The Satchi-Fainaro research laboratory is partially supported by The Association for International Cancer Research (AICR), German-Israel Foundation (GIF), THE ISRAEL SCIENCE FOUNDATION (Grant No. 1309/10), Swiss Bridge Award, and by grants from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics, and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund.

NOTE: All animal procedures were approved and performed in compliance with the standards of Tel Aviv University Sackler School of Medicine Institutional Animal Care and Use Committee (IACUC).
1. Conditioned Media Preparation
<ol>
	<li>Seed 2 x 106 U-87 MG cells in a 10 cm culture plate in a volume of 8 ml Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% pencillin/streptomycin/nystatin solution.</li>
	<li>Incubate cells at 37 &#176;C; 5% CO2 until achieving full confluency at 48 hr.</li>
	<li>Using a 10 ml syringe, withdraw all medium from the plate and filter through a 0.45 &#181;m filter into a 15 ml tube.</li>
	<li>Without washing the cells plate, add 1 ml trypsin solution (0.25%) and incubate.</li>
	<li>When all cells are detached, inactivate the trypsin using at least 5 ml DMEM supplemented with 10% FBS and preform a cell count.</li>
	<li>Count the number of cells using a hemocytometer or other cell-counting device and calculate the number of cells according to the counting method used. 
	NOTE: It will be used later in order to normalize C.M. concentration.</li>
	<li>Transfer the C.M. to a glass round-bottom flask and concentrate under high vacuum rotary evaporator. In order to accelerate the process, increase the bath temperature to 37 &#176;C, if desired. Continue until the C.M. achieves a paste-like texture.</li>
	<li>Resuspend the C.M. from each culture plate in 400 &#181;l saline. When using more than one plate, use equivalent volume. For example, for 2 culture plates, use 800 &#181;l. Use each plate for 4 mice.</li>
	<li>In order to compare different cell lines or treatments, adjust the volume according to the cell ratio received in step 1.6. For example, if one plate contains 1.5x more cells, add 200 &#181;l of saline to the pre-existing 400 &#181;l. This will be the adjusted C.M.</li>
</ol>
2. ECM-mimic Gel Plug Inoculation into Mice
<ol>
	<li>Before inoculating the ECM-mimic gel into mice, prepare the following materials:
	<ol>
		<li>Growth-factors reduced phenol-free ECM-mimic gel (thaw overnight, a day before the experiment, at 4 &#176;C on ice).</li>
		<li>Electric shaver.</li>
		<li>Ketamine and xylazine in order to anesthetize the mice.</li>
		<li>Ice-cold 1,000 &#181;l sterile tips.</li>
		<li>Ice-cold 1 ml syringe and 27 G needles.</li>
	</ol>
	</li>
	<li>Prepare for each mouse a 1.5 ml Eppendorf tube containing 80 &#181;l adjusted C.M. and keep on ice.</li>
	<li>To each tube, add 600 &#181;l ice-cold growth-factors reduced phenol-free ECM-mimic gel that was thawed overnight at 4 &#176;C, on ice. The ECM-mimic gel is liquid in 4 &#176;C and solidifies at body temperature; in order to prevent the ECM-mimic gel from solidifying, use ice-cold 1,000 &#181;l tips. Mix tubes carefully without forming bubbles and keep on ice.</li>
	<li>Anesthetize 6 week old BALB/c mice using ketamine (100 mg/kg) and xylazine (12 mg/kg). Confirm proper anesthesia according to the institutional animal care and use committee, i.e. prior to beginning the experiment. Animals have reached their proper plane of anesthesia when they no longer respond to a firm toe pinch.</li>
	<li>Shave the abdominal area of the mice and disinfect using a surgical scrub/alcohol.</li>
	<li>Use a cold 1 ml syringe and a 27 G needle to inject the contents of the Eppendorf tube subcutaneously. Perform the injection very slowly to prevent bubble formation. Once all volume is administered, do not eject the needle for several minutes, until solidification occurs.</li>
</ol>
3. Evaluation of ECM-mimic Gel Plug Vascularization Using Ultrasound
NOTE: This part of the protocol requires previous knowledge of operating an ultrasound imaging system, or the assistance of an authorized technician.
