title: RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.
abstract: The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3D) models may be more sensitive to certain gene-silencing events than 2D models because of their components of hypoxia, nutrient gradients, and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions. Colorectal cancer (CRC) and breast cancer (BC) models composed of epithelial cells only, deemed single-cell-type tumor spheroids (SCTS) and multi-cell-type tumor spheroids (MCTS), containing fibroblasts were developed for RNAi HTS in 384-well microplates with flat-bottom wells for 2D screening and round-bottom, ultra-low-attachment wells for 3D screening. We describe the development of a high-throughput assay platform that can assess physiologically relevant phenotypic differences between screening 2D versus 3D SCTS, 3D SCTS, and MCTS in the context of different cancer subtypes. This assay platform represents a paradigm shift in how we approach drug discovery that can reduce the attrition rate of drugs that enter the clinic.
authors.affiliation: 1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Fu
authors.affiliation: 1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Fernandez
authors.affiliation: 1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.affiliation: 1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Titus
authors.affiliation: 1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
authors.forename: Eugen
authors.initials: E
authors.lastname: Buehler
authors.affiliation: 1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
authors.forename: Madhu A
authors.initials: MA
authors.lastname: Lal-Nag
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy; D055808:Drug Discovery / Q000379:methods; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D034622:RNA Interference / Q000187:drug effects; D018874:Spheroids, Cellular / Q000187:drug effects*; D059016:Tumor Microenvironment / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents
keywords: RNAi; high-throughput screen; spheroid; three-dimensional culture; tumor microenvironment