Long-term treatment with valganciclovir improves lentiviral suicide gene therapy of glioblastoma

Abstract

Background: Suicide gene therapy for malignant gliomas has shown encouraging results in the latest clinical trials. However, prodrug application was most often restricted to short-term treatment (14 days), especially when replication-defective vectors were used. We previously showed that a substantial fraction of herpes simplex virus thymidine kinase (HSV-TK) transduced tumor cells survive ganciclovir (GCV) treatment in an orthotopic glioblastoma (GBM) xenograft model. Here we analyzed whether these TK+ tumor cells are still sensitive to prodrug treatment and whether prolonged prodrug treatment can enhance treatment efficacy.

Methods: Glioma cells positive for TK and green fluorescent protein (GFP) were sorted from xenograft tumors recurring after suicide gene therapy, and their sensitivity to GCV was tested in vitro. GBM xenografts were treated with HSV-TK/GCV, HSV-TK/valganciclovir (valGCV), or HSV-TK/valGCV + erlotinib. Tumor growth was analyzed by MRI, and survival as well as morphological and molecular changes were assessed.

Results: TK-GFP+ tumor cells from recurrent xenograft tumors retained sensitivity to GCV in vitro. Importantly, a prolonged period (3 mo) of prodrug administration with valganciclovir (valGCV) resulted in a significant survival advantage compared with short-term (3 wk) application of GCV. Recurrent tumors from the treatment groups were more invasive and less angiogenic compared with primary tumors and showed significant upregulation of epidermal growth factor receptor (EGFR) expression. However, double treatment with the EGFR inhibitor erlotinib did not increase therapeutic efficacy.

Conclusion: Long-term treatment with valGCV should be considered as a replacement for short-term treatment with GCV in clinical trials of HSV-TK mediated suicide gene therapy.

Keywords: EGFR; brain tumors; glioblastoma; lentiviral vectors; suicide gene therapy.

Fig. 1

TK+ glioma cells that survive short-term prodrug administration retain sensitivity to GCV in vitro. (A) Immunofluorescence staining with antibodies against human nestin (red) and GFP (green). Scale bar: 50 μm. (B) Cell viability assay showing percent of surviving cells after increasing concentrations of GCV; i.v. TK+: The TK− cells present in the recurrent tumor were transduced with the same lentiviral vector in vitro.

Fig. 2

Long-term administration of valGCV is more efficient compared with short-term GCV treatment in vivo. (A) Representative MRI (T2 rapid acquisition with relaxation enhancement) images are shown. The solid tumor regions are marked with dashed lines. (B) Kaplan–Meier survival analysis: 3-month valGCV treatment has a significant survival benefit compared with the 3-week GCV treatment (P = 0.008).

Fig. 3

Continuous valGCV application eliminates the majority of TK+ tumor cells. (A) Hematoxylin and eosin stainings of recurrences (left panel: recurrence at the primary site in GCV treatment group; right panel: recurrence distant from the primary site in valGCV treatment group). (B) Immunohistochemistry with antibodies for HSV-TK show transduced cells in the recurrent tumor mass. (C) Double immunofluorescent staining using antibodies against human nestin (green) and GFP (red) for the TK.007-eGFP fusion protein. Representative images show TK.007-eGFP+ tumor cells that survived prodrug treatment. Scale bar: 50 μm. (D) Quantification of the remaining tumor cells expressing TK.007-eGFP: percentage of residual TK+ tumor cells in 3-month-long valGCV-treated group is lower compared with the short-term GCV-treatment group (P = 0.042). (E) Proliferative capacity of the remaining TK+ cells is significantly lower compared with TK− cells (P = 0.041) in the TK + GCV group.

Fig. 4

Recurrent tumors after gene therapy are more invasive and less angiogenic compared with primary tumors. (A) Immunostaining with human nestin antibody showing extensive infiltration of glioma cells into corpus callosum in local and distant recurrent tumors, but not in the primary tumor. (B) Quantification of invasive tumor cells outside the solid tumor mass in control tumors and local recurrences (TK + GCV). (C) Hematoxylin and eosin stainings of primary and recurrent tumors showing necroses and microvascular proliferation only in the necrotic tumors. (D) Von Willebrand factor immunostaining reveals reduced angiogenesis in recurrent tumors compared with primary tumors. Scale bar: 50 μm. (E, F) Quantification of vessel area fraction among the groups. Overall recurrent tumors have significantly reduced vessel area compared with primary tumors (P = 0.001).

Fig. 5

EGFR is upregulated in recurrent tumors after suicide gene therapy. Immunohistochemical stainings with antibodies against EGFR (A), PDGFR-A (C), pSTAT3 (E), and Ki-67 (G). Quantification showing percentage of EGFR+ (B), PDGFR-A+ (D) area fraction, and pSTAT3+ cells (F). Scale bar: 50 μm. (H) Quantification of Ki-67+ cells; mean ± SD.

Fig. 6

Combination of suicide gene therapy with erlotinib does not improve therapeutic efficacy. (A) Kaplan–Meier survival analysis does not show a significant difference between double treatment and single treatment arms. (B) Immunohistochemical stainings with antibodies against pEGFR 1068. Scale bar: 50 μm. (C) Quantification showing percentage of pEGFR 1068+ area fraction. (D, E) Immunohistochemical stainings with antibodies against EGFR and quantification of EGFR+ area fraction.