<ol>
	<li>Before starting, have the following materials and instruments ready:
	<ol>
		<li>Electric shaver.</li>
		<li>Depilatory cream.</li>
		<li>Ketamine and xylazine in order to anesthetize the mice.</li>
		<li>Non-targeted microbubbles.</li>
		<li>Activation device.</li>
		<li>US imaging system including a 55 MHz 708 probe and a 3D acquisition micromanipulator.</li>
	</ol>
	</li>
	<li>Measure vascularization within the plugs three weeks post inoculation: 
	NOTE: In the U-87 MG-based animal model, 3 weeks was found to represent the ideal time point for imaging. Under different settings, calibrate vascularization kinetics starting from a few days following inoculation of plugs.
	<ol>
		<li>Anesthetize mice using ketamine (100 mg/kg) and xylazine (12 mg/kg). Confirm proper anesthesia according to the institutional animal care and use committee, i.e. prior to beginning the experiment. Animals have reached their proper plane of anesthesia when they no longer respond to a firm toe pinch.</li>
		<li>Shave the abdominal area of the mice and treat with depilatory cream.</li>
		<li>Fix the mice supinely on the stage of the micro-manipulator.</li>
		<li>Using a 1 ml syringe containing saline and a 30 G needle, place the needle inside the tail vein, fix the needle firmly and loosen the syringe.</li>
		<li>Set up for the Contrast 3D-mode image acquisition session:
		<ol>
			<li>Press &#8220;3D&#8221; to initialize the 3D motor stage and place the transducer in the middle of the desired scanning area.</li>
			<li>Press &#8220;3D&#8221; and input the scan distance and step size, then press &#8220;Scan&#8221;.</li>
			<li>Make sure that the entire plug was scanned in the 3D image data displayed. If not, adjust the transducer so that the entire plug is visible in the scan and repeat step 3.5.2.</li>
			<li>Press &#8220;Contrast&#8221; and push the time gain compensation (TGC) controls to the left to darken the image.</li>
		</ol>
		</li>
		<li>Activate the microbubbles using a vial mixer that is designated for the activation of microbubbles for 45 sec.</li>
		<li>Mix in a 1 ml syringe 50 &#181;l of microbubbles with 50 &#181;l saline.</li>
		<li>Replace the saline-containing loosened syringe with a syringe containing activated microbubbles. Inject the diluted microbubbles into the tail vein of the mice, wait a few seconds for the bubbles to reach a steady state, and then acquire 3D contrast-enhanced cine loop of the plug, using the 3D acquisition motor:
		<ol>
			<li>Press &#8220;3D&#8221; and select &#34;Scan&#34; to acquire 3D contrast-enhanced images and then press &#8220;Cine Store&#8221; to save the image data.</li>
			<li>Press &#8220;3D&#8221; and click &#8220;Destroy 3D&#8221; to destruct the microbubbles.</li>
			<li>Press &#8220;3D&#8221; again and select &#34;Scan&#34; to acquire a post-destruction cine loop, then press &#8220;Cine Store&#8221;.</li>
		</ol>
		</li>
		<li>Generate the overlay image frames pre- and post-destruction of microbubbles:
		<ol>
			<li>In the &#8220;Reference Settings&#8221; on the left side of the screen click &#8220;Create Reference&#8221;.</li>
			<li>Press &#8220;Study Management&#8221; to open the pre-destruction cine loop acquired.</li>
			<li>Click &#8220;Process Cine&#8221; to generate the green contrast overlay, then click &#8220;Load Into 3D&#8221;.</li>
		</ol>
		</li>
		<li>Use the US imaging software in order to quantitate the resulting 3D image for blood volume within the plugs:
		<ol>
			<li>Press &#8220;Scan/Freeze&#8221; and then press &#8220;Mode settings&#8221;.</li>
			<li>Click &#8220;Volume&#8221; and select &#8220;Parallel&#8221;.</li>
			<li>Create a 3D volume of the target tissue by segmenting a series of contours.</li>
			<li>Click &#8220;Finish&#8221; to complete the segmentation. 
			NOTE: The volume of the contrast agent will be displayed on the lower left side of the screen.</li>
		</ol>
		</li>
