Emergence of stem-like stromal cell populations and dynamic changes in stromal-immune cell interactions in response to immunotherapy in glioma

Introduction: There is emerging evidence that stromal cells play critical roles in promoting tumor invasion, angiogenesis, and immune evasion, and that they form complex signaling pathways with tumor and immune cells. However, the essential role of stromal cells in glioma remains poorly understood.

Methods: We performed scRNA-seq on >700,000 tumor, immune, and stromal cells from 22 untreated grade IV gliomas, 4 grade IV gliomas treated with anti-PD1/Lag3 immunotherapy, 3 grade III gliomas, 7 grade II gliomas, and 5 non-neoplastic brain tissue samples. Various scRNA-seq computational methods were implemented to determine the phenotypic composition of stromal cells in the glioma microenvironment and the impact of immunotherapy on stromal cell function.

Results: 46,426 stromal cells were identified across all patients. Across all stromal subtypes, stromal cells in gliomas had significant upregulation of genes conferring stem-like properties compared to those in non-neoplastic tissue. Moreover, there exists a subpopulation of stromal cells with particularly high expression of stemness markers that is exclusively present in gliomas and not non-neoplastic tissue. Analysis of marker genes for this subpopulation in bulk RNA-sequencing and spatial transcriptomics data from different tumor regions indicates that hypoxia plays a key role in the development of this subpopulation. Ligand-receptor interaction analysis revealed that this stemness transition leads to downregulation of MHC I and leukocyte adhesion signaling from stromal cells to CD8 T-cells, and upregulation of extracellular matrix signaling from stromal cells to myeloid and lymphoid cells. Extracellular matrix signaling was further increased following immunotherapy. Gene regulatory network analysis identified the SHOX2 transcription factor as a potential driver of this immunotherapy-specific response.

Conclusion : Large-scale single-cell analysis identified enrichment of stem-like stromal cells in the glioma microenvironment that have the potential to impair CD8 T-cell activation and upregulate extracellular matrix signaling to promote tumor invasion and hinder immune infiltration, a phenomenon that is further augmented following immunotherapy treatment.