sites of interaction between a tumor cell, a macrophage and an endothelial cell (defined as the Tumor MicroEnvironment of Metastasis, TMEM14)13. Furthermore, tumor cell intravasation occurs only at TMEM and is spatially and temporally correlated with vascular permeability13. Single cell resolution of the dynamics of these events was made possible through the use of extended time-lapse multiphoton microscopy of fluorescently labeled cells in the tumor microenvironment.

<table><tbody><tr><td>155 kDa dextran-tetramethylrhodamine isothiocyanate</td><td>Sigma Aldrich</td><td>T1287</td><td>reconstitute at 20 mg/ml in 1x PBS</td></tr><tr><td>70 kDa dextran-Texas Red</td><td>Life Technologies</td><td>D-1830</td><td>reconstitute at 10 mg/ml in 1x PBS</td></tr><tr><td>10 kDa dextran-fluorescein isothyocyanate</td><td>Sigma Aldrich</td><td>FD10S</td><td>reconstitute at 20 mg/ml in 1x PBS</td></tr><tr><td>Qdot 705 ITK Amino (PEG) Quantum Dots</td><td>Life Technologies</td><td>Q21561MP</td><td>Dilute 25 &amp;#956;l in 175 &amp;#956;l of 1x PBS for injection</td></tr><tr><td>MMTV-PyMT mice</td><td>Jackson Laboratory</td><td>2374</td><td></td></tr><tr><td>Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td><td>Jackson Laboratory</td><td>26051</td><td></td></tr><tr><td>Csf1r-EGFP mice</td><td>Jackson Laboratory</td><td>18549</td><td></td></tr><tr><td>1x PBS</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Isoethesia (isoflurane)</td><td>Henry Schein Animal Health</td><td>50033</td><td>250 ml</td></tr><tr><td>Oxygen</td><td>AirTech</td><td></td><td></td></tr><tr><td>1 ml syringe, tuberculin slip tip</td><td>BD</td><td>309659</td><td></td></tr><tr><td>30 G x 1 (0.3 mm x 25&amp;nbsp;mm) needle</td><td>BD</td><td>305128</td><td></td></tr><tr><td>Polyethylene micro medical tubing&amp;nbsp;</td><td>Scientific Commodities Inc</td><td>BB31695-PE/1</td><td>0.28 mm I.D. x 0.64 mm O.D.</td></tr><tr><td>Microscope coverglass</td><td>Corning</td><td>2980-225</td><td>thickness 1.5, 22 x 50&amp;nbsp;mm</td></tr><tr><td>PhysioSuite MouseSTAT pulse oximeter, software and sensors</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>Laboratory tape</td><td>Fisher Scientific</td><td>159015R</td><td></td></tr><tr><td>soft rubber pad</td><td>McMaster-Carr</td><td>8514K62</td><td>Ultra-Soft Polyurethane Film, 3/16&amp;rdquo; Thick, 12&quot; x 12&quot;, 40 Oo Durometer, Plain Back</td></tr><tr><td>hard rubber pad</td><td>McMaster-Carr</td><td>8568K615</td><td>High-Strength Neoprene Rubber Sheet 1/4&quot; Thick, 12&quot; x 12&quot;, 50&amp;nbsp;A Durometer</td></tr><tr><td>Microscope</td><td>Olympus</td><td></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.</td></tr><tr><td>7-Punch set</td><td>McMaster-Carr</td><td>3429A12</td><td>1/4&quot; to 1&quot; Hole Diameter, for Hammer-Driven Hole Punch</td></tr></tbody></table>

extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment

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.

Extended time-lapse intravital microscopy enables single cell resolution imaging of multicellular processes in the tumor microenvironment. By fluorescently labeling tumor cells, macrophages, the vascular space, and visualizing the collagen fiber network using the second harmonic generation signal, multiple compartments in the tumor microenvironment are simultaneously tracked during imaging. Tumor cells labeled with fluorescent proteins can be generated in transgenic mice as has been done ...