		<li>At the end of the procedure, place mice in a warm, clean, dry, quiet environment away from other animals. Provide warmth using a commercially-available surgical heating pad or an incandescent lamp&#160;(50-75 watt), placed 12-14 inches away from rodent. Monitor mice continuously until maintaining upright posture and walking normally.</li>
	</ol>
	</li>
</ol>
4. Evaluation of ECM-mimic Gel Plug Vasculature Morphology Using Fibered Confocal Endomicroscopy
NOTE: this part of the protocol is performed immediately following US imaging and it requires previous knowledge of operating the fibered confocal endomicroscopy imaging system, or the assistance of an authorized technician.
<ol>
	<li>Before starting, have the following materials and instruments ready:
	<ol>
		<li>Surgery tools like forceps, scissors and absorbable sewing threads.</li>
		<li>FITC-conjugated dextran (70 KDa).</li>
		<li>Calibration solutions (three tubes each containing hydrogen peroxide, water or fluorescent solution).</li>
		<li>The imaging system and a probe suitable for imaging of blood vessels according to manufacturer.</li>
	</ol>
	</li>
	<li>Anesthetize mice using ketamine (100 mg/kg) and xylazine (12 mg/kg).</li>
	<li>Administer i.v., via the tail vein, 200 &#181;l of 10 mg/ml FITC-labeled dextran.</li>
	<li>Disinfect the abdominal area using a surgical scrub/alcohol and apply a small incision approximately 1 cm from the plug.</li>
	<li>Place the endomicroscopy probe on top of the plug gently while keeping the plug intact.</li>
	<li>Acquisition:
	<ol>
		<li>Once the probe is set on the plug, press &#8220;Start laser&#8221; using the foot pedal. The fluorescence should be shown on the screen.</li>
		<li>Acquire short movies (up to 30 sec) by pressing &#39;Record&#39; on the screen or &#39;Select&#39; using the foot pedal.</li>
		<li>Between movies, wash the probe by placing the tip in a tube containing water.</li>
		<li>When finished, remove the probe and clean the tip with a cotton tip.</li>
	</ol>
	</li>
	<li>To analyze mean vessel diameter:
	<ol>
		<li>Click on the &#8220;Detect microvessels&#8221; icon in the upper panel to open the vessel detection module window.</li>
		<li>Click &#8220;Preferences&#8221; and mark &#8220;Mean vessel diameter&#8221; in the &#8220;Statistic of interest&#8221; and &#8220;Display histogram bars&#8221; in the &#8220;Display mode&#8221;.</li>
		<li>Click &#8220;Detect&#8221; to display the selected measurements.</li>
	</ol>
	</li>
	<li>To analyze the fluorescent signal intensity in areas adjacent to blood vessels:
	<ol>
		<li>Click on &#8220;Create circle ROI&#8221; in the left panel and create a circle in the area of interest.</li>
		<li>Click &#8220;Compute histogram within ROI&#8221; to compute the values related to this region.</li>
	</ol>
	</li>
</ol>
5. Post-procedural Treatment of Mice
<ol>
	<li>At the end of each imaging procedure, place mice in a warm, clean, dry, quiet environment away from other animals. Provide warmth using a commercially-available surgical heating pad or an incandescent lamp&#160;(50-75 W), placed 12-14 in&#160;away from rodent. Monitor mice continuously until maintaining upright posture and walking normally. &#160;</li>
	<li>For further analysis of plugs&#39; vascularization at additional time points, apply the following recovery conditions:
	<ol>
		<li>Suture the small incision using absorbable sutures.</li>
		<li>Give mice analgesia such as buprenorphine (1 mg/kg) subcutaneously, and return to individual cages.</li>
		<li>Observe all animals to determine if further analgesic treatment is necessary and re-evaluate daily to monitor and assess for distress.</li>
	</ol>
	</li>
	<li>At the end-point of the experiment, euthanize mice by first applying isoflurane, followed by cervical dislocation.</li>
	<li>If additional imaging will be performed in the weeks following the initial imaging steps, all procedures should be kept as sterile as possible.&#160; A surgical skin scrub should be performed, sterile water and sterile cotton tips should be used to clean the endomicroscopy probe, and the probe should be disinfected between each animal using sterile water.&#160;</li>
</ol>

