Basement membrane matrix

HUVEC cells were diluted with serum-free RPMI 1640 and 2×10⁴ cells/well in 100 μl were added to a 96-well culture plate precoated with basement membrane matrix (Corning). The plate was incubated at 37° C for 12 h. Tube formation was visualized under an inverted microscope. The tube structures were randomly selected from three different fields and imaged under a Leica DMI4000B microscope (Leica).

For cell migration assay, the cells were seeded in serum-free RPMI 1640 in the upper chamber. The lower chamber was filled with RPMI 1640 containing 10% FBS. After 12 h, the upper chamber was washed with PBS, scraped on the side of the filter with a cotton swab, and fixed with 4% paraformaldehyde (PFA) for 5 min. Cells adherent to the bottom were stained with hematoxylin for 10 min. The positively stained cells were imaged and examined under a Leica DMI4000B microscope (Leica).
For cell invasion assay, the cells were plated onto the upper chamber precoated with a basement membrane matrix (Corning) filled with serum-free medium, while the bottom chamber was filled with RPMI 1640 supplemented with 10% FBS. Cells were incubated for 16 h. After washing with PBS, the cells in the upper chamber were scraped on the side of the filter with a cotton swab and fixed with 4% PFA for 5 min. Cells adherent to the bottom were stained with hematoxylin for 10 min and then washed 3 times with PBS. The positively stained cells on the underside of the filters were imaged and examined under a Leica DMI4000B microscope (Leica).

The tube or vascular-like structure formation by endothelial cells was assessed on Matrigel Growth Factor-Reduced (Product #356231) Basement Membrane Matrix (Corning, Bedford, MA, USA), as previously described [55 (link)]. Briefly, the Matrigel was thawed overnight at 4 °C and 300 μL of Matrigel was added to each well of the 24-well plate. The plate was then incubated at 37 °C for 30 min, to ensure complete gelation of the matrix. GECs (30,000 cells per well) were seeded on top of the solidified Matrigel layer in 200 μL culture medium, with different stimulations, and incubated at 37 °C. Subsequently, the tube networks were observed using an inverted phase contrast microscope (Eclipse TS100, Nikon), at 40 times magnification, and recorded at 12 h and 20 h after seeding. Five randomly selected non-overlapping fields were photographed for each condition. Each experimental treatment condition was tested in triplicate. The images were analyzed using Angiogenesis Analyzer in ImageJ software and checked by manual counting. The degree of tube formation was quantified by measuring the number of junctions, the number of meshes, and total tube length.

Six-week-old female NSG (NOD.Cg-Prkdcscid ll2rgtm1Wjl/SzJ) mice were purchased from the Centrale Dienst Proefdieren (CDP) breeding facility within the University Medical Center Groningen. Mouse experiments were performed in accordance with national and institutional guidelines, and all experiments were approved by the Institutional Animal Care and Use Committee of the University of Groningen (IACUC-RuG). Ten female NSG mice were injected subcutaneously with U2932 cells in matrigel (Basement Membrane Matrix, 354234, Corning, 50/50, 1 million cells) in each flank and randomly divided into two groups of five. The treatment group received a 50 mg 60-day release tamoxifen pellet (Innovative Research of America) on day zero, which has been shown to raise levels of tamoxifen in serum to 0.07 µM [19 (link)] and was effective in a xenograft mouse model [20 (link)]. Tumor size was measured with caliper up to three times a week.

After dissolving the basement membrane matrix (Corning, Life Sciences, USA) at 4°C, it was infused into a 48-well plate at 100 μL per well and incubated at 37°C for 1 hour. Then, 200 μL of endothelial cell culture medium without FBS containing 2×10⁴ HUVECs were added to the plate. The control group was treated with medium only, and the test group was mixed with 100 μL of exosomes. The total length of tube branches and tubes was calculated using ImageJ software.

Before invasion assays, 30 μl of Basement Membrane Matrix (diluted 1:6 in PBS; Corning Life Science, Lowell, CA, USA) was applied to the bottom of a Transwell chamber (Millipore, Billerica, MA, USA) and placed in a 37 °C incubator for 5 h. The following steps were the same for the migration and invasion assays: cells (6 × 10⁴) in serum-free media were added into the upper well of the Transwell chamber, and 800 μl of DMEM containing 10% FBS was added to the lower chamber, and then, the chamber was placed in a 37 °C incubator. After18h for migration assays or 36 h for invasion assays, cells were fixed with paraformaldehyde for 30 min, and stained for 3 min with crystal violet. Excess stain was washed away with PBS, and the bottom of the upper chamber was gently wiped with a cotton swab. After drying, the chamber was imaged, and the number of invading and migrating cells was calculated.

HUVECs treated with different agents were dissociated and seeded in angiogenesis μ-slides (ibidi, Martin Reid, Germany) precoated with basement membrane matrix (10 μl/well, Corning, Middlesex county, MA, USA) at a density of 1.0 × 10⁴ cells/well. Four to six hours later, images of capillary-like tube structures were acquired by light microscopy. The number and length of intact tubes per field were analyzed using ImageJ.