Characterization of Immune Cell Heterogeneity, Key B Cell Subtypes, and Macrophages Functions Using Multiplex Immunofluorescence and Single-cell RNA Sequencing in Chordomas

INTRODUCTION: Chordomas are rare tumors that arise from remnants of the notochord. The standard of care for chordoma remains only surgery and radiotherapy. Though checkpoint inhibition therapy shows promise, recent immunotherapy trials failed to reach endpoints. This emphasizes the need to comprehensively understand the tumor and peripheral immune landscape of chordomas to establish a rationale for immunotherapeutic intervention. METHODS: We analyzed and quantified the immune cell composition (CD3 + , CD4 + , CD8 + , CD20 + , and CD163 + cells) of 33 chordomas (14 clival, 3 cervical, 2 lumbar, and 14 sacral tumors) through multiplex immunofluorescence. In addition, we investigated the phenotype of tumor-infiltrating and peripheral B cells and myeloid cells from five chordoma patients employing single-cell RNA sequencing. RESULTS: Hierarchical clustering analysis of 33 chordomas identified 3 distinct clusters based on the relative abundance of B cells, T cells, and macrophages. Cluster 1 was characterized by moderate immune cell infiltration, cluster 2 was immune-desertic, and cluster 3 was highly infiltrated by B and T cells. Findings from single-cell transcriptomics revealed that regulatory B cells and plasma cells were the most abundant B cell populations infiltrating tumors. In addition, we found the presence of tumor-infiltrating macrophages with angiogenic and complement-related functions. CONCLUSIONS: Our study provides crucial insights into the immune landscape of chordomas. The identification of distinct clusters based on immune cell infiltration emphasized the heterogeneity of chordomas. The findings from this study may serve as a foundation for future research and the development of targeted immunotherapies for chordomas.