Barcoded viral tracing identifies immunosuppressive astrocyte-glioma interactions

Glioblastoma (GBM) is the most lethal primary brain malignancy¹. Immunosuppression in the GBM tumour microenvironment (TME) is an important barrier to immune-targeted therapies, but our understanding of the mechanisms of immune regulation in the GBM TME is limited². Here we describe a viral barcode interaction-tracing approach³ to analyse TME cell-cell communication in GBM clinical samples and preclinical models at single-cell resolution. We combine it with single-cell and bulk RNA-sequencing analyses, human organotypic GBM cultures, in vivo cell-specific CRISPR-Cas9-driven genetic perturbations as well as human and mouse experimental systems to identify an annexin A1-formyl peptide receptor 1 (ANXA1-FPR1) bidirectional astrocyte-GBM communication pathway that limits tumour-specific immunity. FPR1 inhibits immunogenic necroptosis in tumour cells, and ANXA1 suppresses NF-κB and inflammasome activation in astrocytes. ANXA1 expression in astrocytes and FPR1 expression in cancer cells are associated with poor outcomes in individuals with GBM. The inactivation of astrocyte-glioma ANXA1-FPR1 signalling enhanced dendritic cell, T cell and macrophage responses, increasing infiltration by tumour-specific CD8⁺ T cells and limiting T cell exhaustion. In summary, we have developed a method to analyse TME cell-cell interactions at single-cell resolution in clinical samples and preclinical models, and used it to identify bidirectional astrocyte-GBM communication through ANXA1-FPR1 as a driver of immune evasion and tumour progression.