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.

Glioblastoma (GBM) constitutes the most common primary brain tumor and it remains incurable despite therapeutic advances. The high infiltration/invasion potential of GBM cells is considered to be one of the reasons for the inevitable recurrence of the disease. Radiotherapy (RT) is part of the standard care for patients with GBM, and its benefits on overall survival are extensively reported. However, numerous preclinical studies show that X-ray irradiation can enhance the motility of GBM cells. In the present review, we bring together state-of-the-art research on the impact of radiation on GBM cell motility. The mechanisms through which irradiation impacts the brain tumor microenvironment and the tumor cells themselves, leading to more aggressive/invasive tumors, are described. Finally, we summarize potential pharmacological strategies to overcome this problem. Clinical data validating the occurrence of these processes are urgently needed as they could be of great value for patient outcomes. With this comprehensive review, we expect to highlight the need for methods which allow for monitoring the post-irradiation invasive behavior of GBM in patients.

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.

Radiotherapy (RT) is currently among the standard treatments for lung cancer. However, in vitro studies have revealed that irradiation can increase lung cancer cell motility. This way, RT could potentially enhance the malignancy of solid tumors post-treatment, promoting metastasis. Therefore, there is a continued need to continue evolving RT modalities into safer and more effective treatments. The present study compares the impact of the broad beam (BB) and the spatially fractionated modality of microbeam radiation therapy (MRT) on the motility of A549 lung cancer cells. Our data corroborates previous findings that showed BB irradiation is a promoter of cell motility. For MRT, however, we observed a prevention of cellular migration. A significant reduction in NF-κB expression was observed only when A549 cells were irradiated with MRT, indicating a potential mechanism behind these findings. Finally, our data supports potential issues regarding MRT irradiation of key components of the tumor microenvironment, such as fibroblasts. Co-culturing A549 cells with MRT-irradiated MRC-5 lung fibroblasts led to increased tumor cell invasion, not observed when the fibroblasts received BB irradiation.

Background: Microbeam radiation therapy (MRT) delivers spatially fractionated, high-dose radiation in parallel beam arrays, creating spatial fractionation by alternating peak and valley dose regions. While this approach offers significant potential for tumor control with reduced normal tissue toxicity, the precise biological dose and damage distribution, particularly the relative contributions of direct and non-targeted (bystander) effects, remain incompletely understood. Methods: We employed complementary biodosimetric approaches to quantify DNA damage responses in two-dimensional CHO-K1 cell monolayers and three-dimensional epidermis tissue models following MRT. Biological dosimetry in CHO-K1 monolayers was performed using the cytokinesis-block micronucleus (CBMN) assay for micronuclei (MN) and nucleoplasmic bridges (NPB). In epidermis models, DNA double-strand break (DSB) damage was analyzed by immunofluorescence (IF) with γH2AX+53BP1 DSB markers. Spatial and temporal mapping of damage in tissue sections was performed across microbeam peak and valley regions and compared with physical dosimetry and broadbeam irradiation. Results: In 2D CHO-K1 cells, MN- and NPB-based biological dosimetry closely matched physical dose measurements, with accurate dose reconstruction in valleys but substantial underestimation in peaks due to extensive cell death and loss of scorable nuclei. In 3D epidermis models, MRT induced distinct spatial patterns of DNA damage with pan-γH2AX–positive cells indicating excessive DSB formation at peak regions at early time points, whereas 53BP1 DSB foci predominated in dose valleys. Over 24 h, pan-γH2AX signals declined to sham levels, while 53BP1 foci persisted, particularly in peak regions, indicating DNA damage regions refractory to repair. Comparative analyses revealed that valley cells exposed to 1 Gy MRT radiation exhibited in average significantly more 53BP1 foci at 0.5 h than after uniform 1 Gy broadbeam irradiation, suggesting a bystander contribution from adjacent high-dose peaks. By 24 h, however, damage levels converged between MRT valleys and broadbeam, indicating that bystander-associated excess damage was transient. Conclusion: CBMN-based biological dosimetry reliably quantifies absorbed dose in MRT valleys but is limited in peak regions. 3D epidermis models enable spatial and temporal mapping of DNA damage, revealing persistent complex damage in peak-region cells and provide evidence for transient bystander effects in valley regions. Extending previously established biodosimetric and DNA damage mapping approaches to X-ray MRT, these findings, obtained in one cell line and one tissue model, contribute to the understanding of DSB induction, repair dynamics, and non-targeted effects in MRT.