GAS-Luc2 Reporter Cancer Cell Lines Demonstrating Superior Performance to the Industrial Standard Interferon-gamma ELISA in 2-D and 3-D Ex Vivo Immune Activation and Drug Screening

Interferon-gamma (IFN-γ), a cytokine critical for activating cellular immunity and facilitating anti-tumor responses, has become a key cytokine for assessing the efficacy of cancer immunotherapy drugs. While enzyme-linked immunosorbent assay (ELISA) is widely employed for the detection of IFN-γ, it has shown significant limitations in detecting low levels of IFN-γ during early-stage immune activation. Moreover, it presents serious challenges in accurately quantifying paracrine signaling of IFN-γ secreted by immune cells within 3-D co-culture models. To address the rapidly growing demand for more effective monitoring of immune activation for cancer immunotherapy research and better assessment of immunotherapy drug candidates, we developed immune activation reporter cancer cell lines engineered with a gamma-interferon activation site (GAS)-response element positioned upstream of a luciferase gene. Upon activation of the IFN-γ signaling pathway, these cells express luciferase, allowing for simple detection and quantification to measure immune activation. The cell lines were selected based on a comprehensive protein profiling to ensure endogenous expression of immune checkpoint ligands such as PD-L1, CD155, or B7-H3 for additional benefit in application in immune checkpoint research. To evaluate the system, the reporter cells were stimulated with varying concentrations of IFN-γ, treated with conditioned media from primary T cells, or co-cultured with IFN-γ-producing primary immune cells. Additionally, the reporter cells were co-cultured in 2-D or 3-D systems for the comparison with ELISA. Our data revealed that bioluminescence intensity from the reporter cells increased by approximately 100- to 250-fold in a dose-dependent manner following IFN-γ stimulation. Primary T cell–conditioned media simulated resulted in a 50- to 100-fold increase in bioluminescence intensity. Notably, in both 2-D and 3-D co-culture systems, the reporter cells generated a robust bioluminescent signal even at the IFN-γ concentrations below the detection range of conventional ELISA, highlighting their superior sensitivity and versatility. These reporter cell lines can serve as a valuable standard for early-stage monitoring of immune activation and offer a convenient and highly sensitive platform for evaluating cancer immunotherapy drug candidates.