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.