Objectives: The present study aimed to compare the tumor growth delay between conventional radiotherapy (CRT) and the spatially fractionated modalities of microbeam radiation therapy (MRT) and minibeam radiation therapy (MBRT). In addition, we also determined the influence of beam width and the peak-to-valley dose ratio (PVDR) on tumor regrowth. Methods: A549, a human non-small-cell lung cancer cell line, was implanted subcutaneously into the hind leg of female CD1-Foxn1^nu mice. The animals were irradiated with sham, CRT, MRT, or MBRT. The spatially fractionated fields were created using two specially designed multislit collimators with a beam width of 50 μm and a center-to-center distance (CTC) of 400 μm for MRT and a beam width of 500 μm and 2000 μm CTC for MBRT. Additionally, the concept of the equivalent uniform dose (EUD) was chosen in our study. A dose of 20 Gy was applied to all groups with a PVDR of 20 for MBRT and MRT. Tumor growth was recorded until the tumors reached at least a volume that was at least three-fold of their initial value, and the growth delay was calculated. Results: We saw a significant reduction in tumor regrowth following all radiation modalities. A growth delay of 11.1 ± 8 days was observed for CRT compared to the sham, whereas MBRT showed a delay of 20.2 ± 7.3 days. The most pronounced delay was observed in mice irradiated with MRT PVDR 20, with 34.9 ± 26.3 days of delay. Conclusions: The current study highlights the fact that MRT and MBRT modalities show a significant tumor growth delay in comparison to CRT at equivalent uniform doses.

Microbeam radiation therapy (MRT) is a still pre-clinical form of spatially fractionated radiotherapy, which uses an array of micrometer-wide, planar beams of X-ray radiation. The dose modulation in MRT has proven effective in the treatment of tumors while being well tolerated by normal tissue. Research on understanding the underlying biological mechanisms mostly requires large third-generation synchrotrons. In this study, we aimed to develop a preclinical treatment environment that would allow MRT independent of synchrotrons. We built a compact microbeam setup for pre-clinical experiments within a small animal irradiator and present in vivo MRT application, including treatment planning, dosimetry, and animal positioning. The brain of an immobilized mouse was treated with MRT, excised, and immunohistochemically stained against $\gamma$H2AX for DNA double-strand breaks. We developed a comprehensive treatment planning system by adjusting an existing dose calculation algorithm to our setup and attaching it to the open-source software 3D-Slicer. Predicted doses in treatment planning agreed within 10% with film dosimetry readings. We demonstrated the feasibility of MRT exposures in vivo at a compact source and showed that the microbeam pattern is observable in histological sections of a mouse brain. The platform developed in this study will be used for pre-clinical research of MRT.

Background: Microbeam radiation therapy (MRT) is an advanced preclinical approach in radiotherapy that utilizes spatially fractionated dose distributions by collimating x-rays into micrometer-wide, planar beams. While the benefits of temporal fractionation are well established and widely incorporated into conventional radiotherapy protocols, the interplay between MRT and temporal dose fractionation remains largely unexplored. In this study, we investigate the effects of combining temporal and spatial dose fractionation by assessing clonogenic cell survival following temporally fractionated MRT with varying irradiation angles, compared to conventional broad-beam (BB) irradiation. Methods: A lung tumor cell line (A549) and a normal lung cell line (MRC-5) were irradiated with a total number of four fractions with a 24 h interval between each fraction. We compared a temporally fractionated BB regime to two temporally fractionated MRT schemes with either overlapping MRT fields or MRT fields with a 45° rotation per fraction. Subsequently, the clonogenic cell survival assay was used by analyzing the corresponding survival fractions (SFs). Results: The clonogenic survival of A549 tumor cells differed significantly between microbeam radiation therapy with rotation (MRT + R) and overlapping MRT. However, neither MRT + R nor overlapping MRT showed statistically significant differences compared to the broad-beam (BB) irradiation for A549. In contrast, the normal tissue cell line MRC-5 exhibited significantly higher clonogenic survival following both MRT + R and overlapping MRT compared to BB. Conclusions: This study demonstrates that combining temporal and spatial fractionation enhances normal tissue cell survival while maintaining equivalent tumor cell kill, potentially increasing the therapeutic index. Our findings support the feasibility of delivering temporally fractionated doses using different MRT modalities and provide clear evidence of the therapeutic benefits of temporally fractionated MRT.