Fine-mapping of microbeam radiation therapy-induced DNA damage in 2D cell and 3D epidermis models

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.

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.

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.

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.