Luciferase-expressing Monocyte Reporter Cell Lines as a Predictive Human Cell-Based Model for In Vitro Immune Activation Studies
AACR Annual Meeting 2020
Virtual EventJune 22, 2020
Cancer immunotherapy has emerged as an exciting new approach for cancer treatment, and immuno-oncology is one of the fastest growing fields in oncology. The development of immunomodulatory drugs and biologics dictate a clear need for human cell-based models to evaluate immune activation. THP-1 cell line is one of the best surrogate models for in vitro human monocytes. Additionally, luciferase reporters provide a relatively simple, robust, and highly sensitive means to measure biological processes through in vitro bioluminescence measurements. Here we report the generation of a panel of THP-1 luciferase reporter cell lines that have been transduced with a Luc2P plasmid containing the response element (RE) of various transcription factors, which include NF-κB, GAS, NFAT, ISRE, AP-1, and CRE. After introduction of the pLenti-RE-LUC2P plasmid into the parental THP-1, single cell cloning was performed to isolate stable clones with the best signal-to-noise ratio of luciferase signals upon stimulation. THP-1 NF-kB reporter cells showed greater than 30-fold increase in bioluminescence signals while stimulated by TNFα or LPS. THP-1 GAS reporter cells responded with high sensitivity to IFN-γ which allowed signal fold change to be greater than 100 folds. We also found that IFN-α can stimulate GAS through cross-talk between STAT1 and STAT2 signaling which showed a greater than 10 folds change in bioluminescence signal. In addition, these cell lines were characterized and authenticated using cell morphology, growth kinetics, and STR analysis. The growth of these clones was comparable to that of parental THP-1, and the STR analysis showed that the derived luciferase reporter cell clones were identical to the parental THP-1 cell line STR profile. The selected transcription factors play critical roles in regulating immune reactions, antiviral responses, and inflammation. In addition to allowing the study of specific signaling pathway activity, these THP-1 reporter cell lines can be used to examine various immune response conditions and monocyte activation during immuno-oncology drug discovery. For example, THP-1 NF-κB reporter cells were not only highly sensitive to the stimulation of toll-like receptors and pro-inflammatory cytokine such as TNF-α in a dose-dependent manner, but also responded to the simulation of common damage-associated molecular pattern (DAMP) such as HMGB1, which is released during cancer cell death. Meanwhile, the activation of THP-1 GAS reporter cells were examined not only by cytokine IFN-γ simulation alone, but also were used in co-culture system with activated T cells to evaluate IFN-γ release during various immune-oncology drug treatments. In summary, luciferase-expressing monocytes are valuable tools for studying signal transduction pathway activation, screening of compounds to find activators of signal transduction pathways, and efficacy evaluation of inflammatory effect of new drugs and chemicals.