Matrigel

In total, 24 Transwell chambers (8 μm in pore size) were pre-coated with 20 µL Matrigel (Clontech, Mountain View, CA, USA) and incubated for 20 min (37°C, 5% CO₂), followed by a further coating step with 20 µL Matrigel for 1 h. For VMCUB-1 and UMUC-3, 3 × 10⁴ cells in 100 μL Opti-MEM® Reduced-Serum medium were added to the upper chamber. For BFTC905, 5 × 10⁴ cells were seeded, and 500 μL of conditioned media was added into the lower chamber. Then, 20 h (VMCUB-1 and UMUC-3) to 24 h (BFTC905) later, non-adherent cells were cautiously removed using a cotton swab. BFTC905 were additionally preincubated with CM media (PCM) for 6 days before the experimental setup. The cells in the chambers were washed with PBS (4°C) and fixed for 10 min with ice-cold methanol and then stained with crystal violet for 25 min. After a further washing step and 20 min of drying time, the membrane was removed with a scalpel and embedded in xylol. The membrane was positioned on a microscope slide and coated with DePeX (Serva, Heidelberg, Germany). Three representative images of each membrane were taken with a Zeiss Axiovert 200. The area covered with invaded cells was analyzed using ImageJ, and an average value was calculated. For each cell line, nine differently conditioned media were analyzed, with each of them representing a pool of six donors.

Matrigel (10 mg protein/ml, Clontech, MA, USA; 40 μl) was pipetted into a 96-well culture plate and polymerized for 1 h at 37 °C. HUVECs were harvested after trypsin-EDTA treatment, re-suspended in M199 and then plated onto a layer of Matrigel at a density of 2 × 10⁴ cells/well, followed by the addition of 10uM SPC in the absence or presence of 2.5uM KRO-105714. After the Matrigel cultures were incubated at 37 °C for 24 h, the cultures were photographed (X40).