*4.7. 3D Matrigel Assay*

In 8-well chamber slides (BD Bioscience, Shanghai, China, catalog number: 354108), 30 μL growth factor reduced BD Matrigel (BD Biosciences, catalog number: 354230) was added per well. Slides were placed in a cell culture incubator for at least 15 min to solidify the Matrigel. Next, 2000 C4-2B cells/well or 4000 LNCaP cells/well were seeded in RPMI-1640 medium containing 10% FBS with DMSO or 10 μM enzalutamide (Beyotime Biotechnology) and 2% Matrigel. Media were replenished every 3 days. Chamber slides were incubated for 2 (C4-2B) or 3 (LNCaP) weeks, and then photographed. Image J program (NIH, USA) was used to measure the diameter of each sphere. Spheres with a diameter larger than 80 μm were counted.
