Figure 5. Functional analysis of altered gene expression data in 3D culture, compared to 2D monolayer.

A) Principal component analysis (PCA), illustrating gene sets that distinguish round, mass-, and stellate morphologies. Round phenotype cells included were PrEC, EP156T, and RWPE1. The mass phenotype is represented by LNCaP and 22rV1 cells. Stellate/invasive cells comprise ALVA31, PC3, PC3M, and RWPE-2/w99. B) Ingenuity Pathway Analysis (IPA) of invasion-related clusters 9–12 (Figure 4), illustrating network 2 out of 13 generated. This network is centered on the AKT and PI3-Kinase pathways. Colors correlate to factors of differential expression observed between round and invasive spheroids (red: induced, green: repressed). C) Heatmap, illustrating altered mRNA expression of laminins in 2D and 3D culture. Expression of laminins α5, γ1, β2 and α3 is strongly induced in the invasive transformation of PC-3 and PC-3M cells (days 13+15). D) Continuous expression of various laminins after onset of invasion, as demonstrated by staining with a human-specific antibody (LAM-89) that detects various alpha and beta subunits.