Ultra high dose rate (35 Gy/sec) radiation does not spare the normal tissue in cardiac and splenic models of lymphopenia and gastrointestinal syndrome

Abstract

Recent reports have shown that very high dose rate radiation (35-100 Gy/second) referred to as FLASH tends to spare the normal tissues while retaining the therapeutic effect on tumor. We undertook a series of experiments to assess if ultra-high dose rate of 35 Gy/second can spare the immune system in models of radiation induced lymphopenia. We compared the tumoricidal potency of ultra-high dose rate and conventional dose rate radiation using a classical clonogenic assay in murine pancreatic cancer cell lines. We also assessed the lymphocyte sparing potential in cardiac and splenic irradiation models of lymphopenia and assessed the severity of radiation-induced gastrointestinal toxicity triggered by the two dose rate regimes in vivo. Ultra-high dose rate irradiation more potently induces clonogenic cell death than conventional dose rate irradiation with a dose enhancement factor at 10% survival (DEF₁₀) of 1.310 and 1.365 for KPC and Panc02 cell lines, respectively. Ultra-high dose rate was equally potent in depleting CD3, CD4, CD8, and CD19 lymphocyte populations in both cardiac and splenic irradiation models of lymphopenia. Radiation-induced gastrointestinal toxicity was more pronounced and mouse survival (7 days vs. 15 days, p = 0.0001) was inferior in the ultra-high dose rate arm compared to conventional dose rate arm. These results suggest that, contrary to published data in other models of radiation-induced acute and chronic toxicity, dose rates of 35 Gy/s do not protect mice from the detrimental side effects of irradiation in our models of cardiac and splenic radiation-induced lymphopenia or gastrointestinal mucosal injury.

Figure 1

Ultra-high dose rate (35 Gy/s) RT is more potent than conventional dose rate (0.1 Gy/s) RT in killing tumor cells, with no appreciable difference in modes of cell death. (a) Clonogenic survival curve of KPC cells treated with ultra-high dose rate shows enhancement factor at 10% surviving fraction (DEF₁₀) of 1.310 compared to conventional dose rate RT(0,2,4,6,8 Gy of radiation) (b) Clonogenic survival curve of Panc02 cells treated with ultra-high dose rate shows enhancement factor at 10% surviving fraction (DEF₁₀) of 1.365 compared to conventional dose rate RT(0,2,4,6,8 Gy of radiation) (c,d) The percentage of PBMCs undergoing early apoptosis, late apoptosis, and necrosis at 24 h and 72 h following ultra-high and conventional dose rate RT. Dose enhancement ratio at survival fraction (DER_SF0.1) of 10% was calculated by (radiation dose needed to kill 90% at high dose-rate)/(radiation dose needed to kill 90% with conventional dose rate). The radiation dose was calculated from the linear quadratic model based on the survival fraction at each dose. Data are derived from experiments conducted in sextuplicate.

Figure 2

BALB/c mice undergoing cardiac irradiation with multi-fraction regimen of 2 Gy per day for 5 days or 10 Gy single fraction develop severe lymphopenia irrespective of dose rate. (a) Time trends of flow cytometric CD3, CD4, CD8, and CD19 lymphocyte counts in the peripheral blood of mice undergoing conventional dose rate RT with the multi-fraction regimen. (b) Time trends of flow cytometric CD3, CD4, CD8, and CD19 lymphocyte counts in the peripheral blood of mice undergoing ultra-high dose rate RT with the multi-fraction regimen. (c) Flow cytometric CD3, CD4, CD8, and CD19 counts in the peripheral blood of mice on day 3 following conventional dose rate RT with the single-fraction regimen of 10 Gy. (d) Flow cytometric CD3, CD4, CD8, and CD19 counts in the peripheral blood of mice on day 3 following ultra-high dose rate RT with the single-fraction regimen of 10 Gy. Data are the mean percentages of cells ± SE. *p < 0.05 compared between control and other cohorts. Data are derived from experiment conducted in triplicates. (n = 5 in control, high dose rate and conventional dose rate group).

Figure 3

C57BL/6 mice undergoing splenic irradiation with a multi-fraction regimen of 1 Gy per day for 5 days or a single fraction of 5 Gy experience severe lymphopenia irrespective of dose rate. (a–d) Flow cytometric CD3, CD4, CD8, and CD19 counts in the peripheral blood of mice on day 3 following conventional or ultra-high dose rate RT with the multi-fraction regimen. (e–h) Flow cytometric CD3, CD4, CD8, and CD19 counts in the peripheral blood of mice on day 3 following conventional or ultra-high dose rate RT with a single fraction of 5 Gy. Data are the mean percentages of cells ± SE. *p < 0.05 compared between control and other cohorts. Data are derived from experiment conducted in triplicates. (n = 5 in control, high dose rate and conventional dose rate group).

Figure 4

Ultra-high dose rate (35 Gy/s) RT is more potent than conventional dose rate (0.1 Gy/s) RT in inducing acute gastrointestinal syndrome after a single fraction of 16 Gy of whole abdominal radiation. (n = 5 in high dose rate and conventional dose rate group). The Kaplan Meier curve for the survival data, was determined by the log-rank test. Results with a P value of <0.05 were considered significant.