Abstract B070: Investigating the impact of EGFR alterations on immune cell redistribution and response to immunotherapy in MADR-mEGFRvIII murine model

Abstract Over 50% of GBM cases involve EGFR alterations, yet their impact on the tumor microenvironment (TME) remains unclear. Previous studies suggest EGFR overexpression fosters an immunosuppressive TME dominated by tumor-associated macrophages (TAM), which inhibit immune cell infiltration and activity. This immune "cold" nature of GBM contributes to poor responses to treatments, including immune checkpoint blockade (ICB), which has shown limited effectiveness compared to other malignancies. We hypothesized that EGFR alterations contribute to increased accumulation of immunosuppressive myeloid cells and reduced T cell density in the TME. Targeted EGFR ablation or inhibition using a small molecule inhibitor will reduce myeloid cell populations, promote T and B cell recruitment, and enhance T cell infiltration and activation. To investigate the impact of EGFR inhibition on the GBM tumor microenvironment (TME), we used the MADR-mEGFRvIII murine glioma model, which expresses murine EGFRvIII under a tetracycline-off promoter. The model was established via perinatal electroporation, incorporating PTEN and CDKN2A deletions, and mimics patient GBM. Tumors with active EGFRvIII were implanted intracranially, and samples were collected on days 4, 9, and 18 post-implantation for flow cytometry to profile immune cell composition. Mice with established tumors received treatment with doxycycline for genetic EGFR ablation or ERAS-801, a brain-penetrant small molecule EGFR inhibitor, and were compared to untreated control mice. Brain tissues were zinc-fixed, paraffin-embedded, sectioned, and stained with H&E to confirm tumor presence. Adjacent sections underwent multiplex immunofluorescence (mIF) to visualize and quantify immune cells. Quantitative analysis with HALO Image Analysis Software was used for immune cell densities and localization within the TME. Flow cytometric analysis of tumors, lymph nodes, and spleens collected at different time points showed a dominant infiltration of macrophages within the TME between Day 4 and Day 18 post-implantation. Early EGFR ablation/inhibition at Day 9 reduced TAMs, increased T and B cell infiltration, and improved survival by 50% compared to untreated controls. mIF of brain sections confirmed that MADR-mEGFRvIII tumor’s TME was dominated with myeloid cells, with limited T and B cell infiltration in Vehicle mice, aligning with findings in human GBM. mIF analysis showed reduced tumor cell proliferation and higher immune cell infiltration in the doxycycline and ERAS-801-treated groups compared to Vehicle mice. HALO image analysis confirmed enhanced spatial distribution and co-localization of lymphocytes, indicating improved immune cell activity in the TME following EGFR inhibition. The results demonstrate that EGFR ablation/inhibition reprograms the TME by reducing myeloid cell dominance and enhancing lymphocyte infiltration and activation. Future research will investigate combining EGFR-targeted therapy with ICB to further boost immune responses within the TME. Citation Format: Trinh L Phan, Marissa Pioso, Robert Prins. Investigating the impact of EGFR alterations on immune cell redistribution and response to immunotherapy in MADR-mEGFRvIII murine model [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B070.