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

Summary

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.). 

Abstract

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.

Introduction

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 fac....

Protocol

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

1. Seed 2 x 10⁶ U-87 MG cells in a 10 cm culture plate in a volume of 8 ml Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% pencillin/streptomycin/nystatin solution.
2. Incubate cells at 37 °C; 5% CO₂ until achieving full confluency at 48 hr.
3. Using a 10 ml syringe, withdraw all medium from the plate....

Results

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.......

Discussion

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 th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rotary Vacuum Evaporator	-	-	-
Growth-factors reduced phenol-free Matrigel	BD Bioscience	FAL356231	Thaw overnight, at 4 °C 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 sec 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