Tracing T Cell Activation Cytokines In Reporter Models By Bioluminescent Imaging.

Abstract Introduction: Bioluminescent imaging (BLI) is a valuable tool to visualize cell behaviors spatiotemporally in animal models. Many classical tumor cell lines have been engineered to express luciferase, to allow for accurate and quantitative tracking of orthotopic tumor growth over time. In the era of cancer immunotherapy, contrary to cancer therapies that directly target malignant cells, immuno-oncology (I/O) therapies stimulate the body's immune system to target and attack the tumor, highlighting the need to label vital immune cell lineages or cytokine-producing cells to understand the dynamic changes of immune components in the tumor microenvironment (TME) and interrogate mechanisms of action of I/O treatment. Many transgenic reporter mice have been generated and widely used in mechanistic evaluations of different compounds. Here we report the development and characterization of two cytokine reporter models: IL2-IRES-Venus-Luc and IFNγ-IRES-Venus-Luc. Methods: IL2-IRES-Venus-Luc and IFNγ-IRES-Venus-Luc cytokine reporter mice in the C57BL/6 background were developed using CRISPR/Cas9 technology by micro-injection of reporter gene DNA and Cas9/gRNA complex into the zygotes. The expression of luciferase was restricted by the cell type- or protein-specific promoter. The IFN-γ and IL-2 reporter mice were subcutaneously challenged with MC38-OVA and Hepa 1-6 syngeneic tumor cell lines, respectively. When the mean tumor volume reached ~90mm3, the mice were randomized and administered with 10 mg/kg anti-mouse PD-1 antibody (CrownVivo™) or PBS. Tumor burden was monitored twice weekly, while IFNγ- and IL-2 signal was traced by in-life BLI once/twice weekly. Ex vivo imaging was conducted for the Hepa 1-6 model to quantify the IL-2 signal in the tumor, spleen and tumor draining lymph nodes (TDLN). Several immune cell reporter lines are undergoing validation and the results will be reported at the meeting. Results: Tumor growth kinetics and the response to anti-PD-1 treatment of Hepa 1-6 and MC38-OVA models in reporter mice was consistent with that of wild type (WT) C57BL/6 mice. For the MC38-OVA model engrafted in IFNγ-IRES-Venus-Luc reporter mice, complete regression was achieved for all mice treated with anti-PD-1. The IFNγ signal in MC38 tumors treated with anti-PD-1 increased significantly 48 hours after the first dose, indicating an active and functional immune response in the TME. For Hepa 1-6 model engrafted in IL2-IRES-Venus-Luc reporter mice, anti-PD-1 produced significant antitumor activity (TGI=61%), which was mechanistically supported by a higher IL-2 signal in anti-PD-1 treated tumors and TDLN compared to control animals. Conclusions: We have characterized two important T cell activation reporter lines, which may serve as useful tools for longitudinal tracking of molecular events and monitoring immune cell function in the preclinical evaluation of immunotherapies. Citation Format: Ying Jin, Kaixia Lian, Fengge Li, Ruilin Sun, Annie X. An, Henry Q.x. Li, Davy Xuesong Ouyang. Tracing T cell activation cytokines in reporter models by bioluminescent imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1808.