A single-cell atlas of RNA alternative splicing in the glioma-immune ecosystem

Single-cell analysis has refined our understanding of cellular heterogeneity in glioma, yet RNA alternative splicing, a critical layer of transcriptome regulation, remains underexplored at single-cell resolution. Here, we present a pan-glioma single-cell alternative splicing analysis in both tumor and immune cells through integrating seven SMART-seq2 datasets of human gliomas to uncover overlooked isoform-level regulations shaping glioma progression and immune responses.

Our analysis reveals lineage-specific alternative splicing regulation among glioma cellular states, with the most divergent alternative splicing landscapes observed between mesenchymal and neuronal-like glioma cells. Notably, this includes events in TCF12 exon 15 and PTBP2 exon 10, two key regulators of mesenchymal and neuronal transcriptomes. Inducing TCF12 exon 15 inclusion in glioma cells with a dCasRx-RBM25 system promotes neuronal gene expression, suppresses genes related to extracellular matrix organization, and enhances sensitivity to radiotherapy. Comparison of core and peripheral glioma cells highlights alternative splicing redox co-regulation of TPM1 and ACTN4, key genes involved in cytoskeletal organization. Further analysis of glioma-infiltrating immune cells reveals altered first exon usage of UGP2 in regulatory T cells and an association between MS4A7 alternative splicing in macrophages and clinical response to anti-PD-1 therapy.

This study emphasizes the role of alternative splicing in glioma cellular heterogeneity, highlighting the importance of an isoform-centric approach to better understand the complex biological processes driving tumorigenesis.