The Phenotypes of Proliferating Glioblastoma Cells Reside on a Single Axis of Variation

Abstract

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identifi ed. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are suffi cient to generate the heterogeneity observed in GBM. We performed single-cell/ single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. SIGNIFICANCE: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classicsubtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.

PremalignantNf1, Trp53-null Oligodendrocyte Precursor Cells Become Stalled in a Heterogeneous State of Replication Stress Before Gliomagenesis

“…Bulk and 10cRNA-seq samples were projected onto the first two principal components of the GBM C1 principal components analysis (3). Mouse genes were first converted into their human orthologs using the "biomaRt" package in R. Of the 714 human genes contributing to the principal components, 629 genes had mouse orthologs in our dataset that were used for the projection.…”

“…Subsequent work at the single-cell level suggested that intratumor heterogeneity of advanced gliomas is substantial (4), with the proportion of different cell types (recruited astrocytes vs. microglia) and cell states (proneural vs. mesenchymal) dictating the bulk classification. Glioma-propagating cells are now proposed to reside on a spectrum of proneural-to-mesenchymal cell fate, which is orthogonal to a cell's proliferative state (3). The question of subtyping is also separate from differential expression, as many of the markers delineating a subtype will reflect vestiges of the cell type in which the tumor appeared.…”

“…We sequenced seven samples from tumors and six samples of OPCs from age-matched control mice, detecting 11,748 ± 651 genes at greater than five transcripts per million (TPM). To revisit the molecular subtype of glioma cells in bulk, we used a principal components-based projection that stratifies cells along two axes: proneural-vs.-mesenchymal differentiation and quiescent-vs.-proliferative activity (3). Both control OPCs and mutant tumors clearly resided on the proneural side of the projection, but there was greater variability observed among the tumors (Fig.…”

“…The latest conceptual frameworks support three subtypes (2), two of which encapsulate the proneural vs. mesenchymal regulatory state of GBM tumor cells. The third ("classical") describes mesenchymal tumor cells mixed with astrocytes instead of the microglial recruitment characteristic of mesenchymal GBMs (3). Malignant cells of different subtypes often reside in the same GBM (4), and advanced GBM clones can give rise to cells with OPC-, neural progenitor-, mesenchymal-, and astrocyte-like signatures (5), suggesting that subtypes could reflect dynamic cell states in glioma.…”

“…Boxplots show the median and interquartile range with whiskers indicating the range from n = 6 independent samples of control 150 dpi OPCs. D, Projection of the bulk 150 dpi mutant and control samples onto principal components capturing proneural-mesenchymal state and relative proliferative status(3). E, Volcano plot of differentially expressed genes between tumors and controls.…”