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

The overall goal of this procedure is to visualize the dynamic events within the tumor microenvironment via high resolution, intravital multiphoton mircrospy. This method can help answer key questions about the tumor microenvironment such as which dynamic events are involved in vascular permeability and multicellular interactions. The main advantage of this technique is that the kinetics of the events in the tumor microenvironment can be observed and measured.Before beginning the procedure, turn on all of the microscope and laser components, including the two photon lasers and the detectors. And set the heating box to 30 degree celsius to prewarm the stage. Wipe the microscope stage and stage insert with 70%ethanol.When the stage is dry, place a large drop of water on the 25 x 1.05 NA microscope objective to maintain optical contact with the cover glass and place a number 1.5 thickness cover glass over the insert imaging port. Then, cut a 30 centimeter piece of polyethylene tubing. Place the rubber pad on top of the cover glass.Using forceps, break a 31 gauge needle off of its plastic fitting and insert the blunted end of the needle hub into the tubing. Insert and attached a 31 gauge needle into the other end of the PE tubing. Then, fill a one cc syringe with sterile PBS and attach the syringe to the 31 gauge needle.Use the PBS to flush all of the air out of the tubing and needle. Then, the one one cc syringe with 200 microliters of the appropriate vascular label and another with the appropriate injectable protein. Before beginning the surgery, confirm the proper anesthesia with a lack of response to toe pinch and apply eye ointments to the anesthetized mouse.Next, place the animal on a surgical platform under a heat lamp to increase the tail circulation. After two minutes, sterilize the tail with 70%ethanol and insert the tip of the 31 gauge catheter, two to three millimeters in to the lateral tail vein. Then, place a one centimeter length of lab tape onto the catheter to hold the needle parallel to the vein along the length of the tail.When the catheter has been secured, swab the ventral and tumor surfaces with 70%ethanol. The, lift the skin over the abdomen and use sterile scissors to make an approximately one centimeter subcutaneous incision along the ventral midline of the abdomen, taking care to avoid puncturing the peritoneum. Gently cut the connective tissue attaching the mammary gland and tumor away from the peritoneum to expose the tumor.Then, using the scissors and forceps, gently remove the fascia and fat from the exposed surface of the tumor. When the tumor has been exposed, transfer the mouse to the prewarmed imaging stage. Use lab tapes to secure the cover glass, then place the tumor in the hole of a rubber pad covering the cover slip over the microscope objective.When the animal is in position, fill the rubber pad chamber with PBS to keep the tissue hydrated and to maintain optical contact with the cover slip. Place a pulse oximeter probe collar sensor around the neck to monitor the animal's vital signs and place the heating box over the animal. Then, use the microscope IPs to focus on the fluorescent tumor cells on the surface of the tumor and located area with flowing blood vessels.Once a region of interest has been selected, switch the microscope to multiphoton imaging mode and use the focus adjuster to move the objective to the desired start location. Then, click on the Z position top button to set the upper limit of the z series and visualize the collagen fiber network at the surface of the tumor in the second harmonic generation channel. Then move the objective to the desired imaging depth and click the z position bottom button to set the lower limit of the z series.Next, enter the desired value into the step size fields. Then, switch to the Time Lapse Panel and enter the appropriate lapse time. Once the imaging parameters had been determined, replaced the syringe attached to the catheter with a syringe of vascular labeling dye and slowly administer a maximum of 200 microliters of the dye solution, taking care that the solution enters the vein.After the injection, return the PBS syringe to the catheter and begin the z step time lapse imaging. When all of the images have been obtained, replace the syringe of PBS with the syringe containing the injectable protein, taking care not to introduce any bubbles into the line. Then slowly inject the protein suspension into the mouse and replace the syringe with a syringe containing PBS.Macrophages can be tracked with fluorescently labeled 70 kilodalton and dextran which acummulate in the phagocytic microphages. Dual labeling the tumor cells and the microphages allows the direct interaction with these two cell types to be visualize in real time. To visualize changes in the tumor vascular permeability, quantum dots can also be used to label the vascular space.As they are large enough that they do not readily diffuse through the interarterial spaces. Continuous high resolution imaging facilitates the capture of the vascular permeability events and the quantification of the kinetics of the dextran extravasation and clearance. In this example field of view, the peak of the vacscular di-extravasation is observed between 29 and 34 minutes as evidenced by the extra vascular TMR signal.Injecting additional vascular dyes or proteins to induce vascular permeability can provide insight into the signaling events regulating vascular permeability. Indeed, within the first few seconds of injection, 10 kilodaltons of dextran FITC can be observed extravasating from the vasculature, while the 155 kilodalton in dextran TMR remains confined to the vascular space. Once mastered, this technique can be completed in one hour if it is performed properly.While attempting this procedure, it's important to remember to remove the connective tissue gently without damaging the surface of the tumor and to keep the tumor surface hydrated. Following this procedure of cellular dynamics such tumor cell migration can be visualized to answer additional questions about the directionality of tumor cell migration and intravasation. After watching this video, you should have a good understanding of how to visualize dynamic celluluar events in a tumor microenvironment by intervital multiphoton microscopy.

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Research

JoVE Journal

Medicine

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Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

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