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.).
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.
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 'contrast agent' 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).
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
2. ECM-mimic Gel Plug Inoculation into Mice
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.
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.
5. Post-procedural Treatment of Mice
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 tumor-generating cells (Figure 1).
The morphology of these newly-formed blood vessels is emphasized by fibered confocal endomicroscopy (Figure 2). Blood vessels within plugs loaded with U-87 MG C.M. exhibited morphology typical to the enhanced permeability and retention (EPR) effect for macromolecules such as Dextran-FITC at 70 kDa size used here. Blood vessels were enlarged and highly tangled, with non-continuous and sluggish flow (Figure 2A, C). Also, leakage of blood was observed to the surrounding environment, as shown by high fluorescent signal outside the blood vessels (Figure 2B).
Figure 1: Microbubbles contrast-enhanced US imaging. Following intravenous administration of microbubbles and their destruction, U-87 MG C.M. containing plugs exhibit increased vascularization (in green). Plugs containing C.M. from U-87 MG-derived dormant tumor-generating cells were used as control. Please click here to view a larger version of this figure.
Figure 2: Fiber confocal microscopy imaging. (A) Intravenous administration of 70 KDa Dextran-FITC, enables visualization of blood flow and blood vessels morphology. U-87 MG C.M. containing plugs vasculature consists of leaky, enlarged vessels with blunts ends. Blood vessels leakage is exhibited by fluorescent signal outside the vasculature. Normal vasculature in mouse abdomen was used as control. (B) Fluorescent signal intensity in areas adjacent to blood vessels. The fluorescent signal is exemplified by a spectrum from red to white. Dark red represents low signal intensity, while white represents high fluorescent signal. U-87 MG C.M. containing plugs show a clear fluorescent signal outside the vessels, while no signal is shown outside normal vessels. (C) Mean Vessel Diameter (MVD) of blood vessels within U-87 MG C.M. containing plugs (22.4 µm) is significantly higher compared to the MVD of normal vessels (6.7 µm). Please click here to view a larger version of this figure.
Combining complementary imaging methods allows the acquirement of information on different angiogenesis components' 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 that a pool of factors secreted by fast-growing angiogenic tumors generating cells, without the cancer cells themselves, can not only recruit blood vessels, but also form functional vasculature with EPR typical characteristics. Moreover, this method allows the separation between the impact of the secreted factors and cell-to-cell interaction in tumor microenvironment.
Information obtained from the US and fibered confocal endomicroscopy is however subjected to the limitation of the imaging technique. For instance, while using the fibered confocal endomicroscopy imaging, it is impossible to image a single specific blood vessel over time, or even the same area at the plug.
The ECM-mimic gel plug assay is relatively easy to perform, and this is sustained when loading C.M. with the ECM-mimic gel. However, a critical issue that should be taken under consideration is the C.M. to ECM-mimic gel proportion. High volumes of C.M. might prevent the ECM-mimic gel from solidifying. Therefore, C.M. amount should not exceed approximately 13% of the ECM-mimic gel.
This method can be adjusted for different experimental systems and applications. For example, it can be used to assess anti-angiogenic drugs by loading C.M. from pre- and post-treated cells. Also, other cell lines may be used under these experiment settings. However, calibration is required in order to apply the optimal C.M. concentration. Moreover, the ideal time point for imaging should be optimized according to each model system.
The authors have nothing to disclose.
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.
Name of the Material/Equipment | Company | Catalog number | Comments/Description |
Rotary Vacuum Evaporator | – | – | – |
Growth-factors reduced phenol-free Matrigel | BD Bioscience | FAL356231 | Thaw overnight, at 40C on ice |
Depilatory cream | – | – | – |
Vevo2100 US imaging system | VisualSonics Inc. | – | Requires previous knowledge in operating or the assistance of an authorized technician |
55 MHz 708 probe | VisualSonics Inc. | – | – |
Vevo2100 imaging software | VisualSonics Inc. | – | – |
VialMix | DEFINITY Imaging | VMIX | – |
DEFINITY microbubbles | DEFINITY Imaging | DE4 | Activate for 45 seconds using Vialmix before use |
CellVizio (Fibered confocal endomicroscope) | Mauna Kea Technologies | – | Requires previous knowledge in operating or the assistance of an authorized technician |
ProFlex MiniO/30 probe | Mauna Kea Technologies | – | – |
70 kD FITC-labeled Dextran | Sigma-Aldrich | 46945 | – |
ImageCell software | Mauna Kea Technologies | – | – |
Calibration Kit | Mauna Kea Technologies | – | – |
Dulbecco’s modified Eagle’s medium (DMEM) | Gibco Life Tecchnologies | 41965-039 | |
Fetal bovine serum (FBS) | Biological Industries | 04-007-1A | |
Penicillin-Streptomycin-Nystatin Solution | Biological Industries | 03-032-1B |