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 ...

<ol>
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How tumors make bad blood vessels and stroma.</a> Am J Pathol. 162 (6), 1747-1757 (2003).</li><li>Nagy, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+properties+of+tumor+surrogate+blood+vessels+induced+by+VEGF-A.">Permeability properties of tumor surrogate blood vessels induced by VEGF-A.</a> Lab Invest. 86 (8), 767-780 (2006).</li><li>Egawa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+analysis+of+vascular+permeability+in+mice+using+two-photon+microscopy.">Intravital analysis of vascular permeability in mice using two-photon microscopy.</a> Sci Rep. 3, (2013).</li><li>Yuan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+a+human+tumor+xenograft:+molecular+size+dependence+and+cutoff+size.">Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size.</a> Cancer Res. 55 (17), 3752-3756 (1995).</li><li>Dellian, M., Yuan, F., Trubetskoy, V. S., Torchilin, V. P., Jain, R. 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Chapter. 19, (2013).</li><li>Patsialou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+multiphoton+imaging+reveals+multicellular+streaming+as+a+crucial+component+of+in+vivo.+cell+migration+in+human+breast+tumors.">Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo. cell migration in human breast tumors.</a> Intravital. 2 (2), e25294 (2013).</li><li>Gligorijevic, B., Bergman, A., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+classification+links+tumor+microenvironments+with+tumor+cell+phenotype.">Multiparametric classification links tumor microenvironments with tumor cell phenotype.</a> PLoS Biol. 12 (11), e1001995 (2014).</li><li>Roussos, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mena+invasive+(MenaINV)+promotes+multicellular+streaming+motility+and+transendothelial+migration+in+a+mouse+model+of+breast+cancer.">Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer.</a> J Cell Sci. 13, 2120-2131 (2011).</li><li>Harney, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Imaging+Reveals+Local,+Transient+Vascular+Permeability,+and+Tumor+Cell+Intravasation+Stimulated+by+TIE2hi+Macrophage-Derived+VEGFA).">Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA).</a> Cancer Discov. 5 (9), 932-943 (2015).</li><li>Robinson, B. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Ovchinnikov, D. 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(73), (2013).</li><li>Weis, S., Cui, J., Barnes, L., Cheresh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+barrier+disruption+by+VEGF-mediated+Src+activity+potentiates+tumor+cell+extravasation+and+metastasis.">Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis.</a> J Cell Biol. 167 (2), 223-229 (2004).</li><li>Wyckoff, J., Gligorijevic, B., Entenberg, D., Segall, J., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Multiphoton+Imaging+of+Tumors.">High-Resolution Multiphoton Imaging of Tumors.</a> In Vivo. Cold Spring Harb Protoc. (10), (2011).</li><li>Lathia, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+in+vivo.+evidence+for+tumor+propagation+by+glioblastoma+cancer+stem+cells.">Direct in vivo. evidence for tumor propagation by glioblastoma cancer stem cells.</a> PLoS One. 6 (9), e24807 (2011).</li><li>Nakasone, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer Cell. 21 (4), 488-503 (2012).</li><li>Dreher, M. 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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 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.

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 ...

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

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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JoVE Journal

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8.0K visualizações.  Albert Einstein College of Medicine. 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 proce...