Ab100606

Human MMP2 (ab100606, Abcam, Cambridge, UK) and MMP9 (ab100610, Abcam, Cambridge, UK) ELISA kits were used to measure the level of MMP2 and MMP9. The procedure was followed the manufacturer’s instructions. To summarize, 100 μL of the standard sample, which was the benchmark, and 100 μL of the specific experimental sample to be measured in this experiment were added to the well plates and cultured for 150 min at room temperature. Then, the solution was removed and the plates were washed with distilled water and PBS-Triton. Then, 100 μL of biotinylated (MMP2, MMP9) detection antibody was added to the well plates and mixed carefully for 1 h at room temperature. The solution was removed and the well plates were washed again with distilled water and PBS-Triton. Then, 100 μL of HRP–streptavidin solution was added to each well plate and carefully mixed for 45 min at room temperature. The well plates were washed with distilled water and PBS-Triton again, and 100 μL of TMB one-step substrate reagent was added to each well plate and carefully mixed for 30 min at room temperature without light exposure. Finally, 50 μL of the stop solution was added to each well plate and the absorbance at 450 nm was measured using a microreader. The amount of MMP in the cancer microtissue sample was compared with that in the standard sample.

At day 8 of EVT differentiation, the media supernatants which had been in culture for 2 days were collected, and stored at −80°C. As controls, 0.75 × 10⁵ hTSCs were seeded per well in 2 mL TS medium. After 2 days of culture, the media supernatants were collected and stored at −80°C. The amount of secreted MMP2 was measured using a commercial MMP2 ELISA kit (Abcam, ab100606). Each experiment was performed with two biological replicates, and each biological replicate contains two technical replicates.