Here we report that Prostate Cancer (PCa) cell-lines DU145, PC3, LNCaP and RWPE-1 grown in 3D matrices in contrast to conventional 2D monolayers, display distinct differences in cell morphology, proliferation and expression of important biomarker proteins associated with cancer progression. Consistent with in vivo growth rates, in 3D cultures, all PCa cell-lines were found to proliferate at significantly lower rates in comparison to their 2D counterparts. Moreover, when grown in a 3D matrix, metastatic PC3 cell-lines were found to mimic more precisely protein expression patterns of metastatic tumour formation as found in vivo. In comparison to the prostate epithelial cell-line RWPE-1, metastatic PC3 cell-lines exhibited a down-regulation of E-cadherin and α6 integrin expression and an up-regulation of N-cadherin, Vimentin and β1 integrin expression and re-expressed non-transcriptionally active AR. In comparison to the non-invasive LNCaP cell-lines, PC3 cells were found to have an up-regulation of chemokine receptor CXCR4, consistent with a metastatic phenotype. In 2D cultures, there was little distinction in protein expression between metastatic, non-invasive and epithelial cells. These results suggest that 3D cultures are more representative of in vivo morphology and may serve as a more biologically relevant model in the drug discovery pipeline.