3D Co-culture of Prostate Cell Lines

Human prostate cancer epithelial cell lines, LNCaP, C4-2, PC3, PC3M, and DU145, and a human bone stromal cell line HS27A (ATCC, Manassas, VA) that were used in our previous studies^{61 (link)–63 (link)} were maintained in T-medium (Invitrogen, Carlsbad, CA) with 5% fetal bovine serum (FBS; Invitrogen). Then, the 3D coculture of HS27A cells mixed with equal numbers of LNCaP or C4-2 cells or a monoculture was performed in an RCCS system (Synthecon, Houston, TX) following an established protocol^{21 (link)}. Each condition was performed in duplicate vessels for three independent experiments. Previously established CAFs and BAFs, derived from cancerous and benign/normal regions of the prostate gland, respectively^{21 (link)}, were confirmed as fibroblasts by positive vimentin but negative cytokeratin expression (Supplemental Fig. s6) and were maintained in PrEGM™ Prostate Epithelial Cell Growth Medium (Lonza, Walkersville, MD). All cells were cultured in a 37 °C incubator with 5% CO₂.