Introduction: There has been renewed interest in immune therapy for glioblastoma due to the successes of these treatments in systemic tumors. Preclinical models that accurately recapitulate the immune suppressive properties of human gliomas are essential to evaluate such therapies. GL261 murine glioma cells are widely used as an in vivo model of glioma. The luciferase expressing cell line, GL261 Red-FLuc, has been widely used in the literature to enable non-invasive tumor monitoring in preclinical studies. It is unclear if these cells are equivalent, particularly when applied to investigating tumor immunology.
Methods: C57BL/6 mice (n=20 in each group) underwent stereotaxic, intracranial implantation with GL261 or GL261 Red-FLuc cells at 5x104cells/5uL and were assessed for survival. Immunohistochemistry (IHC) examination of immune cell populations in formalin-fixed paraffin embedded GL261 or GL261 Red-FLuc implanted brains from sacrificed mice was performed. Each parental cell line was assessed in vitro by CFSE proliferation assay, TGF-Beta2 ELISA, and proteome profiler immunoassay for broad cytokine analysis.
Results: Median survival for GL261 implanted mice was 20.9± 1.3 days with all animals progressing to a moribund state, while median survival was not reached for animals implanted with GL261 Red-Fluc cells P=0.001 by Kaplan-Meier analysis. Quantitative IHC analyses of GL261 Red-Fluc cells showed a significant increase in T cell infiltration. Proliferation assays revealed no difference between these cell lines. TGF-Beta2 ELISA showed significantly elevated levels in the GL261 Red-FLuc cells. Proteomic results demonstrated a greater than 2-fold increase in most cytokines analyzed in the GL261 Red-Fluc cells, including inflammatory cytokines such as IFNgamma, CXCL2, and IL-6.
Conclusions: GL261 Red-Fluc cells create a pro-inflammatory microenvironment when implanted intracranially in C57BL/6 which is quite different from the immune microenvironment created by non-transfected GL261 cells. These findings suggest that investigators who seek to evaluate immune therapeutics in a C57BL/6 background may consider utilizing unaltered GL261 cells.
Patient Care: Reliable pre-clinical animal glioblastoma models are critical for the development of therapeutic agents prior to human trials. As immune modulating drugs are investigated for potential glioma treatment, models that accurately reflect the interaction between the tumor microenvironment and host immune system become especially important.
Learning Objectives: 1. Perform survival surgery on mice implanted with either cell line to determine if overall survival differences exist.
2. Determine cytokine expression profile for each cell line.
3. Characterize immune cell populations found at the tumor microenvironment via IHC from sacrificed animal brains injected with either cell line.
References: 1. Nduom, E. K., Weller, M.,&Heimberger, A. B. (2015). Immunosuppressive mechanisms in glioblastoma. Neuro-Oncology, 17(Suppl 7), vii9–vii14. http://doi.org/10.1093/neuonc/nov151
2. Clark, A. J., Safaee, M., Oh, T., Ivan, M. E., Parimi, V., Hashizume, R., Parsa, A. T. (2014). Stable luciferase expression does not alter immunologic or in vivo growth properties of GL261 murine glioma cells. Journal of translational medicine, 12(1), 345.
3. Wei, J., Nduom, E. K., Kong, L. Y., Hashimoto, Y., Xu, S., Gabrusiewicz, K., ...&Ivan, C. (2015). MiR-138 exerts anti-glioma efficacy by targeting immune checkpoints. Neuro-oncology, 18(5), 639-648.