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Cancer cells in vivo are coordinately influenced by an interactive 3D microenvironment. However, identification of drug targets and initial target validations are usually performed in 2D cell culture systems. The opportunity to design 3D co-culture models that reflect, at least in part, these heterotypic interactions, when coupled with RNA interference, would enable investigations on the phenotypic impact of gene function in a model that more closely resembles tumor growth in vivo. Here we describe a high-throughput-compatible method to discover cancer gene functions in a co-culture 3D tumor microtissue model system composed of human DLD1 colon cancer cells together with murine fibroblasts. Strikingly, DLD1 cells in this model failed to expand upon siRNA-mediated depletion of Kif11/Eg5, a member of the mitotic kinesin-like motor protein family. In contrast, these cancer cells proved to be more resistant to Kif11/Eg5 depletion when grown as a 2D monolayer. These results suggest that growth of certain cancer cells in 3D versus 2D can unveil differential dependencies on specific genes for their survival. Moreover, they denote that the high-throughput-compatible, hanging drop technology-based 3D co-culture model will enable the discovery, characterization, and validation of gene functions in key biological and pathological processes.

A High-Throughput–Compatible 3D Microtissue Co-Culture System for Phenotypic RNAi Screening Applications