Reactive astrocytes evolve after brain injury, inflammatory and degenerative diseases, whereby they undergo transcriptomic re-programming. In malignant brain tumors, their function and crosstalk to other components of the environment is poorly understood. Here we report a distinct transcriptional phenotype of reactive astrocytes from glioblastoma linked to JAK/STAT pathway activation. Subsequently, we investigate the origin of astrocytic transformation by a microglia loss-of-function model in a human organotypic slice model with injected tumor cells. RNA-seq based gene expression analysis of astrocytes reveals a distinct astrocytic phenotype caused by the coexistence of microglia and astrocytes in the tumor environment, which leads to a large release of anti-inflammatory cytokines such as TGFβ, IL10 and G-CSF. Inhibition of the JAK/STAT pathway shifts the balance of pro- and anti-inflammatory cytokines towards a pro-inflammatory environment. The complex interaction of astrocytes and microglia cells promotes an immunosuppressive environment, suggesting that tumor-associated astrocytes contribute to anti-inflammatory responses.

Glioblastoma multiforme are highly malignant brain tumours with frequent genetic and epigenetic alterations. The poor clinical outcome of these tumours necessitates the development of new treatment options. Immunotherapies for glioblastoma multiforme including PD1/PD-L1 inhibition are currently tested in ongoing clinical trials. The purpose of this study was to investigate the molecular background of PD-L1 expression in glioblastoma multiforme and to find associated pathway activation and genetic alterations. We show that PD-L1 is up-regulated in IDH1/2 wildtype glioblastoma multiforme compared to lower-grade gliomas. In addition, a strong association of PD-L1 with the mesenchymal expression subgroup was observed. Consistent with that, NF1 mutation and corresponding activation of the MAPK pathway was strongly connected to PD-L1 expression. Our findings may explain different response to PD-L1 inhibition of patients in ongoing trials and may help to select patients that may profit of immunotherapy in the future.

BackgroundIn recent years, PD-1/PD-L1 immune checkpoint inhibitors have improved cancer therapy in many tumor types, but no benefit of immune checkpoint therapy has been found in glioblastoma multiforme (GBM). Based on the results of our earlier work, which showed a reduction of PD-L1 expression in patients treated with temozolomide (TMZ), we aimed to investigate the link between TMZ therapy and the immune control point target PD-L1.MethodsRNA-sequencing data from de-novo and recurrent glioblastoma were analyzed by AutoPipe algorithm. Results were confirmed either in a cell model by two primary and one established GBM cell line and specimens of de-novo and recurrent GBM. PD-L1 and pathway activation of the JAK/STAT pathway was analyzed by quantitative real-time PCR and western blot.ResultsWe found a significant downregulation of the JAK/STAT pathway and immune response in recurrent tumors. The cell model showed an upregulation of PD-L1 after IFNγ treatment, while additional TMZ treatment lead to a reduction of PD-L1 expression and JAK/STAT pathway activation. These findings were confirmed in specimens of de-novo and recurrent glioblastoma.ConclusionsOur results suggest that TMZ therapy leads to a down-regulation of PD-L1 in primary GBM cells. These results support the clinical findings where PD-L1 is significantly reduced in recurrent GBMs. If the target is diminished, it may also lead to impaired efficacy of PD-1/PD-L1 inhibitors such as nivolumab.