P02.31.b Induction of a Chronic Interferon Stimulated Tumor-Associated Myeloid Cell Phenotype Is Directly Orchestrated by Aberrant Tumor-Intrinsic Egfr-Activation in Glioblastoma

Abstract BACKGROUND Glioblastoma (GB) is a lethal primary brain tumor frequently driven by amplification and aberrant activation of the epidermal growth factor receptor (EGFR). Tumor-associated myeloid cells (TAMs) comprise the majority of tumor infiltrating leukocytes (TILs) and secrete inflammatory molecules that promote tumor growth including IL-6, IL-1β, and EGF, a canonical EGFR ligand. These TAMs orchestrate a suppressive tumor immune microenvironment (TIME) with poor T cell infiltration. However, whether EGFR activation plays a direct role in mediating TAM recruitment and phenotype is poorly understood. MATERIAL AND METHODS We leveraged the MADR-mEGFRvIII murine GB model which expresses constitutively active mEGFRvIII under a tetracycline-off promoter. Orthotopic MADR-mEGFRvIII-bearing mice were treated with doxycycline (dox) for one week followed by flow cytometric TIME profiling. Tumor conditioned media (CM) and orthotopic tumor lysates were assessed via ELISA and cytokine array. Lymphocytes (CD4, CD8, NK) were depleted via antibody administration. Magnetically isolated CD45+ TILs from MADR-mEGFRvIII and newly diagnosed (nd) GB patient tumors with known EGFR status underwent bulk or single-cell RNA sequencing. RESULTS Immunophenotyping of TILs from MADR-mEGFRvIII tumors revealed EGFR activation is associated with significantly enhanced F4/80+ myeloid cell infiltration. Assessment of MADR-mEGFRvIII tumor CM identified EGFR-dependent secretion of myeloid chemoattractants CCL2 and CXCL10 in vitro, which was corroborated in tumor lysates from mice. Further, sequencing of TILs revealed EGFR activation is significantly associated with interferon (IFN)-stimulated gene (ISG) expression in both brain-resident microglia and monocytic macrophages. Lymphocyte depletion prior to tumor implantation did not affect the EGFR-driven induction of ISGs in TILs, suggesting type I IFN is responsible for the observed TAM phenotype. Pan-IFNα and IFNβ ELISAs confirmed type I IFN was not present in tumor CM, indicating IFN is secreted by other constituents in the TIME. Importantly, ndGB patient TILs were assessed to determine if EGFR activation is associated with TAM ISG expression in human GB. Indeed, significant differential expression of numerous ISGs including IRF7, IFI44, and MX1 was observed in TAMs from EGFR activated tumors compared to wild type further supporting the concept that EGFR orchestrates the induction of a chronic TAM IFN response in GB. CONCLUSION Our results indicate tumor-intrinsic EGFR activation promotes TAM recruitment via CCL2 and CXCL10 secretion and potentiates robust induction of type I IFN signaling within the TIME, ultimately resulting in a TAM phenotype characterized by ISG expression. Therapeutically targeting this EGFR-driven tumor-TAM crosstalk may be a viable strategy to reduce immunosuppression arising from chronic IFN signaling.