Name: monitoring functionality and morphology of vasculature recruited by factors secreted by fast-growing tumor-generating cells
Uploaded: 2014-11-23T14:00:00
Description: 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

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 ...

<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>

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">
			<td dir="LTR" height="40" style="height:40px;width:255px;">ImageCell software</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;">Calibration Kit</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 align="left" height="40" style="height:40px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td align="left">Gibco Life Tecchnologies</td>
			<td dir="LTR" style="width:179px;">41965-039</td>
			<td></td>
		</tr>
		<tr height="37">
			<td align="left" height="37" style="height:37px;">Fetal bovine serum (FBS)</td>
			<td align="left">Biological Industries&#160;</td>
			<td align="left">04-007-1A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td align="left" height="39" style="height:39px;">Penicillin-Streptomycin-Nystatin Solution</td>
			<td align="left">Biological Industries&#160;</td>
			<td align="left">03-032-1B</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

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