Asymmetric cell division promotes therapeutic resistance in glioblastoma stem cells
Masahiro Hitomi, Anastasia P. Chumakova, Daniel J. Silver, Arnon M. Knudsen, W. Dean Pontius, Stephanie Murphy, Neha Anand, Bjarne W. Kristensen, Justin D. Lathia
Masahiro Hitomi, Anastasia P. Chumakova, Daniel J. Silver, Arnon M. Knudsen, W. Dean Pontius, Stephanie Murphy, Neha Anand, Bjarne W. Kristensen, Justin D. Lathia
View: Text | PDF
Research Article Cell biology Stem cells

Asymmetric cell division promotes therapeutic resistance in glioblastoma stem cells

Abstract

Asymmetric cell division (ACD) enables the maintenance of a stem cell population while simultaneously generating differentiated progeny. Cancer stem cells (CSCs) undergo multiple modes of cell division during tumor expansion and in response to therapy, yet the functional consequences of these division modes remain to be determined. Using a fluorescent reporter for cell surface receptor distribution during mitosis, we found that ACD generated a daughter cell with enhanced therapeutic resistance and increased coenrichment of EGFR and neurotrophin receptor (p75NTR) from a glioblastoma CSC. Stimulation of both receptors antagonized differentiation induction and promoted self-renewal capacity. p75NTR knockdown enhanced the therapeutic efficacy of EGFR inhibition, indicating that coinheritance of p75NTR and EGFR promotes resistance to EGFR inhibition through a redundant mechanism. These data demonstrate that ACD produces progeny with coenriched growth factor receptors, which contributes to the generation of a more therapeutically resistant CSC population.

Authors

Masahiro Hitomi, Anastasia P. Chumakova, Daniel J. Silver, Arnon M. Knudsen, W. Dean Pontius, Stephanie Murphy, Neha Anand, Bjarne W. Kristensen, Justin D. Lathia

×

Figure 1

A plasma membrane–green fluorescence protein (PM-GFP) reporter system allows the reliable evaluation of cell division mode and reveals functional differences in asymmetrically divided cells.

Options: View larger image (or click on image) Download as PowerPoint
A plasma membrane–green fluorescence protein (PM-GFP) reporter system al...
(A) Confocal microscopy captured asymmetrically (left) and symmetrically (right) dividing T4121-PM-GFP cancer stem cells (CSCs) in telophase. Scale bar: 20 μm. CSCs expressing a lipid raft marker, PM-GFP (green), were stained with Hoechst 333342 for DNA (cyan), with cholera toxin B (CTB, red), another marker of lipid rafts, and with a specific antibody for surface CD133 (yellow). (B) Quantification of asymmetry percentage during mitosis reveals a significant correlation between asymmetry of PM-GFP and CD133 by Pearson’s correlation coefficient analysis (P < 0.00001). Each dot represents 1 cell division. Blue dots indicate divisions with cosegregation of PM-GFP and CD133 to the same progeny. Divisions exhibiting segregation of each marker to opposite progeny are shown in red. (C) Time-lapse microscopy recording detected asymmetric PM-GFP inheritance between T4121-PM-GFP CSC daughter cells: darker cell (d) and brighter cell (b), at the time of mitosis. Progeny were traced, and their SOX2 levels were quantified by immunofluorescence after time-lapse recording. Scale bars: 20 μm. (D) Pearson’s correlation coefficient analysis demonstrated a significant association between the degree of PM-GFP asymmetry at the time of mitosis and SOX2 expression asymmetry of corresponding progeny at the end of the 3-day -time-lapse microscopy (P = 0.03). (E) FACS analysis of cells released from S phase synchronization. Once-divided cells (green shaded) exhibited a CellTrace signal intensity that was half the value of the nondivided cells (gray shaded). Gated divided cells were then sorted based on their PM-GFP signal. As asymmetric divisions constituted 10%–15% of divisions in T4121-PM-GFP cells, the top and bottom 5% of PM-GFP cells (PM-GFP–high and PM-GFP–low) were sorted as asymmetrically divided, and the cells in the middle fraction of the PM-GFP distribution (PM-GFP–mid) were selected as progeny of symmetric division. (F) CD133 immunofluorescence intensity was quantified for each sorted faction cell normalized by DNA content. PM-GFP–low, PM-GFP–mid, and PM-GFP–high populations expressed CD133 at significantly different levels, with the highest CD133 mean level in PM-GFP–high and the lowest in PM-GFP–low (***P < 0.000001, 1-way ANOVA). Bars indicate mean expression levels. (G and H) Cell viability of PM-GFP–low, PM-GFP–mid, and PM-GFP–high populations after 3-day exposure to 100 μM temozolomide (TMZ, with 2 biological replicates) (G), or 3 days after 2 Gy γ-irradiation (H). PM-GFP–high cells had a significantly higher relative viability (mean ± SEM, ***P < 0.000001, 1-way ANOVA).