The timing of neural stem cell-based virotherapy is critical for optimal therapeutic efficacy when applied with radiation and chemotherapy for the treatment of glioblastoma

Abstract

Glioblastoma multiforme (GBM) remains fatal despite intensive surgical, radiotherapeutic, and chemotherapeutic interventions. Neural stem cells (NSCs) have been used as cellular vehicles for the transportation of oncolytic virus (OV) to therapeutically resistant and infiltrative tumor burdens throughout the brain. The HB1.F3-CD human NSC line has demonstrated efficacy as a cell carrier for the delivery of a glioma tropic OV CRAd-Survivin-pk7 (CRAd-S-pk7) in vitro and in animal models of glioma. At this juncture, no study has investigated the effectiveness of OV-loaded NSCs when applied in conjunction with the standard of care for GBM treatment, and therefore this study was designed to fill this void. Here, we show that CRAd-S-pk7-loaded HB1.F3-CD cells retain their tumor-tropic properties and capacity to function as in situ viral manufacturers in the presence of ionizing radiation (XRT) and temozolomide (TMZ). Furthermore, for the first time, we establish a logical experimental model that aims to recapitulate the complex clinical scenario for the treatment of GBM and tests the compatibility of NSCs loaded with OV. We report that applying OV-loaded NSCs together with XRT and TMZ can increase the median survival of glioma bearing mice by approximately 46%. Most importantly, the timing and order of therapeutic implementation impact therapeutic outcome. When OV-loaded NSCs are delivered prior to rather than after XRT and TMZ treatment, the median survival of mice bearing patient-derived GBM43 glioma xenografts is extended by 30%. Together, data from this report support the testing of CRAd-S-pk7-loaded HB1.F3-CD cells in the clinical setting and argue in favor of a multimodality approach for the treatment of patients with GBM.

Keywords: Adenovirus; Chemotherapy; Gene therapy; Glioma; Neural stem cell; Radiation; Temozolomide; Virotherapy.

Figures

Figure 1.

Characterization of surface markers and migration of loaded NSCs treated with XRT-TMZ. (A): Surface marker expression of irradiated and chemotherapy-treated NSCs at 24 hours as analyzed by fluorescence-activated cell sorting (FACS). Shown are representative FACS plots (left) and the percentage of positive and mean fluorescent intensity of the surface markers of untreated compared with treated neural stem cells (right). (B): Transcription level of surface receptors associated with NSC migration at 12 and 24 hours after XRT-TMZ treatment. Relative mRNA transcripts were analyzed by quantitative real-time polymerase chain reaction and were compared with untreated NSCs. (C): Functional migration of XRT-TMZ-treated NSCs at 48 hours after treatment. The percentage of distance change was greater for XRT-TMZ-treated NSCs than untreated control NSCs. *, p < .05; ***, p < .001. Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; NSC, neural stem cell; PE, phosphatidylethanolamine; Rx, radiation therapy-temozolomide therapy; SSC, side scatter; TMZ, temozolomide; uPAR, urokinase plasminogen activator receptor; VEGFR, vascular endothelial growth factor receptor; XRT, radiation therapy.

Figure 2.

Evaluation of CRAd-Survivin-pk7 (CRAd-S-pk7) replication in neural stem cells (NSCs) treated with XRT-TMZ. Viral replication of CRAd-S-pk7 was measured by quantitative real-time polymerase chain reaction and presented as a number of viral E1A copies per nanogram of DNA from infected NSCs. (A, B): Viral replication was evaluated daily up to 96 hours after treatment with 0, 10, 50, or 100 μM TMZ (A) and 0, 2, or 4 Gy of XRT (B). (C): CRAd-S-pk7 viral titer levels 96 hours after XRT-TMZ treatment of infected NSCs. Treatment with both XRT and TMZ slightly reduced viral titer levels at high doses of TMZ, but no change was observed when treated with TMZ concentrations closer to physiologically relevant levels. *, p < .05. Abbreviations: DMSO, dimethyl sulfoxide; IU, infectious units; TMZ, temozolomide; XRT, radiation therapy.

Figure 3.

Antitumor effects of CRAd-Survivin-pk7 (CRAd-S-pk7)-loaded NSCs and their combination with XRT-TMZ against glioma cell lines in vitro. (A): Cytotoxicity of patient-derived GBM43 tumor cells 96 hours after coculture with CRAd-S-pk7-loaded NSCs at the NSC to GBM43 cell ratios of 1:0, 1:2, 1:5, 1:10, or 1:50. Top: Representative light microscope pictures of GBM43 viability. Bottom: Mean luciferase intensity values represented as the percentages of viable glioma cells compared with control. (B): U251 and U87 glioma cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 96 hours after treatment. The addition of CRAd-S-pk7 (50 infectious units) to conventional XRT-TMZ therapy reduced the percentage of glioma cell viability in both tested cell lines. The IC50 values of TMZ for U251 and U87 cells when treated with XRT-TMZ decreased by 31 and 15 μM, respectively, when OV was added. **, p < .01; ***, p < .001. Abbreviations: NSC, neural stem cell; OV, oncolytic virus; TMZ, temozolomide; XRT, radiation therapy.

