Abstract 5225: Engineered signaling reporter immune cell lines for monitoring immune activation and multidirectional interactions among cancer cells, immune cells, and tumor microenvironmental cells

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While immunotherapies targeting T cells have achieved remarkable success, a significant subset of patients remain unresponsive or experience relapse, often attributed to the immunosuppressive nature of the tumor microenvironment. Emerging evidence suggests that other immune cell types, such as B cells and myeloid cells, play crucial roles in modulating the efficacy of cancer immunotherapies. However, there remains a clear lack of widely accessible immunological models capable of accurately representing the intricate multidirectional interactions among cancer cells, adaptive and innate immune cells, and other tumor microenvironmental cells. To address this gap, we engineered signaling reporter cell lines of T cell, B cell, or myeloid origin to express luciferase reporter protein in response to the activation of nuclear factor of activated T cells (NFAT) or nuclear factor kappa B (NF-κB) signaling pathway. The cell lines also endogenously express high level of T cell checkpoints (PD-1, TIGIT, and/or GITR) or myeloid checkpoints (SIRPα, Siglec-10, LILRB1, and/or B7-1) for additional application in immune checkpoint research. To validate luciferase expression, NFAT luciferase reporter T cell lines were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, while NF-κB luciferase reporter myeloid cell lines were activated with tumor necrosis factor alpha (TNF-α) or conditioned media from T cells. A B cell-derived NF-κB reporter cell line (BDCM-NFκB-Luc2) with high basal luciferase activity was treated with an NF-κB signaling inhibitor. These reporter cells were subsequently evaluated in various co-culture conditions with primary immune cells, cancer cells, and/or other tumor microenvironmental cells to study immune cell crosstalk. Luciferase assays were performed for convenient quantification of luminescence intensity under all conditions. The results demonstrated that stimulations leading to NFAT or NF-κB signaling activation yielded dose-dependent increase in luciferase expression. In contrast, inhibition of the signaling led to dose-dependent decrease in luciferase expression. Furthermore, co-culture of reporter cell lines with various other immune and cancer cells significantly enhanced luminescence signals, with increases of up to 200-fold. In summary, these newly developed luciferase reporter immune cell lines provide a robust ex vivo model for evaluating cancer immunotherapies. These cell lines enable sensitive and reproducible monitoring of complex interactions among cancer cells, innate and adaptive immune cells, and other tumor microenvironmental cells, offering a valuable standard for the assessment of combinatorial immune responses in the tumor microenvironment. Citation Format: Hyeyoun Chang, Alicia Walker, John Foulke, Luping Chen, Fang Tian, Zhizhan Gu. Engineered signaling reporter immune cell lines for monitoring immune activation and multidirectional interactions among cancer cells, immune cells, and tumor microenvironmental cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5225.

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