Single-cell multi-omics and TCR sequencing to identify early tumor cavity homing and phenotypic evolution of CAR-T cells in glioblastoma.

Abstract

2554 Background: Chimeric antigen receptor T-cell (CAR-T) therapy has shown remarkable efficacy in hematological malignancies but remains largely ineffective in glioblastoma. A systematic understanding of early CAR-T cell trafficking and phenotypic adaptation within the tumor immune microenvironment may provide critical insights into this limitation. Methods: We performed single-cell multi-omics profiling using the 10x Chromium Single-Cell 5' Immune Profiling platform on infused CAR-T cells and matched tumor cavity cerebrospinal fluid (CSF) obtained via an Ommaya reservoir at day 3 post-infusion. Paired transcriptome/TCR data enabled analyses of composition, clonal tracking, and phenotypic transitions within CAR-T populations following early tumor cavity homing. Results: Single-cell profiling of the infused CAR-T product revealed a T cell–dominant population, primarily composed of CD8⁺ T cells (62.24%) and CD4⁺ T cells (34.63%). Analysis of tumor cavity CSF demonstrated a heterogeneous immune landscape, including CD4⁺ T cells (32.79%), CD8⁺ T cells (20.83%), dendritic cells, monocyte/macrophage populations, and other innate immune subsets. Gene set variation analysis across all immune cell populations showed enrichment of memory-associated programs in the CSF, while exhaustion-related signatures were not prominent, indicating a relatively non-exhausted immune state at this early time point. TCR-based clonal tracking identified 188 CAR-T cells in the CSF that originated from the infusion product, corresponding to an early homing rate of 2.19%. These homed CAR-T cells were predominantly derived from cytotoxic and proliferating CD8⁺ T cell subsets, as well as effector CD4⁺ T cells. Notably, cytotoxic CD8⁺ CAR-T cells exhibited pronounced clonal expansion in the CSF, whereas proliferating CD8⁺ CAR-T cells underwent phenotypic transitions toward cytotoxic states following tumor cavity infiltration. In parallel, activated CD4⁺ CAR-T cells preferentially adopted memory-like phenotypes. Consistently, TCR-tracked CAR-T cells in the CSF displayed significantly stronger memory-associated transcriptional signatures compared with their clonal counterparts in the infusion product (CSF vs. product, -0.187 vs -0.088，p = 2.48 × 10⁻⁹). Conclusions: This study provides a single-cell and clonal-level view of early CAR-T cell trafficking and phenotypic adaptation within the glioblastoma tumor cavity. Early tumor cavity infiltration is a selective process driven by cytotoxic and proliferative CAR-T subsets and occurs in the context of preserved memory-associated programs with limited exhaustion. These findings suggest that the limited efficacy of CAR-T therapy in glioblastoma is likely to stem from barriers that emerge at later stages following initial tumor entry instead of immediate CAR-T dysfunction.