Nanoscale topology of γH2AX and 53BP1 foci in U87 cancer cells and normal NHDF after high-LET radiation-induced DSB repair

DNA damage repair is essential for maintaining genomic integrity, thereby preventing diseases like cancer. Traditionally, radioresistance has been linked to the ability of cells to repair DNA double-strand breaks (DSBs) accurately. Recent research emphasizes the critical role of spatial chromatin organization and its dynamic reorganization in regulating repair and gene expression. In this study, we have employed single-molecule localization microscopy (SMLM) and Python-based mathematical methods of statistics and topology to locally analyze the spatial organization of γH2AX and 53BP1 foci in ¹⁵N-ion irradiated normal human dermal fibroblasts (NHDF) and highly radioresistant U87 glioblastoma cells over extended post-irradiation (PI) periods with nanoscale resolution. Our findings reveal that U87 cancer cells fail to regulate chromatin changes at DSB sites during and after repair. Specifically, Ripley's statistics and cluster analysis showed that both NHDF and U87 cells exhibit smaller, denser, and better separated γH2AX nano-foci surrounded by 53BP1 nano-foci. Mathematical topology approaches, including persistent homology, revealed that γH2AX nano-foci (clusters) have lower topological similarity compared to the more conserved 53BP1 nano-foci during the 24-hour repair period. These findings support the non-random, functional spatial organization of DSB repair (nano)foci and demonstrate its preservation in cancer cells. However, principal component analysis of persistent images showed that γH2AX- and 53BP1 nano-foci in normal fibroblasts exhibit stable, closed cycles, while U87 cells display chaotic, open shifts in topology in the 2D latent space. Combined with DSB repair kinetics measurements, this observation indicates that although U87 cells rejoin DSBs similar to normal cells, they experience more pronounced, dysregulated chromatin alterations during repair, ultimately failing to restore it to its pre-irradiation state. These alterations correlate with topologically more variable DSB sites, slower (more challenging) repair focus formation but faster repair once foci are established, compared to NHDF cells. More disorganized repair and persistent topological alterations likely contribute to genetic instability of cancer cells after irradiation and the development of radioresistant clones, posing challenges for effective radiotherapy.