<ol>
	<li>Kibbey, M. C., Grant, D. S., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+SIKVAV+site+of+laminin+in+promotion+of+angiogenesis+and+tumor+growth:+an+in+vivo+Matrigel+model.">Role of the SIKVAV site of laminin in promotion of angiogenesis and tumor growth: an in vivo Matrigel model.</a> J Natl Cancer Inst. 84, 1633-1638 (1992).</li><li>Passaniti, 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=A+simple,+quantitative+method+for+assessing+angiogenesis+and+antiangiogenic+agents+using+reconstituted+basement+membrane,+heparin,+and+fibroblast+growth+factor.">A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor.</a> Lab Invest. 67, 519-528 (1992).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10, 588-612 (2006).</li><li>Auerbach, R., Lewis, R., Shinners, B., Kubai, L., Akhtar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+assays:+a+critical+overview.">Angiogenesis assays: a critical overview.</a> Clin Chem. 49, 32-40 (2003).</li><li>Johns, A., Freay, A. D., Fraser, W., Korach, K. S., Rubanyi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+estrogen+receptor+gene+prevents+17+beta+estradiol-induced+angiogenesis+in+transgenic+mice.">Disruption of estrogen receptor gene prevents 17 beta estradiol-induced angiogenesis in transgenic mice.</a> Endocrinology. 137, 4511-4513 (1996).</li><li>Adini, 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=Matrigel+cytometry:+a+novel+method+for+quantifying+angiogenesis+in+vivo.">Matrigel cytometry: a novel method for quantifying angiogenesis in vivo.</a> J Immunol Methods. 342, 78-81 (2009).</li><li>Matsumura, Y., Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+concept+for+macromolecular+therapeutics+in+cancer+chemotherapy:+mechanism+of+tumoritropic+accumulation+of+proteins+and+the+antitumor+agent+smancs.">A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs.</a> Cancer Res. 46, 6387-6392 (1986).</li><li>Lindner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+in+medical+imaging:+current+applications+and+future+directions.">Microbubbles in medical imaging: current applications and future directions.</a> Nat Rev Drug Discov. 3, 527-532 (2004).</li><li>Kuliszewski, M. 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=Molecular+imaging+of+endothelial+progenitor+cell+engraftment+using+contrast-enhanced+ultrasound+and+targeted+microbubbles.">Molecular imaging of endothelial progenitor cell engraftment using contrast-enhanced ultrasound and targeted microbubbles.</a> Cardiovasc Res. 83, 653-662 (2009).</li><li>Stieger, S. M., 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=Ultrasound+assessment+of+angiogenesis+in+a+matrigel+model+in+rats.">Ultrasound assessment of angiogenesis in a matrigel model in rats.</a> Ultrasound Med Biol. 32, 673-681 (2006).</li><li>Erez, N., Truitt, M., Olson, P., Arron, S. T., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-kappaB-Dependent.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent.</a> Manner. Cancer Cell. 17, 135-147 (2010).</li><li>Segal, E., Satchi-Fainaro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+development+of+polymer+conjugates+as+anti-angiogenic+agents.">Design and development of polymer conjugates as anti-angiogenic agents.</a> Adv Drug Deliv Rev. 61, 1159-1176 (2009).</li><li>Satchi-Fainaro, R., 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=Prospective+identification+of+glioblastoma+cells+generating+dormant+tumors.">Prospective identification of glioblastoma cells generating dormant tumors.</a> PLoS One. 7, 44395 (2012).</li></ol>

