Characterization of the tumor microenvironment after combined radio- and immunotherapy in a murine glioblastoma model

Recent therapeutic approaches for glioblastoma (GBM) focus on GBM cells eradication by local radiotherapy (RT) and targeting the tumor microenvironment (TME), e.g. by excluding myeloid-derived suppressor cells (MDSCs) by blocking the CXCR4/CXCL12-signal axis. The effect of RT combined with antagonization of CXCL12 (SDF-1) by a novel Spiegelmer immune modulator on survival, cellular TME and cytokine production was investigated here the first time in an immune-competent murine model as the main objective of the study. Further, the survival effects of a second immune modulator neutralizing CCL2 were evaluated. Immune-competent 7-12 week-old female wild-type C57BL/6J mice were intracerebrally implanted with 1.5x105 murine GFP-expressing glioma cells (GL261) and survival was monitored up to 100 days. Three different immune modulators (anti-PD-1 antibody (6x 250 µg ip.), CXCL12 and CCL2 antagonists (both 20 µg/g body weight s.c. every 2nd day)) were tested in various combinations (13 groups) with or without half-brain irradiation (1 x 12 Gy). To monitor tumor growth, animals (n = 8-15) were observed weekly via MRI (1 Tesla, T1-weighted) and after a maximum of 100 days, brains were examined for complete tumor regression (MRI, fluorescence signal, immunohistochemistry). To investigate the effects of the combination of CXCL12 antagonist and RT on the immune response, tumor tissue, spleen and blood from animals randomized into four groups (untreated control, NOX A12, RT, RT with NOX-A12) were analyzed by flow cytometry 20 days after tumor inoculation. Besides, the concentrations of 31 cytokines and chemokines in tumor and plasma were measured by bead-based multiplex immune assay and receptor profiles after RT were determined for the CD8+ effector cells, CD4+FOXP3+ Tregs and CD11b+Gr1+ MDSCs. Survival data were statistically analyzed by the Kaplan-Meier method, other data using a one-way ANOVA test or an unpaired t-test with a significance limit of 5% (p < 0.05). As monotherapy, both Spiegelmers had no positive effect ...