Matrigel™ Assay for Anti-Angiogenic and Vascular-Disrupting Compounds

The Matrigel™ (BD Biosciences, North Ryde, Australia) assay was used to determine the anti-angiogenic and vascular-disrupting properties of propranolol alone or in combination with chemotherapeutic agents, as previously described [38 (link)]. Briefly, 24-well plates were coated at 4°C with 270μL of a Matrigel™ solution (1:1 dilution in cell culture medium), which was then allowed to solidify for 1 h at 37°C before cell seeding. For the anti-angiogenesis assay, endothelial cells were treated with different drug solutions 20 min after seeding on Matrigel™ and photographs were taken after 8h drug incubation using the 5X objective of an Axiovert 200M fluorescent microscope coupled to an AxioCamMR3 camera driven by the AxioVision 4.7 software (Carl Zeiss, North Ryde, Australia). For the vascular-disruption assay, endothelial cells were first allowed to undergo morphogenesis and form capillary-like structures for 6 h before drug treatment was initiated. Photographs were then taken using the same microscope device after 2h drug incubation. The anti-angiogenic and vascular-disrupting activities of the compounds were then quantitatively evaluated by measuring the total surface area of capillary tubes formed in at least 10 view fields per well using AxioVision 4.7 software. Time-lapse videomicroscopy was also employed to further evaluate the effects of propranolol on the anti-angiogenic activity of chemotherapeutic agents, as previously described [38 (link)]. Cells were constantly maintained at 37°C and 5% CO₂ and photographs were taken every 5 min from the beginning of drug treatment and for 9 h.