EMP2 是调节胶质母细胞瘤中巨噬细胞诱导吞噬作用的潜在免疫治疗靶点

Glioblastoma (GBM) is the most aggressive primary brain tumor. Despite advances in immunotherapy for other solid tumors, T-cell checkpoint blockades have failed to improve overall survival in GBM patients, largely due to low T-cell infiltration. In contrast, tumor-associated macrophages (TAMs) comprise 82-97% of the immune cell population in newly diagnosed GBM. TAMs, however, often fail to engulf tumor cells, partly due to the phagocytosis inhibitory “do not eat me" signals such as CD47, which interacts with macrophage SIRPalpha to suppress phagocytosis. Although, CD47 blockade enhances phagocytosis in preclinical models, clinical trials of anti-CD47 therapies such as magrolimab were halted due to increased mortality and limited efficacy in acute myeloid leukemia patients, highlighting the need for alternative phagocytosis-inducing therapies. To identify novel tumor-expressed genes that inhibit macrophage phagocytosis, we performed a genome-wide CRISPR screen using two rounds of coculture of radioresistant GBM tumor cells (MGG18-RR) and tumor-conditioned human peripheral blood (hPBMCs)-derived macrophages. Tumor-conditioned hPBMCs macrophages were considered more reliable, as our cytometry imaging results revealed distinct polarization states compared to unpolarized (M0) macrophages. In the first round, MGG18-RR GBM cells were co-cultured with tumor-conditioned hPBMCs-derived macrophages at a 1:5 ratio for 7 days, with media refreshed every 2 days. Parallel tumor-only cultures served as controls. Tumor cells that survived this first co-culture were pooled and subjected to a second 7-day co-culture with freshly differentiated and polarized macrophages. After the second round, the remaining tumor cells were collected, and genomic DNA was extracted for library prep and sequencing. Among the top 100 hits, several known regulators of phagocytosis, including CD47 and KRAS were enriched, validating the reliability of the screen. We identified Epithelial Membrane Protein 2 (EMP2) as a novel tumor-expressed gene that suppresses macrophage phagocytosis. EMP2 showed high expression in glioma cells in publicly available single-cell sequencing data for GBM patients. EMP2 is highly expressed in GBM and associated with tumor progression and poor survival. Functional validation using the pHrodo in-vitro phagocytosis assay demonstrated a significant increase in the engulfment of EMP2-knockout GBM cells (JX14P-RT) by both hPBMCs-derived macrophages and RAW246.7 macrophages (p < 0.05), compared to wild-type tumor cells, suggesting that EMP2 acts as an anti-phagocytic regulator in GBM. Ongoing studies aim to validate that EMP2 knockout enhances macrophage phagocytosis in syngeneic murine GBM model. Collectively, our findings highlight EMP2 as a novel regulator of macrophage-mediated tumor clearance and a promising therapeutic target for macrophage-based immunotherapy in GBM.