3035 - Optimizing Spatial Fractionation with Sensitizing Agents and Stromal Cell Immune Function

Purpose/Objective(s): Spatially fractionated radiation therapy (SFRT) is the administration of heterogeneous doses in a peak (high dose) and valley (low dose) pattern and is normal tissue sparing. We hypothesized that radiosensitizing agents would increase direct and bystander cell killing in valley dose regions and contribute to improved effectiveness of adding SFRT to conventional therapy regimens.

Materials/Methods: Murine SCCVII squamous cell carcinoma cells or 2H11 murine endothelial cells were plated in 24 well plates and after overnight incubation were treated with 5 Gy broad beam (BB) or using a honeycomb pattern collimator with peak dose areas of 1.7 mm in diameter and center to center distance of 2.5 mm. The tumor cells were also treated with hyperthermia or DNA protein kinase inhibitor (small molecule, DA-143) just before irradiation. For the hyperthermia experiment, plates were sealed and heated in a 42 °C water bath for one hour. For the DNA PK inhibitor groups, 0 (DMSO), 0.1, or 1 µM of DA-143 was added 30 minutes before radiation. The plates were then irradiated with 5 Gy broad beam or 5 Gy equivalent GRID (peak dose of ~10 Gy and valley dose of ~1.0 Gy). At 2.5 h after radiation treatment, cells were trypsinized, counted and re-plated in sparse numbers with or without the same concentration of inhibitor in 6 well plates and allowed to form single cell colonies for 6 days before staining and subsequent counting of colonies. A portion of the harvested cells were collected and lysed for qPCR gene expression analysis of the adhesion molecule ICAM-1.

Results: 42 °C for one hour had a minimal effect on clonogenic survival, reducing survival by only 12%. However, when combined with radiation, mild hyperthermia enhanced the normalized radiation-induced cell killing effect by 1.7-fold (5 Gy BB) and 1.2-fold (5 Gy GRID).Thus far in our studies, DNA-PK inhibitor DA-143 was tested at valley dose (1.1 Gy), mean dose (5 Gy), and peak dose (13.1 Gy) levels. Treatment with 0.1 µM DA-143 increased radiation-induced cell killing by 1.9-fold at 1.1 Gy and 1.7-fold at 5 Gy and eliminated all colonies at 13.1 Gy. The endothelial cells demonstrated increased ICAM1 expression after both types of radiation exposure, with maximal induction of ICAM-1 expression after a 5 Gy GRID exposure. However, tumor cells responded to GRID or BB irradiation with decreased ICAM-1 expression.

Conclusion: Hyperthermia or DNA-PK inhibition may sensitize tumor cells treated with SFRT depending on peak or valley dose level. The expression of ICAM-1 by endothelial cells appears to be maximally induced by GRID exposure compared to conventional BB, suggesting beneficial effects on immune system activity. Further work to optimize sensitizing drugs in combination with GRID and to employ GRID as a primer for improved immune response against irradiated tumors is ongoing in our laboratory.