Improved killing of human high-grade glioma cells by combining ionizing radiation with oncolytic parvovirus H-1 infection

Abstract

Purpose: To elucidate the influence of ionizing radiation (IR) on the oncolytic activity of Parvovirus H-1 (H-1PV) in human high-grade glioma cells.

Methods: Short term cultures of human high-grade gliomas were irradiated at different doses and infected with H-1PV. Cell viability was assessed by determining relative numbers of surviving cells. Replication of H-1PV was measured by RT-PCR of viral RNA, fluorescence-activated cell sorter (FACS) analysis and the synthesis of infectious virus particles. To identify a possible mechanism for radiation induced change in the oncolytic activity of H-1PV we performed cell cycle analyses.

Results: Previous irradiation rendered glioma cells fully permissive to H-1PV infection. Irradiation 24 hours prior to H-1PV infection led to increased cell killing most notably in radioresistant glioma cells. Intracellular levels of NS-1, the main effector of H-1PV induced cytotoxicity, were elevated after irradiation. S-phase levels were increased one day after irradiation improving S-phase dependent viral replication and cytotoxicity.

Conclusion: This study demonstrates intact susceptibility of previously irradiated glioma-cells for H-1PV induced oncolysis. The combination of ionizing radiation followed by H-1PV infection increased viral cytotoxicity, especially in radioresistant gliomas. These findings support the ongoing development of a clinical trial of H-1PV in patients with recurrent glioblastomas.

Figure 1

Effects of late parvovirus H-1 (H-1PV) infection on human high-grade glioma cells surviving ionizing radiation (IR). Short-term cultures of human gliosarcoma NCH-37, human glioblastoma NCH-82, and human glioblastoma NCH-89 were seeded at 30,000 cells/well, irradiated with 10 Gy and infected with H-1PV at an MOI of 5 PFU/cell 9 days post-IR (MOI 5) and compared to mock-infected cells surviving IR (MOCK). The short-term culture of in vivo irradiated recurrent glioblastoma NCH-307 was seeded at 30,000 cells/well and infected with H-1PV at low (MOI = 5 PFU/cell: MOI 5) or high (MOI = 100 PFU/cell: MOI 100) virus doses and compared to mock-infected cells. All experiments were performed in three independent assays. Viability (%) was assessed as the number of living treated cells over the number of living untreated cells three days post (mock-) infection. Error bars represent the respective standard error. (*) indicates significant differences (P < .001) between the number of living infected and the corresponding number of living MOCK-infected cells.

Figure 2

Effects of ionizing radiation (IR), parvovirus H-1 (H-1PV) infection, and combination of IR and H-1PV infection on human high-grade glioma cells. Short-term cultures of human gliosarcoma NCH-37 (a), human glioblastoma NCH-82 (b), and human glioblastoma NCH-89 (c) were seeded at 30,000 cells/well, irradiated with 5 Gy, 10 Gy, or 20 Gy, and infected with H-1PV at an MOI of 5 PFU/cell 24 hours post-IR (IR→H-1) or cells were infected with H-1PV at an MOI of 5 PFU/cell and irradiated with 10 Gy 24 hours post-infection (H-1→IR). Effects on cell survival were compared to single treatment with IR or single treatment with H-1PV. Control cells were mock-infected and transported to the accelerator but not exposed to IR (0 Gy). All experiments were performed in three independent assays. Viability (%) was assessed as the number of living treated cells over the number of living untreated cells three days post (mock-) infection. Error bars represent the respective standard error. Significant differences (P < .05) between single treatment and combination treatment groups are indicated by brackets.

Figure 3

Long-term effect of combined ionizing radiation (IR) and parvovirus H-1 (H-1PV) infection on human high-grade glioma cells. Short-term cultures of human glioblastoma NCH-89 were irradiated with 20 Gy, and infected with H-1PV at an MOI of 5 PFU/cell 24 hours post-IR. 3-week postseeding photographs of culture dishes were taken at a magnification of 400× and no surviving cells could be found (a). In comparison, after mono treatment with H-1PV at an MOI of 5 PFU/cell or after mono treatment with IR (data not shown), proliferating cell clones could be observed (b).

Figure 4

Replication of parvovirus H-1 (H-1PV) in irradiated human high-grade glioma cells. FACS analysis of intracellular cytotoxic parvoviral protein NS-1 in short-term cultures of human gliosarcoma NCH-37 (a), human glioblastoma NCH-82 (b), and human glioblastoma NCH-89 (c) after infection with H-1PV. To analyse the influence of radiation therapy, glioma cells were irradiated with 10 Gy or transported to the accelerator but not exposed to IR (0 Gy). For early infection experiments, cells were infected at an MOI of 5 PFU/cell 24 hours post-IR (MOI 5-E) or mock-infected (MOCK); for late infection experiments cells were infected at an MOI of 5 PFU/cell 9 days post-IR (MOI 5-L). The short-term culture of in vivo irradiated recurrent glioblastoma NCH-307 (d) was infected with an MOI of 5 PFU/cell (MOI5) or mock-infected (MOCK). All cell cultures were harvested 24 hours p.i. and the percentage of cells positive for intracellular NS-1 was determined by FACS-analysis. (e) Detection of H-1PV RNA by RT-PCR. All cell lines except for NCH-307 were irradiated with 10 Gy. 24 hours post-IR and 24 postseeding for NCH-307, respectively, cells were infected with H-1PV at an MOI of 5 PFU/cell (MOI5). RNA was isolated 24 hours p.i. amplified by RT-PCR and compared with RNA of mock-infected cells (MOCK). For positive control, RNA of H-1PV infected unirradiated highly susceptible RG2 rat glioma cells was used.

Figure 5

Effects of ionizing radiation (IR) on the cell cycle of human high-grade glioma cells. Short-term cultures of human gliosarcoma NCH-37, human glioblastoma NCH-82, and human glioblastoma NCH-89 were irradiated with 10 Gy, and cell cycle analyses were performed 24 hours post-IR (a, middle column) or 48 hours post-IR (a, right column). Control cells (a, left column) were transported to the accelerator but not exposed to IR (ctrl.). (b) IR induced increase of S-Phase fraction in percent of unirradiated control cells.