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High‐throughput drug screening enables the discovery of new anticancer drugs. Although monolayer cell cultures are commonly used for screening, their limited complexity and translational efficiency require alternative models. Three‐dimensional cell cultures, such as multicellular tumor spheroids (MCTS), mimic tumor architecture and offer promising opportunities for drug discovery. In this study, we developed a neuroblastoma MCTS model for high‐content drug screening. We also aimed to decipher the mechanisms underlying synergistic drug combinations in this disease model. Several agents from different therapeutic categories and with different mechanisms of action were tested alone or in combination with selective inhibition of prostaglandin E2 by pharmacological inhibition of microsomal prostaglandin E synthase‐1 (mPGES‐1). After a systematic investigation of the sensitivity of individual agents and the effects of pairwise combinations, GFP‐transfected MCTS were used in a confirmatory screen to validate the hits. Finally, inhibitory effects on multidrug resistance proteins were examined. In summary, we demonstrate how MCTS‐based high‐throughput drug screening has the potential to uncover effective drug combinations and provide insights into the mechanism of synergy between an mPGES‐1 inhibitor and chemotherapeutic agents.

High‐throughput drug screening enables the discovery of new anticancer drugs. Although monolayer cell cultures are commonly used for screening, their limited complexity and translational efficiency require alternative models. Three‐dimensional cell cultures, such as multicellular tumor spheroids (MCTS), mimic tumor architecture and offer promising opportunities for drug discovery. In this study, we developed a neuroblastoma MCTS model for high‐content drug screening. We also aimed to decipher the mechanisms underlying synergistic drug combinations in this disease model. Several agents from different therapeutic categories and with different mechanisms of action were tested alone or in combination with selective inhibition of prostaglandin E2 by pharmacological inhibition of microsomal prostaglandin E synthase‐1 (mPGES‐1). After a systematic investigation of the sensitivity of individual agents and the effects of pairwise combinations, GFP‐transfected MCTS were used in a confirmatory screen to validate the hits. Finally, inhibitory effects on multidrug resistance proteins were examined. In summary, we demonstrate how MCTS‐based high‐throughput drug screening has the potential to uncover effective drug combinations and provide insights into the mechanism of synergy between an mPGES‐1 inhibitor and chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

DNA mismatch-repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5'-UTRs and 3'-UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI-H (microsatellite instability-high) tumors. The microsomal prostaglandin E synthase-1 (mPGES-1) gene product, mPGES-1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES-1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES-1 gene 3' untranslated region (3'-UTR), and analyzed its mutation frequencies in MSI-H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI-H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES-1 mRNA levels in MSI-H and MSS colorectal cancer cell lines. Three of four previously designated "MSI-H" cell lines showed higher mPGES-1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES-1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES-1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence.

DNA mismatch‐repair gene mutations, with consequent loss of functional protein expression, result in microsatellite instability (MSI). Microsatellite sequences are found in coding regions and in regulatory regions of genes (i.e., 5′‐UTRs and 3′‐UTRs). In addition to being a surrogate marker of defective mismatch repair, deletion or insertion microsatellite sequences can dysregulate gene expression in MSI‐H (microsatellite instability‐high) tumors. The microsomal prostaglandin E synthase‐1 (mPGES‐1) gene product, mPGES‐1, participates in prostaglandin E2 (PGE2) production. Moreover, mPGES‐1 is often overexpressed in human colorectal tumors, and is thought to contribute to progression of these tumors. Here we identified a dinucleotide repeat, (GT)24, in the mPGES‐1 gene 3′ untranslated region (3′‐UTR), and analyzed its mutation frequencies in MSI‐H and microsatellite stable (MSS) tumors. The (GT)24 repeat exhibited instability in all MSI‐H tumors examined (14), but not in any of the MSS tumors (13). In most cases, (GT)24 repeat instability resulted in insertion of additional GT units. We also determined mPGES‐1 mRNA levels in MSI‐H and MSS colorectal cancer cell lines. Three of four previously designated “MSI‐H” cell lines showed higher mPGES‐1 mRNA levels compared to MSS cell lines; correlations between elevated mPGES‐1 mRNA levels and microsatellite (GT)24 repeat characteristics are present for all six cell lines. Our results demonstrate that mPGES‐1 is a target gene of defective mismatch repair in human colorectal cancer, with functional consequence.