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. 2014 Sep;16(9):1210-9.
doi: 10.1093/neuonc/nou026. Epub 2014 Mar 16.

Convection-enhanced Delivery of Etoposide Is Effective Against Murine Proneural Glioblastoma

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Free PMC article

Convection-enhanced Delivery of Etoposide Is Effective Against Murine Proneural Glioblastoma

Adam M Sonabend et al. Neuro Oncol. .
Free PMC article

Abstract

Background: Glioblastoma subtypes have been defined based on transcriptional profiling, yet personalized care based on molecular classification remains unexploited. Topoisomerase II (TOP2) contributes to the transcriptional signature of the proneural glioma subtype. Thus, we targeted TOP2 pharmacologically with etoposide in proneural glioma models.

Methods: TOP2 gene expression was evaluated in mouse platelet derived growth factor (PDGF)(+)phosphatase and tensin homolog (PTEN)(-/-)p53(-/-) and PDGF(+)PTEN(-/-) proneural gliomas and cell lines, as well as human glioblastoma from The Cancer Genome Atlas. Correlation between TOP2 transcript levels and etoposide susceptibility was investigated in 139 human cancer cell lines from the Cancer Cell Line Encyclopedia public dataset and in mouse proneural glioma cell lines. Convection-enhanced delivery (CED) of etoposide was tested on cell-based PDGF(+)PTEN(-/-)p53(-/-) and retroviral-based PDGF(+)PTEN(-/-) mouse proneural glioma models.

Results: TOP2 expression was significantly higher in human proneural glioblastoma and in mouse proneural tumors at early as well as late stages of development compared with normal brain. TOP2B transcript correlated with susceptibility to etoposide in mouse proneural cell lines and in 139 human cancer cell lines from the Cancer Cell Line Encyclopedia. Intracranial etoposide CED treatment (680 μM) was well tolerated by mice and led to a significant survival benefit in the PDGF(+)PTEN(-/-)p53(-/-) glioma model. Moreover, etoposide CED treatment at 80 μM but not 4 μM led to a significant survival advantage in the PDGF(+)PTEN(-/-) glioma model.

Conclusions: TOP2 is highly expressed in proneural gliomas, rendering its pharmacological targeting by intratumoral administration of etoposide by CED effective on murine proneural gliomas. We provide evidence supporting clinical testing of CED of etoposide with a molecular-based patient selection approach.

Keywords: convection-enhanced delivery; etoposide; glioma; proneural; topoisomerase.

Figures

Fig. 1.
Fig. 1.
Topoisomerase II gene expression in human glioblastoma and mouse glioma samples. (A) Bar plot representation of expression array data for TOP2A and TOP2B for samples from different human glioblastoma subgroups according to Verhaak et al (data obtained from TCGA). *Significant difference in comparison with other groups, ANOVA P < .00001. (B) TOP2A and TOP2B expression determined by microarray data for samples of PDGF+PTEN−/− as well as PDGF+PTEN−/−p53−/− mouse PN glioma tumor specimens and normal mouse brain. Red color indicates higher expression, black intermediate expression, and green lower expression. (C) TOP2A (left) and TOP2B (right) expressions were longitudinally characterized by qRT-PCR on PDGF+PTEN−/− mouse PN gliomas. Days post i.c. injection for retroviral-mediated tumor induction (dpi). *Significant difference in expression in comparison with mouse brain (P < .05). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fig. 2.
Fig. 2.
Topoisomerase IIB transcript levels correlate with susceptibility to etoposide among human cancer cell lines. One hundred and thirty-nine human cancer cell lines from CCLE (columns) were sorted from high to low AUC derived from dose response curves to etoposide performed by Basu et al (higher AUC signifies resistance, whereas lower AUC signifies susceptibility to etoposide) represented in the top row. TOP2B transcript levels for each of the cell lines obtained from CCLE are represented in the bottom row. A significant inverse correlation was found between AUC for etoposide and TOP2B expression among these cell lines (P < .05). To plot gene expression for TOP2B and AUC for etoposide, both of these data were normalized independently using z-score transformation. Yellow represents higher values and black lower values.
Fig. 3.
Fig. 3.
Proneural glioma cell lines are highly sensitive to etoposide, and this sensitivity correlates with transcript levels of TOP2B. (A) Cell viability was determined on dose response curves for etoposide in murine glioma cell line GL261, in 3 separate PDGF+PTEN−/− murine PN glioma cell lines (JF, JM3, PTENYB) and in a PDGF+PTEN−/−p53−/− PN glioma cell line 48 h (top) and 72 h (bottom). (B) TOP2A (left) and TOP2B (right) transcript levels were compared between GL261, PDGF+PTEN−/− (JF cell line from panel A), and a PDGF+PTEN−/−p53−/− mouse glioma cell line, evaluated by qRT-PCR. PDGF+PTEN−/− and PDGF+PTEN−/−p53−/− glioma cell lines were previously classified as PN based on their gene expression profile. *Represents significant difference in expression with GL261 as a reference (P < .05). IC50, half-maximal inhibitory concentration.
Fig. 4.
Fig. 4.
In vivo survival study of mice treated with 680 μM etoposide or phosphate buffered saline (controls) for 7 days utilizing CED. Pumps were implanted and treatment was initiated when animals reached 106 luciferase bioluminescent (p/sec/cm2) signaling. (A) Kaplan–Meier plot shows that survival of mice bearing PDGF+PTEN−/−p53−/− cell-based tumors treated with etoposide (n = 12) is significantly prolonged compared with the control group (n = 9) (log-rank test P < .001). (B) PDGF+PTEN−/−p53−/− tumors treated with etoposide showed lower bioluminescence signal than that seen in control tumors. Survival was measured starting at day of treatment with i.c. implantation of CED pump.
Fig. 5.
Fig. 5.
Survival experiments for retrovirally induced PDGF+PTEN−/− PN gliomas treated with a 7-day course of etoposide CED shows a dose response relationship. (A) Retrovirally induced PDGF+PTEN−/− PN gliomas treated with 4 μM etoposide for 7 days upon reaching 106 bioluminescence (p/sec/cm2) for 2 consecutive imaging dates had no significant survival benefit over control mice (n = 30) (log-rank test P > .05), as demonstrated by the Kaplan–Meier plot (left), and no significant difference in luminescence (right). (B) Retrovirally induced PDGF+PTEN−/− PN gliomas treated with 80 μM etoposide for 7 days had a significant survival benefit over control mice (n = 28) (log-rank test P < .01), as demonstrated by the Kaplan–Meier plot (left), and a significantly lower luminescence signal following treatment. *Represents significant difference in bioluminescence between groups (P < .05).

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