Intratumoral heterogeneity of glioblastoma is attributed to precancerous cells derived from neural stem cells

Neural stem cells (NSCs) in the subventricular zone (SVZ) have been identified as cells-of-origin carrying driver mutations in glioblastoma (GBM), but there is a lack of understanding how a few NSCs with driver mutations chronologically transform into distant tumor with transcriptional heterogeneity. We used single cell RNA sequencing (scRNA-seq) to show the precancerous cells arising from mutation-harboring NSCs emerge prior to tumor formation and lead to intratumoral heterogeneity in our spontaneous, somatic mouse model as well as in patients’ tumor-free SVZ tissues. In mouse GBM model, the SVZ and corpus callosum (CC) tissues were microdissected for precancerous stage, and tumor tissues were used for the study. Mutation-harboring cells with tdTomato signal were enriched with fluorescent-activated cell sorting (FACS) and analyzed using scRNA-seq. For human specimens, freshly frozen samples of pathologically normal and tumor-free SVZ tissues, as well as tumors, were obtained from three GBM patients with EGFRvIII, PTEN mutations. Nuclei were isolated by FACS and analyzed using single nucleus RNA-seq.