Incorporating Metabolic Competence into High-Throughput Profiling Assays

One limitation of previous high-throughput transcriptomics (HTTr) chemical screening studies using TempO-Seq is that most of the in vitro models used on those studies lack instrinsic capabilites to metabolize xenobiotics. This study incorporated an in vitro metabolism platform, aliginate immobilization of metabolic enzymes (AIME), into the dosing procedures for HTTr screening to address this limitation. The 384-well, lid-based platform is incubated for 2 hours on standard microplates containing chemicals, metabolic co-factors and cell culture medium to make "conditioned" medium. This medium is used to treat cells rather than treated them with parent chemicals directly, and the AIME platform does not impact downstream procedures for preparation of cell lysates that are compatible with the TempO-Seq assay. The AIME platform was used concurrently in negative or "nomet" (water + alginate) and positive or "met" (hepatic S9 + alginate) metabolic conditions. This proof-of-concept experiment screened three previously studied estrogenic reference chemicals representing three "shifts" with metabolism in the VM7Luc4E2 breast cell line in an estrogen receptor transactivation assay: bioinactivation, bioactivation or no change with metabolism. Our results indicated that the TempO-Seq assay could detect changes in bioactivity with metabolism. The established workflow can be used in future studies to improve human relevance in our chemical screening efforts. Overall design: The AIME platform was used in a "nomet" and "met" condition in VM7Luc4E2 breast cells. Cells were treated with three estrogenic reference chemicals at eight concentrations, one estrogen receptor active control (17B-estradiol) at one concentration and a vehicle control (DMSO). Each TC plate was split into twelve equal sections that were randomized uniquely, and one of these sections on each plate was analyzed using the human whole transcriptome TempO-Seq assay. One section on three independent assay plates was sequenced per AIME condition.