956 Glioma-neuronal circuit remodeling induces regional immunosuppression

BackgroundGlioblastomas remodel neuronal circuits.1 Moreover, direct synaptic connections between neurons and glioblastoma cells and glioblastoma- and neuron-derived paracrine growth factors promote glioblastoma growth and invasion.2–4 In converse, glioblastoma cells cause neuronal hyperexcitability and neuronal circuit hypersynchrony through the tumor-derived synaptogenic factor Thrombospondin-1 (TSP-1/Thbs1). Intriguingly, TSP-1 is expressed not only by glioblastoma cells but also by myeloid cells within the tumor. However, the contributions of immune cell components in glioma-neuronal interactions remain unknown. In this study, we test the hypothesis that neuronal activity-dependent glioblastoma proliferation is mediated through the crosstalk between glioblastoma cells, neurons, and immune cells and explore the therapeutic vulnerabilities.MethodsWe investigated the differences in transcriptional programs between high- and low-functional connectivity regions (termed HFC and LFC, respectively) through single-cell RNA-sequencing (sc-RNAseq) analysis of patient surgical specimens. We used spatially-resolved RNA-sequencing in a preclinical glioblastoma model SB28. Using bulk RNA-sequencing and flow cytometry, we investigated the significance of TSP-1 through CRISPR-Cas9 knockout in SB28 glioblastoma cells in vitro and in vitro. Furthermore, we evaluated the mechanism of interactions between excitatory neuronal activity and immune regulation using bone-marrow-derived macrophage (BMDM) and cortical neurons in vitro. Finally, we evaluated the effect of neuronal activity-oriented therapy on the tumor-associated macrophages (TAMs) in mice bearing intracerebral SB28 tumors.ResultsThrough human sc-RNAseq analysis, we discovered a strong association between glioma-neuronal circuit remodeling and regional immunosuppression, in which HFC-derived cells presented remarkable downregulation of several key inflammatory pathways, such as IFNG and TNFa-NFkB (figure 1). Spatial transcriptomics revealed the negative correlations between synaptic activities and inflammatory responses (figure 2). Characterization of TSP-1-WT and KO tumors in vivo demonstrated the significance of TSP-1 in enhancing neuro-synaptic activities but suppressing immune responses, including the differences in TAM polarization patterns and T-cell compositions (figure 3). In vitro assays demonstrated that neuron-secreted factors, but not TSP-1, induced an anti-inflammatory, M2-like phenotype in BMDMs, whi