AAV-mediated Siglecf elimination on TAMs reverses TMZ therapy-induced T cell dysfunction in glioblastoma

Glioblastoma (GBM) is the most aggressive primary tumor of the brain, characterized by extensive intratumor heterogeneity, and exhibits inevitable recurrence despite conventional therapy. This study investigates the immunosuppressive mechanisms underlying GBM recurrence following temozolomide (TMZ) therapy, with a specific focus on macrophage-associated pathways driving immune evasion. By integrating single-cell RNA sequencing and mass cytometry time-of-flight analyses in orthotopic mouse GBM models, we revealed extensive transcriptional and phenotypic remodeling of the tumor microenvironment after TMZ treatment, including reduced malignant cell proliferation, activation of cytokine-mediated signaling, pronounced T-cell exhaustion, and a shift of tumor-associated macrophages (TAMs) toward immunosuppressive sialic acid-binding Ig-like lectin F (Siglec-F, encoded by Siglecf) positive subsets. Functional validation demonstrated that expression of Siglec-F inhibited macrophage phagocytosis and enhanced immunosuppression, whereas Siglecf-targeted intervention using adeno-associated virus (AAV) restored T-cell activation and improved antitumor efficacy. Analyses of human GBM datasets confirmed the enrichment of SIGLEC5⁺ (human homolog of murine Siglecf) TAMs in recurrent tumors, correlating with adverse prognosis. Collectively, our findings suggest a TMZ-induced immunosuppressive reprogramming of GBM microenvironment and implicate Siglec-F/SIGLEC5⁺ TAMs as potential mediators of immune escape and promising therapeutic targets to overcome chemoresistance.