Single-cell based elucidation of molecularly distinct GBM states and drug sensitivity

Glioblastoma heterogeneity and plasticity remain controversial, with proposed subtypes representing the average of highly heterogeneous admixtures of independent transcriptional states. Single-cell, protein-activity-based analysis identified four novel, molecularly distinct subtypes that successfully harmonize across multiple GBM datasets, including previously published bulk and single-cell profiles and single cell profiles from seven orthotopic PDX models, representative of prior subtype diversity. From these studies, GBM emerges as the plastic equilibrium of single tumor cells coexisting in two mutually-exclusive developmental states, with additional stratification provided by their proliferative potential. Consistent with these findings, all previously proposed subtypes could be recapitulated by mixtures of single cells representing these newly identified states. Most critically, drug sensitivity was predicted and experimentally confirmed as highly state-dependent, both in single-cell assays from patient-derived explants and in PDX models treated with clinically relevant drugs, suggesting that successful therapeutic strategies will likely require combinations of multiple drugs targeting these distinct tumor states. Single-cell RNA-sequencing of control and drug-treated slice cultures of human glioma surgical specimens.