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Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. Intracranially injected GL261 cells are widely used as an immunocompetent animal model of glioma, but it is common practice to transfect these with luciferase to facilitate tumor monitoring during treatment. Our group has previously shown that the luciferase-expressing GL261 Red-FLuc cells create an inflammatory response when implanted intracranially. Now, we additionally explore the inflammatory response of GL261-Luc2 cells and demonstrate a similar host immune response occurs with this model as well. In our in vivo evaluation, C57BL/6 mice underwent stereotaxic, intracranial implantation with GL261, GL261 Red-FLuc or GL261-Luc2 cells at doses of 5x104cells/5mL or 3x105cells/5uL.MRIs were performed to monitor relative tumor growth. To assess intrinsic differences between cell lines, in vitro cytokine profiles were evaluated by proteome microarray. Kaplan-Meier survival analyses demonstrated median survival for mice implanted with GL261 cells at 5x104cells was 18 to 21 days. The GL261-Red FLuc implanted mice cells did not reach median survival at either tumor dose with greater than 60% of mice termed long-term survivors. Finally, mice injected with GL261-Luc2 cells at 3x105cells reached median survival at 23 days, but median survival was significantly prolonged for mice implanted with GL261-Luc2 at a dose of 5x104cells (37 days, with 40% becoming long-term survivors) compared to GL261 implanted mice. MRIs reveal differences in tumor growth that correspond with the differences in median survival between groups. In addition, proteomic analyses revealed significantly elevated inflammatory cytokines such as IFN-gamma, IL-7 and TNF-alpha in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Further immune characterization is ongoing. Our data suggests that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators which stimulate the tumoricidal function of immune cells in the tumor microenvironment.

Immunocompetent murine models of glioma, such as GL261, are essential to assess immune therapy efficacy. The GL261 cell line expressing firefly luciferase (GL261RFluc), has been used to enable non-invasive tumor monitoring in preclinical studies. However, it is unclear if the GL261 and GL261RFluc cells are equivalent, particularly when applied to tumor immunology. C57BL/6 mice (n=20 per group, repeated once) underwent stereotaxic, intracranial implantation with GL261 or GL261RFLuc cells at 5x10^4cells/5uL and were assessed for survival. GL261 and GL261RFluc cell lines were assessed by CFSE proliferation assay. TGF-Beta2 ELISA and proteome profiler immunoassay were performed on equivalent cell culture lysates for cytokine analysis. Mice were implanted with GL261 or GL261RFluc cell lines, sacrificed at day 10, and brains were either disassociated, FACS stained, and sorted for immune cell surface antigens (n=4 each group) or formalin-fixed and paraffin embedded for immunohistochemistry (IHC, n=6 each group). 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 GL261RFluc cells (P=0.001). Quantitative IHC analyses of brains implanted with GL261RFluc cells showed significant increases in CD4 and CD8 cells but decrease in FoxP3 positive cells. Proliferation assays were equivalent. TGF-Beta2 ELISA showed significantly elevated levels in the GL261RFLuc cells. Proteomic profiler results demonstrated differential cytokine expression greater than 2-fold for over 25% of cytokines evaluated. The detected increases in chemoattractants CCL2 and CCL5 were particularly pronounced in GL261RFLuc cells. FACS analysis showed a trend of increased PD-L1 positive cells in brains implanted with GL261 cells, but this did not reach significance. GL261RFluc cells create an inflammatory immune microenvironment when implanted intracranially in C57BL/6 mice compared to GL261 cells. These findings suggest that investigators should avoid GL261RFluc cells when evaluating immune therapeutics in a C57BL/6 background.