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 3

Alteration of the p75NTR axis modifies CSC phenotypes after differentiation.

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Alteration of the p75NTR axis modifies CSC phenotypes after differentiat...
(A) Experimental design to assess self-renewing capacity after differentiation induction. T4121-PM-GFP CSCs were subjected to 3 days of pretreatment with either CSC medium (containing epidermal growth factor [EGF] and fibroblast growth factor 2 [FGF2]), CSC medium with the addition of nerve growth factor (NGF), 10% FBS containing differentiation medium without EGF and FGF, or medium with 10% FBS, along with EGF and NGF. The cells were then plated for limiting-dilution assay in CSC medium containing EGF and FGF2 for 2 weeks and assessed for self-renewal (sphere forming) capacity. (B) Self-renewal capacity of T4121-PM-GFP cells after 3-day exposure to these conditions. (C) Self-renewal capacity of T4121-PM-GFP cells after differentiation using 10% FBS with and without growth factor stimulation (EGF, NGF, or combination). (D) Immunoblotting showing expression of p75NTR and SOX2 in T4121-PM-GFP cells expressing p75NTR knockdown shRNAs (KD1 and KD2) compared with nontargeting (NT) shRNA. (E) Self-renewal capacity of T4121-PM-GFP cells in stem cell medium (NB) alone, in the presence of 10% FBS (NB + FBS), or with FBS with EGF and NGF together (FBS + EGF + NGF). Nontargeting shRNA (NT) transduced cells were compared with knockdown shRNA (KD1) transduced cells. (B, C, and E) Estimated stem cell frequencies are shown with 95% CI (numbers in parentheses). ****P < 0.000001, **P = 0.0144. (F) Expression levels of phospho-EGFR and SOS2 in T4121-PM-GFP cells generated through different modes of cell divisions were determined for individual cells by quantitative immunofluorescence. After cell sorting, divided daughter cells of three groups — cells with asymmetrically depleted PM-GFP (low), symmetrically divided cells with mid levels of PM-GFP (mid), and those with asymmetrically enriched PM-GFP (high) — were cultured in differentiation-inducing serum containing medium supplemented with EGF and NGF. Left panel is quantified phospho-EGFR levels after culturing for a day, and the right panel shows levels of SOX2 expression on day 1 and 3 (**P < 0.001 as calculated by 1-way ANOVA). SOX2 expression levels on Day 3 were analyzed by Wilcoxon test. Each dot represents an individual cell.