The authors have nothing to disclose.

Combining complementary imaging methods allows the acquirement of information on different angiogenesis components&#39; microvessel density, functionality and morphology. Loading C.M. on ECM-mimic gel plugs generates a tumor microenvironment, which is separated from other cell populations, like endothelial cells, that are recruited and extravasate into the plug.
By performing, in parallel, microbubbles contrast-enhanced US imaging and fibered confocal endomicroscopy imaging, we can conclude th...

The matrigel plug angiogenesis assay was first described by Kibbey et al. in 1992, where it was used to evaluate angiogenesis stimulation by the peptide SIKVAV (Ser-Ile-Lys-Val-Ala-Val)1. In contrast to other in vivo angiogenesis assays, like the mouse corneal angiogenesis or chick chorioallantoic membrane assays, this assay is relatively easy to perform2. The injected ECM-mimic gel may contain cells, pro-angiogenic or an anti-angiogenic compounds and/or factors. When evaluating the activity of an anti-angiogenic compound, the plug normally contains a mixture of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), and the anti-angiogenic substance can be administered directly into the plug or systemically3,4.
The ECM-mimic gel is injected subcutaneously where it solidifies within minutes. Assessment of blood vessel recruitment in the resulting plug is typically performed by measuring hemoglobin levels within the plug, by fluorescence signal measurement following injection of fluorescein isothiocyanate (FITC)-labeled dextran, by immunostaining of histology sections for endothelial-specific markers or by fluorescence-activated cell sorting (FACS)2,5,6. However, this assessment only allows end-point analysis and lacks information about the functionality and morphology of blood vessels. In addition, measurements of plasma volume, either by hemoglobin levels or by using dextran-FITC as an indicator, may be misleading since blood content is affected by the size of blood vessels and the extent of stagnant pools of blood. This is especially crucial when the recruited vasculature is characterized by the enhanced permeability and retention (EPR) effect7.
We herein propose a new, more precise, method for visualization of the recruited blood vessels by combining two complementary imaging modalities. High-resolution microbubbles contrast-enhanced ultrasound (US) combined with intravital fibred-confocal endomicroscopy can provide information not only about blood vessel density, but also on their morphology and functionality. Moreover, this analysis can be performed at several time points thereby enabling monitoring of angiogenesis kinetics. US is a widely-used imaging modality which possesses high spatial resolution for blood vessels imaging following intravenous (i.v.) injection of microbubbles that remain exclusively in the vascular compartment8-11. The microbubbles are gas-filled microbubbles that produce a strong echogenic signal when excited with an US pulse and thus serve as a good contrast agent. 3D images of the plug are acquired using the US imaging system software following microbubbles destruction. The resulting images are composed from image frames of pre- and post-destruction of microbubbles and reflect the difference in video intensity in color. These overlayed images are automatically displayed by the software. Consequently, the percent of functional vessels within the plug can be quantified. High resolution fibered confocal endomicroscopy serves as a complementary imaging modality by reporting on blood vessels morphology. In this minimally invasive method, images are acquired following i.v. administration of FITC-conjugated dextran12. The fluorescent polymer dyes the bloodstream and thus serves as a &#39;contrast agent&#39; for blood vessels morphology and blood flow. Moreover, since the injected polymer is at a size of 70 KDa, it can only cross leaky vessels as found in tumor vasculature. This will result in high background from the FITC-labeled dextran outside the blood vessels. Consequently, fibered confocal endomicroscopy can be used to visualize typical characteristics of the EPR effect in tumor neovasculature- enlarged, leaky, highly-tangled vessels, with blunt ends.
This system model was described in the comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant clone13. Vascularization within the plugs was evaluated three weeks post ECM-mimic gel inoculation. It was shown that the vascularization within plugs containing C.M. from U-87 MG fast-growing tumor-generating cells was significantly increased in terms of number, density and functionality, when compared with vascularization within plugs containing C.M. from the dormant tumor-generating cells. Hence, the authors were able to conclude that C.M. isolated from U-87 MG fast-growing tumor-generating cells contains higher levels of pro-angiogenic factors, compared with the C.M. from dormant tumor-generating cells. These factors stimulate the formation of functional blood vessels by positively affecting all steps of the angiogenic cascade (i.e., proliferation, sprouting, migration and finally, formation of tubular structures of endothelial cells).

<table border="0" cellpadding="0" cellspacing="0" width="864">
	<tbody>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Name of the Material/Equipment</td>
			<td dir="LTR" style="width:179px;">Company</td>
			<td dir="LTR" style="width:179px;">Catalog number</td>
			<td dir="LTR" style="width:251px;">Comments/Description&#160;</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Rotary Vacuum Evaporator</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Growth-factors reduced phenol-free Matrigel</td>
			<td dir="LTR" style="width:179px;">BD Bioscience</td>
			<td dir="LTR" style="width:179px;">FAL356231</td>
			<td style="width:251px;">Thaw overnight, at 40C on ice&#160;</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Depilatory cream&#160;</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Vevo2100 US imaging system</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.&#160;</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">Requires previous knowledge in operating or the assistance of an authorized technician</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">55 MHz 708 probe</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Vevo2100 imaging software</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">VialMix&#160;</td>
			<td dir="LTR" style="width:179px;">DEFINITY Imaging</td>
			<td dir="LTR" style="width:179px;">VMIX</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">DEFINITY microbubbles</td>
			<td dir="LTR" style="width:179px;">DEFINITY Imaging</td>
			<td dir="LTR" style="width:179px;">DE4</td>
			<td dir="LTR" style="width:251px;">Activate for 45 seconds using Vialmix before use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">CellVizio (Fibered confocal endomicroscope)</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">Requires previous knowledge in operating or the assistance of an authorized technician</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">ProFlex MiniO/30 probe</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">70 kD FITC-labeled Dextran</td>
			<td dir="LTR" style="width:179px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:179px;">46945</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
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monitoring functionality and morphology of vasculature recruited by factors secreted by fast-growing tumor-generating cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

Implanting ECM-mimic gel plugs with C.M. from fast-growing angiogenic tumor-generating cell line U-87 MG, results in extensive angiogenesis. This vasculature can be extensively explored by using two complementary imaging methods.
Microbubbles contrast-enhanced US imaging reveals extensive recruitment of functional blood vessels into the plug by illustrating extensive blood flow within U-87 MG C.M. loaded plugs, as opposed to undetectable blood flow within plugs containing C.M. from dormant tum...

Watch this Scientific Journal Video about Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells at JoVE.com

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

monitoring-functionality-morphology-vasculature-recruited-factors

Research

JoVE Journal

Biology

9.7K Views.  Tel Aviv University. We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.). 

Video: Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (&bull;OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.1 When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA1 and RNA2 with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein1 and RNA-protein complexes. Equilibrium3 and kinetic4 transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer.5
In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H2O2 (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O2 (we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...