Bi-directional immunosuppressive interactions between astrocytes and glioblastoma cells identified by barcoded viral tracing [Human Single Cell Dataset]

Glioblastoma (GBM) is the most lethal primary brain malignancy. Immunosuppression in the GBM tumor microenvironment (TME) presents a major barrier to immune-targeted therapies. However, our understanding of mechanisms of immunomodulation in the GBM TME is still limited. To address this challenge, we developed a viral barcode interaction tracing approach for the analysis of cell-cell communication in the TME in GBM samples and preclinical models with single-cell resolution, which we combined with single-cell and bulk RNA-seq analyses of GBM clinical samples and pre-clinical models, human organotypic GBM slice cultures, in vivocell-specific CRISPR/Cas9-driven genetic perturbations and human and mousein vitroexperimental systems to identify a novel bi-directional communication pathway between ANXA1 expressed inastrocytes and its receptor FPR1 expressed by GBM cells that limits anti-tumor immune responses. ANXA1 expression in astrocytes and FPR1 expression in cancer cells are associated with shorter survival and earlier recurrence in GBM patients. The inactivation of astrocyte-glioma ANXA1/FPR1-signaling enhanced tumor specific CD4+ and CD8+ T-cell responses, increasing tumor infiltration by neoantigen-specific CD8+ T cells while limiting transcriptional modules linked to T-cell exhaustion; ANXA1/FPR1-signaling inactivation also boosted tumor-associated macrophage responses. Using pre-clinical models and human GBMin vitroexperimental systems, we established that FPR1 inhibits immunogenic necroptosis in tumor cells, while ANXA1 retrograde signaling suppresses NF-kB and inflammasome activation within astrocytes. In summary, we developed a new barcoding approach to analyze TME cell-cell interactions with single-cell resolution in clinical samples and pre-clinical models, and used it to identify a novel pathway of bidirectional ANXA1–FPR1 astrocyte-GBM communication which promotes immunoevasion and cancer progression.