Aims: Treatment for glioblastoma involves resection and post-operative IR and chemotherapy. Unfortunately, significant populations of resistant glioma stem cells remain after treatment, these cells resist IR/chemotherapy in part because they are quiescent/slow-cycling. As glioma stem cells sit on top of the cellular hierarchy in glioblastoma, the proliferation of these cells post-therapy reconstitutes the tumour. Multiple pathways are known to regulate quiescence in healthy neural stem cells. It is unclear whether these pathways are conserved in quiescent glioma stem cells and whether these pathways are therapeutically tractable. To address these questions, we set out to implement novel 1) bioinformatic, 2) in vitro and 3) in vivo tools.
Methods: We used single-cell RNA sequencing data and trajectory inference tools to compare the gene expression changes that occur as quiescent neural stem cells and glioma stem cell leave quiescence. Secondly, we tested a number of these pathways through functional in vitro assays using patient-derived 2D glioma stem cell cultures and organoids. Thirdly, to track quiescent glioma stem cells in vivo, we generated a novel somatic cell electroporation model of brain cancer, based on combining deletion of the tumour suppressors Nfi, Pten, P53 with the G0-reporter mVenus-P27K–.
Results: We found that activation signatures of quiescent neural stem cells and glioma stem cells are largely correlated and functionally conserved. We identified pathways that could both deepen quiescence or inhibit quiescence. Our in vivo quiescence tracking model successfully distinguished between proliferating tumour cells (fluorescing red) and quiescent glioma stem cells (fluorescing green). The behaviour of these different populations during early vs late gliomagenesis, response to temozolomide, and at the tumour core vs invasive edge revealed distinct behaviour.
Conclusions: Overall, these data reveal conservation of major signalling pathways controlling quiescence in health and disease and suggest new therapeutic opportunities.