2803 - Clonogenic Cell Survival Following In Vitro Irradiation Using Open vs. 2-Dimensional GRID Field Spatially Fractionated Radiotherapy of Murine Lymphoma, Breast and Renal Carcinoma

H. R. Han¹, V. Pachipulusu², H. Zhang³, Y. Zhu², A. L. Epstein², and L. Lukas³; ¹Department of Radiation Oncology, Los Angeles General Medical Center, Los Angeles, CA, ²Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, ³Department of Radiation Oncology, Keck School of Medicine, University of Southern California, Los Angeles, CA

Purpose/Objective(s): In vitro cell survival analysis of different cell lines following spatially fractionated radiotherapy using 2-dimensional GRID collimators allows investigation of cell death that may result from local bystander effects without immune responses that occur in vivo. We aim to compare GRID and open field irradiation for in vitro killing of cancer cell lines of varying radiosensitivity. Materials/

Methods: Clonogenic assays of murine 4T1 breast cancer, Renca renal adenocarcinoma, and A20 B cell lymphoma cell lines were performed in 6-well plates. Each well was seeded with 1000 cells and incubated overnight. The following day, the cells were irradiated with 250 kV x-rays in open or GRID (3mm thick brass plate, uniform 2 mm apertures) fields. Radiation doses were 1, 2, 3, 5, 7, 10, 12, 15 Gy for 4T1 and Renca and these doses with additional 0.3 and 0.5 Gy for A20 cells. Three samples of each cell type were irradiated with each radiation dose for each field. Both unirradiated control and irradiated cell plates were incubated for 5-7 days before manual counting of visible cell colonies under the microscope. The means of three samples for each dose and field were calculated. The percentage of colony survival following irradiation was compared to that of unirradiated control. Two-tailed nested t test was used to compare the means of colony survival by open vs GRID fields for each cell line.
Results: Across 8 total doses from 1 to 15 Gy, 4T1 (p=0.62) and Renca (p=0.41) cells showed no statistically significant colony survival differences between open and GRID fields. Across 10 total doses from 0.3 to 15 Gy, A20 cells showed no statistically significant difference between open and GRID fields, p=0.18. For all three cell lines, GRID resulted in fewer surviving colonies in low doses < 1 Gy, while > 1 Gy yielded fewer colonies with open field. For 4T1, there were <10% of surviving colonies after irradiation > 7 Gy using both open and GRID fields. For Renca, there were <10% of surviving colonies following > 7 Gy using open field, but colonies survived up to 12 Gy with GRID field. For A20, there were little to no colonies following > 3 Gy using open field and > 5 Gy using GRID. A20 was most sensitive to low doses of radiation, with surviving colonies down to < 60% at even the lowest 0.3 Gy using both fields.
Conclusion: The difference in cell survival following open field vs GRID radiotherapy was not statistically significant across all three cell lines despite differences in the radiosensitivity of these cells. Further studies are warranted to investigate the mechanism of bystander effects, such as cytokines or cytotoxic contents released by irradiated cells that affect nearby unirradiated cells, that might have contributed to the cell-killing comparability of GRID with open field radiotherapy.