The effects of extra high dose rate irradiation on glioma stem-like cells

Abstract

Radiation therapy is an integral part of treatment for patients with glioblastoma. New technological advances in linear accelerators have made extra-high dose rate irradiation possible. This shortens patient treatment time significantly compared to standard dose rate irradiation, but the biologic effects of extra high dose rate irradiation are poorly understood. Glioma stem-like cells (GSCs) are resistant to standard radiation and contribute to tumor progression. Here, we assess the therapeutic effect of extra high dose rate vs. standard dose rate irradiation on GSCs. GSCs were exposed to 2, 4 and 6 Gy X-irradiation at dose rates of 4.2 Gy/min or 21.2 Gy/min (400 monitoring units (MU)/min or 2100 MU/min). We analyzed cell survival with cell growth assays, tumorsphere formation assays and colony formation assays. Cell kill and self-renewal were dependent on the total dose of radiation delivered. However, there was no difference in survival of GSCs or DNA damage repair in GSCs irradiated at different dose rates. GSCs exhibited significant G1 and G2/M phase arrest and increased apoptosis with higher doses of radiation but there was no difference between the two dose rates at each given dose. In a GSC-derived preclinical model of glioblastoma, radiation extended animal survival, but there was no difference in survival in mice receiving different dose rates of radiation. We conclude that GSCs respond to larger fractions of radiation, but extra high dose rate irradiation has no significant biologic advantage in comparison with standard dose rate irradiation.

Fig 1. Irradiation set-up and cell viability assay.

A. The cell culture dish (red arrow) was placed on solid water to improve dosimetry. B. The cell culture dish was placed at the field center and its position confirmed with crosshairs and the light field. C. Solid water bolus of 1 cm thickness was placed over the cell culture dish to improve dosimetry. (D) 387 GSCs, (E) 3359 GSCs and (F) 4121 GSCs were irradiated to doses of 2, 4 and 6 Gy X- irradiation at dose rates of 400 MU/min or 2100 MU/min. Data presented as mean ± SD. NS, not significant.

Fig 2. Tumorsphere formation of GSCs treated with different dose rates of radiation.

Representative bright field images of 387 GSCs (A), 3359 GSCs (C) and 4121 GSCs (E) irradiated at the indicated dose and dose rates (400 MU/min or 2100 MU/min). Quantification of the surviving fraction of 387 GSCs (B), 3359 GSCs (D) and 4121 GSCs (F) is shown. There was no statistically significant effect on tumorsphere formation between dose rates at each radiation dose level. Scale bar measures 1000 μm. NS, not significant.

Fig 3. Colony formation assay of GSCs treated with different dose rates of irradiation.

Bright field images of 387 GSCs (A), 3359 GSCs (C) and 4121 GSCs (E). Quantification of colony numbers in 387 GSCs (B), 3359 GSCs (D) and 4121 GSCs (F) is shown. Data presented as mean ± SD. *, p<0.05; NS, not significant.

Fig 4. γ-H2AX foci formation in GSCs.

(A) Representative immunofluorescence images of 387 GSCs harvested at 30 min, 24 hrs and 48 hours after irradiation with the indicated dose and dose rates. (B) Quantification of γ-H2AX foci from three independent experiments presented as mean ± SD. There was no statistically significant difference in γ-H2AX foci between dose rates at each radiation dose level. Scale bar 20 μm. NS, not significant.

Fig 5. Cell cycle analysis and apoptosis marker expression in irradiated GSCs.

(A) 387 GSCs, (B) 3359 GSCs and (C) 4121 GSCs were irradiated to doses of 2, 4 and 6 Gy X-ray at dose rates of 400 MU/min or 2100 MU/min. Cells were collected and fixed with ethanol 24 hrs after irradiation. Three independent experiments were performed for statistical analyses. Data presented as mean ± SD. Statistical significance was calculated in comparison with non-irradiated controls (*, p<0.05; **, p<0.01). There was no statistically significant effect in cell cycle phase between dose rates at each radiation dose level. Apoptosis markers cleaved-Parp and cleaved-Caspase 3 were detected in irradiated GSCs (D). 387, 3359 and 4121 GSCs were subjected to doses of 2, 4 and 6 Gy X-irradiation at dose rates of 400 MU/min or 2100 MU/min. Cells were collected and cell lysates were prepared 24 hrs after irradiation.

Fig 6. Radiation therapy increases the overall survival in an orthotopic GSC-derived model of glioblastoma.

Kaplan-Meier survival curve of animals treated with different dose rates of radiation or control. Mantel-cox test was used to analyze the data. Control N = 5; 1500 MU/min N = 6; 400 MU/min N = 5. Control V.S. irradiated: *, p<0.05; 1500 MU/min vs. 400 MU/min: NS, not significant.