GLIOMA-261 LUCIFERASE-EXPRESSING CELL LINE STIMULATES AN IMMUNOGENIC RESPONSE SIGNATURE IN AN IMMUNOCOMPETENT MURINE MODEL

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.