A new 3D culture system to identify disease-relevant genes in colorectal cancer. Homo sapiens

It is increasingly appreciated that properties of cultured epithelial cells differ dramatically in 2D compared to 3D, and the latter more faithfully recapitulates in vivo behavior. By studying a battery of human colorectal cancer (CRC) cell lines in type-1 collagen, we have found that HCA-7 cells form colonies with two distinctive and persistent morphological and functional properties. We observed predominantly single-layered polarized cysts (cystic colonies, CC) and a smaller fraction displaying disorganized solid masses (spiky colonies, SC) that were highly invasive in vivo. Despite overall genomic similarity, CC and SC exhibited distinct and dynamic patterns of gene expression in 3D that could largely be attributed to global changes in histone methylation. One of the most upregulated genes in CC was the prostaglandin-degrading enzyme HPGD that exhibits tumor-suppressive activity by metabolizing PGE2 to 15-keto-PGE2. HPGD protein and 15-keto-PGE2 were elevated in CC in 3D as was HPGD protein in nude mouse xenografts. The most upregulated gene in SC was versican (VCAN) and VCAN protein was markedly increased in SC conditioned medium and in SC nude mouse xenografts. Comparative ChIP analysis between CC and SC in 3D revealed extensive reprogramming of chromatin modifications across the VCAN locus in the absence of copy number changes, suggesting an epigenetic basis for overexpression of VCAN in SC cells. Analysis of a CRC tissue microarray revealed that epithelial, but not stromal, VCAN staining correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. We demonstrate the utility of this 3D system to identify disease-relevant genes in CRC.