IMMU-65. Single-cell analysis of the immune microenvironment of cerebrospinal fluid in leptomeningeal metastasis

Abstract Leptomeningeal metastasis (LM) occurs when tumor cells spread into the cerebrospinal fluid (CSF)-filled leptomeninges. 5-10% of solid tumor patients develop LM. Survival is generally less than a year, and therapeutic options are limited. Several aspects of the LM immune microenvironment remain unexplored, and their understanding will be key in the development of novel immunotherapies targeting LM. We investigated the immune microenvironment and characterized T cell responses in the CSF of patients with confirmed LM (n = 16) and tumor patients without LM (n = 5). The LM cohort comprised patients with diverse primary tumors, including carcinomas, hematological, and glial tumors. By combined single-cell RNA and T cell receptor (TCR) sequencing, we generated a transcriptomic immune landscape from LM CSF samples. We identified different myeloid and lymphoid immune cell populations, including various subtypes of CD4 and CD8 T cells. The majority of CD8 T cells displayed an effector cytotoxic phenotype characterized by the expression of genes such as GZMA, NKG7 and PRF1. Analysis of the distribution of the most clonally expanded TCRs revealed an enrichment in effector cytotoxic CD8 T cells, indicating a CD8-mediated anti-tumor response. Furthermore, differential gene expression analysis of effector cytotoxic CD8 T cells and effector CD4 T cells in LM vs. control samples revealed upregulation of genes related to interferon response (ISG15, MX1, IFI6, STAT1) and antigen presentation (TAP1, HLA-C, CD74). Overall, this data demonstrates that T cells in the CSF exhibit cytotoxic activity, supporting the rationale for exploring immunotherapeutic strategies to treat LM.