BackgroundChordomas are rare osseous neoplasms with a dismal prognosis when they recur. Here we identified cell surface proteins that could potentially serve as novel immunotherapeutic targets in patients with chordoma.MethodsFourteen chordoma samples from patients attending Xuanwu Hospital Capital Medical University were subjected to single-cell RNA sequencing. Target molecules were identified on chordoma cells and cancer metastasis-related signalling pathways characterised. VEGFR-targeting CAR-T cells and VEGFR CAR-T cells with an additional TGF-beta scFv were synthesised and their in vitro antitumor activities were evaluated, including in a primary chordoma organoid model.ResultsSingle-cell transcriptome sequencing identified the chordoma-specific antigen VEGFR and TGF-beta as therapeutic targets. VRGFR CAR-T cells and VEGFR/TGF-beta scFv CAR-T cells recognised antigen-positive cells and exhibited significant antitumor effects through CAR-T cell activation and cytokine secretion. Furthermore, VEGFR/TGF-beta scFv CAR-T cells showed enhanced and sustained cytotoxicity of chordoma cell lines in vitro compared with VRGFR CAR-T cells.ConclusionsThis study provides a comprehensive single-cell landscape of human chordoma and highlights its heterogeneity and the role played by TGF-beta in chordoma progression. Our findings substantiate the potential of VEGFR as a target for CAR-T cell therapies in chordoma which, together with modulated TGF-beta signalling, may augment the efficacy of CAR-T cells.