Glioblastoma scRNA-seq shows treatment-induced, immune-dependent increase in mesenchymal cancer cells and structural variants in distal neural stem cells

Glioblastoma is a treatment-resistant brain cancer. Its hierarchical cellular nature and its tumor microenvironment (TME) before, during, and after treatments remain unresolved. Here, we used single-cell RNA sequencing to analyze new and recurrent glioblastoma and the nearby subventricular zone (SVZ). We found 4 glioblastoma neural lineages are present in new and recurrent glioblastoma with an enrichment of the cancer mesenchymal lineage, immune cells, and reactive astrocytes in early recurrences. Cancer lineages were hierarchically organized around cycling oligodendrocytic and astrocytic progenitors that are transcriptomically similar but distinct to SVZ neural stem cells (NSCs). Furthermore, NSCs from the SVZ of patients with glioblastoma harbored glioblastoma chromosomal anomalies. Lastly, mesenchymal cancer cells and TME reactive astrocytes shared similar gene signatures which were induced by radiotherapy in a myeloid-dependent fashion in vivo. These data reveal the dynamic, immune-dependent nature of glioblastoma’s response to treatments and identify distant NSCs as likely cells of origin. Single-Cell RNA sequencing and analysis Brain metastasis samples were harvested under a protocol approved by the Montreal Neurological Hospital’s research ethics board (protocol NEU-10-066). All patients gave consent. Surgeries were performed at the Montreal Neurological Hospital. Samples were obtained from within the contrast-enhancing area of the tumor and just outside of the contrast-enhancing region. To extract brain tumour cancer stem (BTCS) cells, fresh tumour specimens were washed 3 times in sterile PBS containing 1% penicillin and streptomycin (PBS+PS). Using scalpels, specimens are minced into fragments of less than 1mm in size and digested in 4ml collagenase solution (1mg/ml; final; Sigma) containing DNAse (50U/ml final; Biorad) and MgCl2 (1mM final) for 1h at 37°C. The digested specimens were washed once with sterile PBS+PS, and large undigested samples were removed with a 70μm strainer and cells were centrifuged for 10 min at 1300 rpm to remove collagenase solution. Cell pellets were then resuspended in 21ml PBS+PS and 9ml of percoll and a centrifugation step at 15000 rpm for 30min at 4C was done to separate cells from blood and debris. Cells were then transferred to a 50ml tube and counted (using trypan blue) before being centrifuged at 1400 rpm for 10 min, then cell pellets were resuspended in PBS +0.04%BSA to a final concentration of 1000 cells/ul, and 100ul of the sample was sent for single-cell capture. *************************************************************** Raw data files not provided as there are privacy concerns regarding sequencing file (BAM, FASTQ) ***************************************************************