High cell density promotes NED of prostate cell lines cultured in 3D conditions independent of AR status. A, Analysis of NED marker expression in LNCaP and LAPC-4 cells seeded on Alvetex® scaffold at increasing density (0.5 × 106, 1.0 × 106, and 1.5 × 106). Western blot (upper) and qRT-PCR (lower) analysis of the NED markers γ-enolase (ENO2) and tubulin β-III (TUBB3). Activity of AR was examined by detection of expression of the androgen-regulated protein DcR2 at protein and mRNA level (TNFRSF10D) and PSA at mRNA level (KLK3) in 3D conditions. B, Western blot (upper) and qRT-PCR (lower) analysis of NED markers in the C4-2 cell line (LNCaP androgen-independent clone) in 3D conditions. Activity of AR was confirmed by detection of DcR2 at both protein and mRNA level (TNFRSF10D) and PSA at mRNA level (KLK3). C, Changes in protein (upper) and mRNA (lower) levels of NED markers after 3D cultivation of AR-negative prostate cell lines BPH-1, CAFTD03, PC3, DU-145, and PC3 cells stably transfected with AR (PC3-AR). qRT-PCR data are presented as mean ± S.D. of two independent experiments except for results for C4-2, which are from one experiment. Triangle represents increasing seeding density in 3D conditions on Alvetex (0.5 × 106, 1.0 × 106, and 1.5 × 106, respectively).