Single-cell Bottlenecks and Dead-ends During Glioma Premalignancy

Cancer evolves from premalignant clones that accumulate mutations and adopt unusual cell states to achieve transformation. Tracking a cancer cell-of-origin through the cell-state alterations of premalignancy could provide clues for early-detection and cancer-prevention strategies. Previously we pinpointed the oligodendrocyte precursor cell (OPC) as a cell-of-origin for glioma. However, the early adaptations and cell-state changes of mutant OPCs during premalignancy are unknown. Using a genetically engineered mouse model (GEMM) of inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection and determined gene-expression changes in two ways: global changes in gene expression were measured by differential analysis of wild-type and mutant OPCs after bulk RNA sequencing; cell-to-cell state variations were identified by a fluctuation analysis, called stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. We chose two time points for the analysis. At 12 days after Nf1-Trp53 deletion, while bulk differences were mostly limited to increases in mitotic hallmarks and decreases in ribosome biosynthesis, stochastic profiling of mutant OPCs revealed a spectrum of stem-progenitor, proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days after Nf1-Trp53 deletion, while bulk sequencing detected very few differentially expressed transcripts, stochastic profiling revealed multiple cell states that are absent from glial tumors, suggesting that they marked dead-ends for gliomagenesis. In parallel, we identified cells without dead-end markers but abundantly expressing key effectors of nonsense-mediated decay and homology-dependent DNA repair. This suggests that resolution of replication stress may pose a considerable bottleneck for glioma initiation in premalignant mutant OPCs.