Figure 4.

In vivo efficacy of CRAd-Survivin-pk7 (CRAd-S-pk7)-loaded NSCs and XRT-TMZ treatment against human-derived glioma xenografts. Intracranial GBM43 (3.5 × 105 cells per animal) was established, and the animals were treated for 5 consecutive days beginning on day 6 after tumor cell implantation. (A): Survival of animals treated with escalating doses of intraperitoneally administered TMZ (0, 5, 10, or 30 mg/kg). (B): Survival of animals treated with XRT (2 Gy) or a combination of XRT (2 Gy) and TMZ (2.5, 5, 10, or 30 mg/kg). (C): Survival of animals treated with the optimized dose of 2 Gy XRT and 5 mg/kg TMZ in addition to 5 × 105 or 3 × 106 NSCs loaded with 50 infectious units of CRAd-S-pk7. The addition of 5 × 105 or 3 × 106 loaded NSCs to XRT-TMZ treatment increased the median survival of glioma-bearing mice by 7 and 11 days, respectively. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: ND, not determined; ns, no significance; NSC, neural stem cell; TMZ, temozolomide; XRT, radiation therapy.

Figure 5.

Optimization of combination therapy in vitro. (A): Cytotoxicity of U251 and U87 glioma cell lines and GBM39 patient-derived cell line treated with Rx-OV or OV-Rx. Left: The percentage of viability of glioma cells measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 96 hours after treatment. Right: Representative light microscope pictures of U87 glioma cell viability (magnification, ×10). (B): Percentage of apoptotic GBM43 cells at 48 hours after treatment with Rx-OV or OV-Rx treatment protocols as measured by the expression of active caspase-3-positive tumor cells by FACS (bottom). Top: Representative FACS plots. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: DMSO, dimethyl sulfoxide; OV-Rx, ionizing radiation-temozolomide therapy 24 hours after oncolytic virus; PE, phycoerythrin; Rx-OV, oncolytic virus 24 hours after ionizing radiation-temozolomide therapy.

Figure 6.

Scheduling of CRAd-Survivin-pk7 (CRAd-S-pk7)-loaded NSC administration in vivo. (A): Survival of animals treated with both therapeutic scheduling protocols. Intracranial GBM43 (3.5 × 105 cells per animal) was established, and the animals received an intratumoral (IT) injection of loaded NSCs (5 × 105) on day 5 followed by 5 consecutive days of XRT-TMZ (2 Gy and 5 mg/kg) therapy beginning on day 6 or alternatively XRT-TMZ therapy starting on day 6 for 5 consecutive days followed by an IT injection of loaded NSCs on day 12. A 9-day preferential median survival was observed in mice that received upfront NSC-based oncolytic therapy. (B): Fluorescent microscopy of mouse brain tissue bearing GBM43 xenografts (left). Top: Anti-cleaved caspase-3 (green). Bottom: Overlay; anti-cleaved caspase-3 (green) and anti-4′,6-diamidino-2-phenylindole (blue). Magnification, ×20. Scale bar = 50 μm. For each treatment group, five images were taken using the ×20 objective, and the number of positive cells was quantified per field of view (right). *, p < .05; **, p < .01; ***, p < .001. Abbreviations: FOV, field of view; NSC, neural stem cell; OV-Rx, XRT-TMZ therapy 24 hours after oncolytic virus-loaded NSCs; Rx-OV, oncolytic virus-loaded NSCs 24 hours after XRT-TMZ therapy; TMZ, temozolomide; XRT, radiation therapy.

Figure 7.

Radiosensitizing effect of CRAd-Survivin-pk7 (CRAd-S-pk7) infection on glioma. (A): Protein expression of the Mre11-Rad50-NBS1 complex proteins Rad50 (153 kDa) and Mre11 (81 kDa) at 12, 24, 36, and 48 hours after infection with 50 infectious units of CRAd-S-pk7 or ONYX-015. Western blots show that Rad50 and Mre11 protein expression are reduced at both 36 and 48 hours after infection with CRAd-S-pk7 but not ONYX-015. (B): Immunofluorescent staining of radiation induced γH2AX foci under a confocal laser microscope. Top: Anti-γH2AX (green); bottom: Dapi (blue). Magnification, ×63. Scale bar = 20 μm. (C): Quantification of γH2AX foci resolution over 72 hours after XRT treatment. The number of γH2AX foci per cell was counted and grouped according to the following range of foci per cell: 0–50 (red arrows), 51–100 (yellow arrows), 101–200 (blue arrows), and >200 (orange arrows). Left: Time effect was determined by ordinal logistic regression analysis. The number of γH2AX foci was significantly resolved over time in XRT-OV-treated cells (p = .020), whereas there was no significant change in the number of foci over time in OV-XRT-treated cells (p = .386). Right: Representative overlay images of each range of foci per cell (anti-γH2AX, green, and anti-DAPI, blue). Magnification, ×63. Scale bar = 20 μm. Abbreviations: Dapi, 4′,6-diamidino-2-phenylindole; OV-XRT, radiation therapy 24 hours after oncolytic virus; XRT-OV, oncolytic virus 24 hours after radiation therapy.