Abstract 2455: Development of a bioluminescence reporter mouse model for tracking and quantifying Ly6G+ neutrophils in vivo
Authors: Chalishazar Milind D. , Solban Nicolas , Kopinja Johnny , Linn Doug , Cristescu Razvan , Zhou Heather , Rosahl Thomas , Long Brian , Zhang Weisheng , Hostetler Eric 
Published: June 2022

Immunotherapy has revolutionized the way cancer is treated; however, despite tremendous success, not all patients benefit from immunotherapy. Identifying biomarkers predictive of effective treatment response to current and novel immunotherapies is of utmost importance. Multiple studies have indicated neutrophil-lymphocyte ratio (NLR) may be a predictive biomarker of immunotherapy response. We analyzed hematological parameters across multiple preclinical murine tumors to determine if NLR predicts tumor growth and response to treatment. The hematological survey identified NLR to be strongly correlated to tumor weights, with larger tumors exhibiting higher NLRs. In addition, mouse tumors that have been characterized as resistant or partially responsive to immunotherapy had a higher NLR than tumors responsive to immunotherapy. To track neutrophils in vivo, we generated a BAC transgenic mouse wherein firefly luciferase (Luc2), diphtheria toxin receptor (DTR), and enhanced green fluorescence protein (eGFP) genes were expressed under the endogenous Ly6G promoter. The Ly6G promoter enables tracking of Ly6G+ neutrophils using luciferase-based bioluminescence imaging (BLI), depletion of Ly6G+ neutrophils by diphtheria toxin treatment, and the ability to perform flow analyses using the eGFP marker. In vivo and ex vivo BLI demonstrated that the Ly6G+ cells were primarily expressed in bone marrow, blood, spleen, and the digestive tract. To ensure the specificity of the bioluminescence signal, we isolated Ly6G+ and Ly6G- immune cells and performed in vitro BLI. In vitro BLI confirmed that bioluminescence was exclusive to Ly6G+ cells. To confirm that the Luc2-DTR-eGFP (LDG) reporter cassette expression did not affect neutrophil activity and function, we quantified the level of myeloperoxidase (MPO), a marker of neutrophil activation, in the phorbol myristate acetate (PMA)-induced ear edema mouse model. Neutrophil activity was measured using luminol enabled BLI of MPO. Luminol-BLI following PMA-induced ear edema demonstrated similar MPO activity between wildtype and transgenic mice. Finally, we investigated if the LDG reporter cassette impacted tumor growth. Measures of tumor burden in this transgenic line recapitulated our hematological findings. Tumors with higher NLR showed higher tumor BLI signal when compared to tumors with lower NLR ratio, demonstrating that the LDG reporter cassette did not impact tumor growth in the transgenic mice and importantly, providing a tool to track Ly6G+ cells in tumors. Our results demonstrate that NLR in tumor correlates with response to immunotherapy across multiple tumor types. Here we describe the development of a transgenic mouse to track intratumoral Ly6G+ cells. These transgenic mice provide a valuable tool for profiling oncology compounds, including those targeting immune cells, and for understanding the mechanism of action of such agents.

Citation Format: Milind D. Chalishazar, Nicolas Solban, Johnny Kopinja, Doug Linn, Razvan Cristescu, Heather Zhou, Thomas Rosahl, Brian Long, Weisheng Zhang, Eric Hostetler. Development of a bioluminescence reporter mouse model for tracking and quantifying Ly6G+ neutrophils in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2455.