Cancer immunotherapy harnesses and amplifies the host's anti-tumor immune responses to eradicate malignant cells. Tumor neoantigens serve as ideal targets due to high specificity and immunogenicity. Circular RNA (circRNA), with a stable covalently closed structure, can be engineered as a novel vector for neoantigen expression. Here, we developed a circRNA-based vaccine encoding multiple melanoma neoantigens (circRNA). circRNAsignificantly suppressed tumor growth in both prophylactic and therapeutic murine models and induced robust anti-tumor immunity. Mechanistically, using single-cell RNA sequencing, we elucidated the antitumor mechanisms and alterations in the tumor and immune microenvironment following vaccination. circRNAimmunization nearly eliminated the Ccl6Mel subcluster-highly active in chemokine signaling, leukocyte recruitment, and antigen presentation pathways. While residual Cdk1Mel and Cdhr4Mel subclusters exhibited downregulation of immune-response-related pathways. Furthermore, circRNAvaccination reshaped myeloid cell composition, markedly increasing infiltration of monocytes/macrophages (Monocyte/MФ) with antitumor phenotypes, including classical inflammatory monocytes (Mono-Vcan), Macro-C1qc, and activated M1-like macrophages (Macro-Ifng). Chemokine signaling was enriched in Ccl6Mel and Monocyte/MФ clusters. Moreover, circRNAenhanced T cell-mediated antitumor responses, elevating T cell receptor (TCR) clonotype diversity and expansion of TCR clonotypes especially in CD4effector (CD4 Eff) and CD8cytotoxic (CD8 Cyto) T cells. Notably, Tigit was identified as the most highly expressed checkpoint receptor in CD8exhausted T cells (CD8 Tex), and combining circRNAwith anti-TIGIT/PD1 blockade produced synergistic antitumor effects. Collectively, this study systematically elucidates the therapeutic potential and immune mechanisms of the neoantigen circRNA vaccine in melanoma, providing a mechanistic framework for developing personalized RNA vaccines and rational combination immunotherapies. MNA MNA MNA + + + MNA + MNA + + + MNA
