Barrier to progress: diffuse intrinsic pontine glioma (dipg) induces a tightened blood-brain barrier phenotype.

Abstract DIPG is an incurable paediatric brainstem tumour. Treatments have been significantly hindered by the necessity for drugs to penetrate the blood-brain barrier (BBB). We, and others, have shown that the BBB remains intact in DIPG, and it is thought that the BBB in the brainstem may be even less permeable in the presence of DIPG tumor cells. We performed in vivo studies in orthotopic DIPG mouse models that showed higher efficacy of mTOR inhibitor, temsirolimus, in cortex-injected DIPG compared to brainstem-injected DIPG. Analysis showed significantly increased survival, higher temsirolimus levels and decreased proliferating tumour cells in the cortex. Subsequently, the effect of DIPG on brain vasculature was interrogated by single-cell RNA sequencing. Microvascular endothelial cells were isolated from the brainstem and cortex, where DIPG cells or Matrigel were implanted. Transcriptomic analysis of DIPG-injected brainstem uncovered significant downregulation of inflammatory pathways, especially P38MAPK, downregulation of antigen presentation, and increased transporter Mfsd2a, suggesting a tightened BBB. Apoptotic signalling pathways were significantly upregulated, particularly MCL-1. Further analysis into cortex-injected DIPG revealed downregulation of interferon β pathways, suggesting a leakier phenotype. To overcome the tightened barrier in the brainstem, MCL-1 inhibitor S63845, was investigated as a BBB modulator. S63845 significantly reduced transendothelial electrical resistance, increased tracer dye leakage in an in vitro BBB transwell model with DIPG and decreased protein expression of claudin-5. In vivo, S63845 increased permeability to 3kDa Texas-red dextran after a single dose in DIPG mouse models. Overall, this study revealed that DIPG directly affects endothelial cell pathways associated with BBB permeability, resulting in a tightened BBB and reduced therapeutic efficacy. S63845 represents a promising BBB modulator that can be easily administered and may facilitate the delivery of therapeutics through the constricted BBB. This discovery has the potential to enhance the activity of anti-DIPG therapeutics that were previously considered impenetrable.