Differential temporal dynamics of Wnt/β-catenin transcriptional activity following minibeam and conventional irradiation

Minibeam radiation therapy (MBRT) improves the therapeutic index by sparing normal tissues while maintaining tumour control, yet the biological mechanisms underlying these effects remain poorly understood. We investigated whether minibeam and conventional irradiation are associated with different temporal profiles of Wnt/β-catenin transcriptional activity, a mechanosensitive signalling readout. TOP-TCF luciferase reporter activity and cell viability were assessed following MBRT or conventional radiotherapy (CRT) in A431, RG2 and MDCK cell models. Dose- and time-dependent responses were evaluated at 24, 48 and 72 h post-irradiation. Wnt/β-catenin transcriptional activity showed delayed, dose-dependent and strongly cell-context-dependent responses. Across the three cell lines, MBRT generally produced lower reporter activity than CRT at later measured time points and/or higher doses. These differences were accompanied by distinct cytotoxicity profiles, with CRT generally inducing greater PI positivity than MBRT. MBRT and CRT were associated with different magnitudes and temporal profiles of Wnt/β-catenin transcriptional activity. Because the irradiation modalities also differed in dose rate and cytotoxicity, the present data do not establish that spatial dose heterogeneity itself caused these signalling differences. The findings provide an initial framework for further mechanistic and spatially resolved studies.