Background Glioblastoma (GBM), a universally lethal primary brain tumor with a median survival of less than one-year, has failed to respond in clinical trials of multiple immunotherapies. This intractability may be due to GBM’s highly suppressive brain tumor immune microenvironment (TIME) which is dominated by myeloid cells alongside poor T-cell infiltration. The epidermal growth factor receptor (EGFR) is aberrantly activated in over half of GBM patients, and is known to directly impair cytotoxic T-cell infiltration in non-small cell lung cancer. However, the impact of tumor intrinsic aberrant EGFR activation on TIME composition and anti-tumor immunity in GBM remains poorly understood.
Methods We leveraged a novel orthotopic murine EGFRvIII-expressing glioma model wherein the constitutively active mEGFRvIII gene is under a tetracycline-off system (MADR-mEGFRvIII). Tumor cells were implanted intracranially in female C57BL/6J mice, allowed to engraft, randomized and treated with ERAS-801, a highly brain-penetrant EGFR inhibitor, doxycycline, or vehicle. Experimental endpoints include assessment of survival outcomes, and flow cytometric profiling of tumor infiltrating leukocytes (TILs) and tumor-draining lymph nodes.
Results Immunophenotyping of TILs from orthotopic MADR-mEGFRvIII tumors revealed a significant temporal increase in F4/80+ macrophages from 11.64% and 15.61% of live CD45+ cells at days four and nine post-implantation respectively (n=5) to 21.13% at day 18 (n=7). CD8+ T-cell infiltration increased 2.4-fold between days four and 18, but notably was accompanied by increased PD-1 median fluorescent intensity within the tumor but not tumor-draining lymph nodes, suggesting T-cells become exhausted within the brain TIME. Enrollment in pharmacologic EGFR inhibition via ERAS-801 at day nine post-implantation extended animal survival by 50% (Log-rank test p=0.0082) compared to vehicle controls (n=7/arm) but failed to significantly improve survival in animals enrolled at days 14 or 18 when tumors displayed increased macrophage infiltration and T-cell exhaustion. Evaluation by western blot confirmed in vivo ERAS-801 administration fully ablated activation of EGFR and downstream kinases. Immunophenotyping TILs following one week of EGFR ablation revealed a significant 4.6-fold reduction in intratumoral macrophages and enhanced T and B lymphocyte infiltration. Lastly, MADR-mEGFRvIII conditioned media polarized bone marrow-derived macrophages to an immunosuppressive M2 (CD206+ CD163+) phenotype in vitro, which was subsequently impaired by pre-treatment of the tumor culture with ERAS-801 or doxycycline.
Conclusions Collectively, our work suggests aberrant EGFR signaling supports the development of an immunosuppressive TIME driven by macrophage infiltration and T-cell exhaustion in GBM, and furthermore that EGFR inhibition has an immunostimulatory effect on the microenvironment beyond direct oncogene ablation.
Acknowledgements This work was supported by the UCLA SPORE in Brain Cancer. The authors would like to thank Ms. Amanda Anil for assistance with animal studies.
Ethics Approval All mouse experiments in this study were approved by UCLA’s institutional animal care and use committee (IACUC) D16–00124.
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