Abstract PS1-09-22: Single-cell RNA Sequencing Reveals Intrathecal Treatment Sensitivity and Regulatory Mechanisms in Breast Cancer Leptomeningeal Metastasis

Abstract Background: Breast cancer leptomeningeal metastasis (BCLM) is a highly aggressive and rapidly progressive form of advanced metastasis associated with an extremely poor prognosis. Although intrathecal therapy represents a critical local treatment modality, significant inter-individual variability in treatment sensitivity exists, and its underlying mechanisms remain unclear. This study aims to comprehensively characterize the tumor microenvironment (TME) in the cerebrospinal fluid (CSF) of BCLM patients using single-cell RNA sequencing (scRNA-seq), and to identify key cellular subpopulations and molecular pathways associated with intrathecal treatment sensitivity. Methods: Cerebrospinal fluid samples from eight BCLM patients were collected at the Second Affiliated Hospital of Dalian Medical University and categorized into three groups: pre-treatment, post-treatment sensitive, and post-treatment resistant. Four normal CSF samples from the GEO database were included as controls. All samples were sequenced using the 10X Genomics platform, resulting in a total of 80, 274 high-quality cells for downstream analysis after quality control. Results: Dimensionality reduction and clustering identified seven major immune cell types: T cells, monocytes/macrophages, neutrophils, dendritic cells, NK cells, plasma cells, and B cells, with significant differences in their distribution across treatment states. A substantial population of epithelial cells, including tumor cells, was also observed and further classified into seven functionally heterogeneous subpopulations. Copy number variation (CNV) analysis stratified epithelial cells into high- and low-malignancy subtypes, with the high-malignancy subtype exhibiting enhanced proliferative and migratory potential, otentially contributing to chemotherapy resistance. Some epithelial subsets were also present in normal samples, suggesting a non-malignant origin. Compared to controls, BCLM CSF showed a marked reduction in cytotoxic T cells and NK cells, along with an increase in immunosuppressive Tregs. Monocytes/macrophages were significantly enriched and displayed a bias toward M2 polarization. Downregulation of ribosomal protein genes was observed across multiple immune subsets, indicative of an immunosuppressive TME. Among immune populations, monocytes/macrophages exhibited the most distinct transcriptomic alterations across treatment groups and were divided into 12 functionally heterogeneous subclusters. Notably, the C3S100A9+ subcluster was significantly enriched in the treatment-sensitive group, with high expression of inflammatory markers, ribosomal proteins, and regulatory lncRNAs. Functional enrichment analysis revealed its involvement in inflammatory cytokine activation, antigen uptake and presentation, cell adhesion, and multiple layers of immune signaling, indicating a highly activated immune phenotype. Pseudotime trajectory analysis positioned this subcluster at the terminal differentiation state along the treatment-sensitive trajectory. Cell-cell interaction analysis demonstrated enhanced ligand-receptor interactions between this subcluster and highly malignant tumor cells, implicating roles in adhesion, metabolism, and immune activation. These findings suggest that this subpopulation may contribute to intrathecal treatment response by reshaping the immune microenvironment. Conclusions: These findings highlight the potential of C3S100A9+ monocyte-macrophage subcluster as both a predictive biomarker and a therapeutic target. Future studies will integrate transcription factor regulatory network analysis to further elucidate its underlying mechanisms, providing novel insights for precision therapy in BCLM. Citation Format: J. Gao, H. Li, S. Zhao, M. Li, L. Xu. Single-cell RNA Sequencing Reveals Intrathecal Treatment Sensitivity and Regulatory Mechanisms in Breast Cancer Leptomeningeal Metastasis abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32 (4 Suppl): Abstract nr PS1-09-22.