CHARACTERIZATION OF AN INDUCIBLE p65 KNOCKOUT MOUSE MODEL FOR STUDYING GLIOBLASTOMA

Abstract

Glioblastoma (GBM) is a malignant primary brain tumor that results in patient death within two years following diagnosis. The GBM tumor microenvironment has an important impact on the formation, progression, and drug resistance of this lethal disease. The GBM tumor microenvironment is composed of a variety of cell types that can support tumor growth like microglia. Microglia are immune cells that can switch from a classical, tumoricidal M1 phenotype to an alternative, tumor-promoting M2 phenotype. However, there are limited studies about the specific effect(s) that microglia have on GBM tumors. Given the importance of the tumor microenvironment to GBM tumor progression, targeting key signaling pathways in tumor-associated cells like microglia could be an effective way to reduce tumor progression. One signaling pathway involved in causing different cancers is the nuclear factor-kappa B (NF-κB) pathway, and it has been implicated in M1 to M2 phenotype polarization. Therefore, this study focuses on the characterization of the p65fl/fl/CX3CR1creER/+ mouse model, which is an inducible p65 knockout mouse model for studying how inhibition of canonical NF-κB signaling in microglia affects GBM tumors. After tamoxifen is administered to the mouse, p65, a transcription factor of the canonical NF-κB pathway, should be deleted in microglia due to the activation of a Cre recombinase (CreER) under the control of the CX3CR1 promoter. Characterization of this mouse model is necessary to determine if the p65 gene is effectively deleted in microglia from these mice. Liquid chromatography-mass spectrometry analysis has shown that the orally administered tamoxifen was present and quantifiable in the brains of tamoxifen-treated mice to induce recombination. Based on data from PCR and western blots, tamoxifen given to the p65 knockout mice induced partial deletion of the p65 gene in isolated microglia when compared to control groups. Preliminary flow cytometry data suggested that p65 deletion in microglia occurred in p65fl/fl/CX3CR1creER/+ mice that received GBM implantations and tamoxifen treatment compared to a vehicle control group. Overall, the data from this study suggests that the p65 gene is partially deleted in microglia from the tamoxifen-treated p65 knockout mice compared to the vehicle control mice.