New approaches for studying radiobiological effects of kilovoltage X-rays in vivo and in vitro

Introduction: X-ray microbeam radiation therapy (MRT) as a novel tumor treatment strategy deposits high doses in spatially fractionated X-ray beamlets promising reduced normal tissue toxicity, compared to conventional irradiation, and a better tumor control. Radiobiological studies of MRT with kilovoltage X-rays are mainly performed at synchrotron radiation facilities with high costs and space requirements. The Munich Compact Light Source (MuCLS) is a laboratory-sized and cost-effective source based on inverse Compton scattering of infrared laser photons [1]. Currently the most widely accepted method for assessment of treatment efficiencies is tumor growth delay with subcutaneous tumors in the hind leg of small animals. However, a new model is required for MRT with kilovoltage X-ray beams which only allow for short penetration depths. Therefore, we successfully developed a setup for a growth delay study in a tumor-bearing mouse ear model for investigation of MRT at the MuCLS. In addition, we successfully established a protocol to isolate tumor cells from irradiated tumors for evaluation of radiobiological effect on cellular level.
Materials & Methods: The dose rate of the MuCLS was improved with the installation of a polycapillary collimation optic. A W-Air collimator was inserted to get a collimated X-ray beam with 50 μm wide microbeams and a center-to-center distance of 350 μm. We implemented the mouse ear tumor model with a human head and neck cancer cell line FaDu [2] suspended in extracellular matrix and subcutaneously injected into the right ear of NMRI (nu/nu) mice. After reaching a size of 2x2 mm2 tumors were irradiated using doses of either 3 and 5 Gy with 25 keV X-rays at the MuCLS. Tumor growth delay was determined with a caliper over a follow-up period of 30 days and compared between MRT, homogeneous and control mice. Animals were sacrificed when tumors reached the 15-fold initial volume. A single tumor cell suspension was prepared from excised tumors for in-vitro studies. The analysis of radiosensitivity by colony formation assay and stable chromosomal aberrations by two-color fluorescence in-situ hybridization is still on-going.
Results: We successfully installed a setup at the MuCLS which allows irradiation of tumors in small animals and implemented a xenograft tumor model in mouse ears. Homogeneously irradiated tumors showed a growth delay at 5 Gy compared to control mice. There was no tumor growth delay after MRT and homogeneous irradiation at 3 Gy. Tumor cells from irradiated tumors were successfully isolated and cultured. Preliminary data shows an increased radiosensitivity of tumor cells originating from homogeneously and MRT-irradiated tumors compared to control tumor cells.
Conclusion: This innovative approach allowed the irradiation of tumors in a mouse ear model at a novel laser-based X-ray source, the MuCLS. Homogeneous irradiation at MuCLS induced a tumor growth delay at 5 Gy. In addition, we successfully validated a protocol for tumor cell isolation for investigations of radiation-induced effects.
Supported by the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics.
References:
[1] Eggl et al., J. Synchrotron Rad. (2016) 23: 1137
[2] Beyreuther et al., PLos One (2017)