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
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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

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Figure 2

EGFR and p75NTR cosegregate during asymmetric cell division.

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EGFR and p75NTR cosegregate during asymmetric cell division. (A) Immunof...
(A) Immunofluorescence staining of an asymmetrically divided T4121-PM-GFP cell in late telophase. Scale bar: 20 μm. PM-GFP (green), EGFR (red), and p75NTR (yellow) are shown, and DNA is stained with Hoechst 333342 (blue). (B and C) Quantification of percentage of asymmetry during mitosis reveals a correlation between the asymmetry of PM-GFP and EGFR (B) and PM-GFP and p75NTR (C). Each dot represents 1 cell division. Divisions with cosegregated PM-GFP and EGFR/p75NTR on the same daughter cell are marked in blue. Divisions that exhibited segregation of these markers on opposite daughter cells are marked in red. Calculated Pearson’s correlation coefficient demonstrated a significant association (P < 0.000001) between PM-GFP reporter asymmetry and that of EGFR (B) and p75NTR (C). (D and E) Immunofluorescence staining quantification of EGFR (D) and p75NTR (E) expression in sorted symmetrically and asymmetrically divided cells. EGFR/p75NTR signal intensity per cell was normalized to DNA intensity per cell. PM-GFP–low, PM-GFP–mid, and PM-GFP–high populations all expressed significantly different EGFR and p75NTR expression levels, with the highest expression level in PM-GFP–high and the lowest in PM-GFP–low (***P < 0.000001) as calculated by 1-way ANOVA. Bars indicate mean values. (F) Immunofluorescence staining of asymmetrically dividing cells in late telophase from 4 different glioma stem cell specimens. Scale bar: 20 μm. CTB, used as lipid raft marker (green); p75NTR (red); and EGFR (yellow) are shown. DNA was stained with Hoechst 333342 (cyan). (G) EGFR asymmetry in mitotic cells in human GBM tumors was captured after IHC. Scale bar: 10 μm. After staining signal was classified (green marking), the daughter cell with higher staining was defined by blue region of interest (ROI), and the other with less staining by red ROI. Quantitative analysis on 26 mitotic cells that exhibited asymmetric EGFR staining area (green classifier) and intensity (brown staining under classifier) detected significant difference of EGFR staining between the daughter cells (Mean ± SEM, P < 0.001 as calculated by 1-way ANOVA). (H) Quantification of asymmetry percentage during late telophase reveals a correlation between asymmetry of EGFR and p75NTR in 4 different non–PM-GFP–expressing glioma stem cell specimens. Divisions where EGFR and p75NTR cosegregated on the same daughter cell are marked in blue. Divisions that exhibited segregation of these markers on opposite daughter cells are marked in red. Pearson’s correlation coefficients were calculated and demonstrated a significant association (P < 0.000001) between asymmetry of p75NTR and that of EGFR.