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, 14 (2), 338-350

Adult Human Glioblastomas Harbor Radial Glia-like Cells

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Adult Human Glioblastomas Harbor Radial Glia-like Cells

Rong Wang et al. Stem Cell Reports.

Abstract

Radial glia (RG) cells are the first neural stem cells to appear during embryonic development. Adult human glioblastomas harbor a subpopulation of RG-like cells with typical RG morphology and markers. The cells exhibit the classic and unique mitotic behavior of normal RG in a cell-autonomous manner. Single-cell RNA sequencing analyses of glioblastoma cells reveal transcriptionally dynamic clusters of RG-like cells that share the profiles of normal human fetal radial glia and that reside in quiescent and cycling states. Functional assays show a role for interleukin in triggering exit from dormancy into active cycling, suggesting a role for inflammation in tumor progression. These data are consistent with the possibility of persistence of RG into adulthood and their involvement in tumor initiation or maintenance. They also provide a putative cellular basis for the persistence of normal developmental programs in adult tumors.

Keywords: brain tumor; cancer stem cells; glioblastoma; inflammation; radial glia; scRNASeq; tumor mitosis.

Figures

Figure 1
Figure 1
RG-like Cells in Human Adult GBMs (A) Representative confocal images of GBM sections. Cells in M phase are labeled with phospho-histone 3 (pH3) and their radial fiber-like processes are detected with pVIM. (B) Percentage of pVIM+ and pH3+ cells in human GBMs (n = 21 tumors). (C) Quantification of co-expression of pVim with an array of RG markers, by immunochemistry (IHC). (D) Schematic flow of the development of 3D explants from patient GBM samples. IHC demonstrates the expression of pH3 and pVim during cell division; arrows points to mitotic cells with or without process (labeled RG-like or non-RG-like). (E) Co-expression of RG-like markers in pVim+ cells in explants. Scale bars, 10 and 5 μm (A), 20 and 10 μm (D and E).
Figure 2
Figure 2
Human RG-like Mitotic Behavior and Division Planes Frames from timelapse microscopy show four major types of mitotic behavior in explants. (A) Human oRG-specific MST pattern: the cell body moves along the process and accomplishes a fast movement over a significant distance before cytokinesis (dotted white lines); ventricular RG-specific interkinetic nuclear migration: cells move along the process up and down during mitosis; arrow heads point to the nucleus and the stars to the daughter cells; stationary divisions occur without significant movement (dotted line). (B and C) Real-time images show a cell undergoing MST (arrowheads) in (B). Immediate fixation shows the same two daughter cells visible in the last frame (200) and identified with stars. Both cells are immunopositive for pH3, pVIM, and BLBP while only one daughter cell retains the elongated fiber process as shown in (C). Dotted contour around the RG-like fiber in (B). (D and E) Similar approach identifies a cell without process undergoing stationary division in (D) and negative for RG markers in (E). (F) RG cells in explants undergoing division along a horizontal plane. Schematic figures define the cleavage planes (top panel). (G and H) Immunohistochemistry on brains injected with GBM cells expressing CD133 or CD133/GLAST (G). Strong tropism toward the subventricular zone (SVZ) in the CD133/GLAST group, quantified as percent of human cells in V (ventricle), SVZ, and Cx (cortex) in (H). (I and J) Immunohistochemistry of tumor cells in the SVZ region shows RG-like morphology in GFP-labeled human cells lining the SVZ, and expressing SOX2 in (I) and GLAST/BLBP in (J). Scale bars, 10 μm (A), 20 μm (C, D, E, F, and I), 100 μm (G), 20 and 10 μm (J). DP, double-positive or CD133 +/GLAST+. Time in (A)–(D) in minutes.
Figure 3
Figure 3
Single-Cell Transcriptional Analysis of GBM Cells Reveals a Population of RG-like Cells (A) EMD heatmaps to identify RG-like clusters in the analyses of individual patient data. (B) RG-like cells in individual patient data are segregated into non-cycling (orange) and cycling (green) based on EMD scores for cell-cycle gene sets (top panel). Diffusion maps on raw data using all genes demonstrate the distribution of RG-like cells in individual patients. Each dot represents a cell and each axis represents a diffusion component. The cells are color coded based on the clusters they belong to. (C) Expression distribution of RG markers (HOPX, IL6ST, LIFR, and BLBP) are shown for individual tumors. Orange and blue lines represent the expression of a specific gene in the indicated cluster and its expression in the rest of the data, respectively. (D) Immuno-FISH on sections from four patients show colocalization of pVim with the EGFR probe. (E) CNV analysis of chromosome 7 in RG- and non-RG-like cells (n = minimum of 50 nuclei per tumor). Scale bar, 8 μm.
Figure 4
Figure 4
Inflammation Regulates Cell-Cycle States of RG-like Cells (A) Immunohistochemistry for GLAST and protein synthesis (OP-puro) in growth factor (GF) and GF-free conditions. (B) IL-1b treatment activates RG-like cells coexpressing GLAST+LIFR+ by stimulating protein synthesis (OP-puro) and the expression of EGFR. Quantification of general protein synthesis and expression of EGFR protein in the indicated cell groups (right panel), p = 0.0008 (OP-puro), p = 0.019 (EGFR). (C) RNA-FISH showing that IL-1b activates the transcription of EGFR in RG-like cells and while CRYAB expression is maintained. (D) Treatment with IL-1b for 72 h significantly increases Ki67 in GLAST+LIFR+ cells, signifying entry into the cell cycle (n = 6, triplicate from two patients). Scale bars, 20 μm (A, B, and D), 50 μm (C). (E and F) IHC for GLAST (E) in a set of matching primary (left) and recurrent (right) tumors quantified in (F). n = 4 pairs, Mann-Whitney.

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