Abstract
Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.
Highlights
Several cell-based migration assays are being optimized for more high-throughput image screening[9,10,11,12,13,14,15,16,17,18,19,20], but they are not yet compatible to screen 1000s of compounds across a broad range of concentrations
We report a simple, phenotypic, fluorescent chemotaxis-dependent aggregation assay in a 1536-well plate format that utilizes the unique chemotactic properties of Dictyostelium
Chemotaxis/aggregation is quantified using Dictyostelium carrying a stable GFP reporter[31] that is only expressed in multicellular aggregates (Fig. 1A)
Summary
Several cell-based migration assays are being optimized for more high-throughput image screening[9,10,11,12,13,14,15,16,17,18,19,20], but they are not yet compatible to screen 1000s of compounds across a broad range of concentrations. We report a simple, phenotypic, fluorescent chemotaxis-dependent aggregation assay in a 1536-well plate format that utilizes the unique chemotactic properties of Dictyostelium. While the described Dictyostelium chemotaxis-dependent aggregation assay system offers unique advantage for HTS, we recognize that the readout is indirect. Compounds that affect development independently of chemotaxis, signal-response, and motility are eliminated in directed screens. >4-log) concentrations each, using the described Dictyostelium chemotaxis-dependent aggregation assay system, we identified two compounds that inhibit chemotaxis in mammalian cells. The Dictyostelium aggregation assay is a first assay suitable for HTS of chemotaxis/migration inhibitors in large libraries of small molecules, and we have described a novel technological approach to identify lead compounds in chemotaxis for mechanistic studies and new therapeutics
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