Heightened levels of circulating cell-free dna contribute to peripheral immunosuppression during gbm progression

Abstract Glioblastoma (GBM) is an incurable and aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immune modulatory therapies. Using the murine GL261 model of GBM, we recapitulated major features of peripheral immunosuppression observed in patients including T cell lymphopenia. We determined that peripheral immunosuppression in GBM is multifaceted and involves release of novel non-steroidal soluble factors with large molecular weight that are found in sera of glioma-bearing mice. These high molecular weight species potently inhibits T cell proliferation in vitro despite the presence of strong T cell stimulation. Our goal was to determine the identity of the immunosuppressive species in serum of glioma-bearing mice. We performed state-of-the-art comparative high throughput mass spectrometry on fractionated and multiplexed sera isolated from control and glioma-bearing mice. This proteomics analysis demonstrated histones as a top highly abundant protein in serum of glioma-bearing mice compared to naïve controls. Cell-free DNA has been recently identified as an immune suppressive factor in circulation and can be bound to histones. We, therefore, assessed cell-free DNA levels in the serum of glioma-bearing mice. We determined that sera isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded and single stranded cell-free DNA compared to healthy controls. Reducing the DNA content in serum, using DNAse treatment, partially improved the defect in T cell proliferation observed when T cells are exposed to serum isolated from glioma-bearing mice. These data indicate that heighted levels of circulating DNA in serum contributes to peripheral immunosuppression in GBM. These findings provide a tractable approach to investigate the cellular source and immunosuppressive effects of cell-free DNA in murine models of GBM and to devise strategies to reverse immunosuppression with the hope of improving outcomes in patients.