DNA double-strand breaks (DSB) are among the most harmful DNA lesions induced by ionizing radiation (IR). Although the induction and repair of radiation-induced DSB is well studied for acute irradiation, responses to DSB produced by chronic IR exposures are poorly understood, especially in human stem cells. The aim of this study was to examine the formation of DSB markers (γH2AX and phosphorylated kinase ATM, pATM, foci) in human mesenchymal stem cells (MSCs) exposed to chronic gamma-radiation (0.1 mGy/min) in comparison with acute irradiation (30 mGy/min) at cumulative doses of 30, 100, 160, 240 and 300 mGy. A linear dose-dependent increase in the number of both γH2AX and pATM foci, as well as co-localized γH2AX/pATM foci (“true” DSB), were observed after an acute radiation exposure. In contrast, the response of MSCs to a chronic low dose-rate IR exposure deviated from linearity towards a threshold model, for γH2AX, pATM foci and γH2AX/pATM foci, with an indication of a “plateau”. The state of equilibrium between newly formed DSB at a low rate during the protracted exposure time and the elimination of a fraction of DSB is proposed as a mechanistic explanation of the non-linear DSB responses following a low dose-rate irradiation. This notion is supported by the observation of the elimination of a substantial fraction of DSB 6 h after the cessation of the exposures. Our results demonstrate non-linear dose responses for γH2AX and pATM foci in human MSCs exposed to low dose-rate IR and showed the existence of a threshold, which may have implications for radiation protection in humans.

Rapidly evolving laser technologies have led to the development of laser-generated particle accelerators as an alternative to conventional facilities. However, the radiobiological characteristics need to be determined to enhance their applications in biology and medicine. In this study, the radiobiological effects of ultrashort pulsed electron beam (UPEB) and X-ray radiation in human lung fibroblasts (MRC-5 cell line) exposed to doses of 0.1, 0.5, and 1 Gy are compared. The changes of γH2AX foci number as a marker of DNA double-strand breaks (DSBs) were analyzed. In addition, the micronuclei induction and cell death via apoptosis were studied. We found that the biological action of UPEB-radiation compared to X-rays was characterized by significantly slower γH2AX foci elimination (with a dose of 1 Gy) and strong apoptosis induction (with doses of 0.5 and 1.0 Gy), accompanied by a slight increase in micronuclei formation (dose of 1 Gy). Our data suggest that UPEB radiation produces more complex DNA damage than X-ray radiation, leading to cell death rather than cytogenetic disturbance.

Hypoxia is a key microenvironmental factor shaping tumor cell survival and plasticity, but its cellular consequences depend on the cancer subtype. Here, we compare breast cancer cell lines with varying metastatic potential (MP) in response to CoCl₂-induced hypoxia (100 μM, 20 h), analyzing the results immediately (T0) and after 24 h (T1). Plasma membrane damage was measured by the number of propidium iodide-positive cells. Hypoxia pathway activation was evaluated using HIF-1α immunofluorescence, while phenotypic changes were assessed through quantitative morphometric analysis. Chemically induced hypoxia promoted a delayed increase in membrane failure in low-MP cells, consistent with reduced tolerance to hypoxia-mimetic stress. In contrast, high-MP cultures showed a lower PI-positive fraction at T1, consistent with enhanced survival under these conditions. This divergence was accompanied by higher basal and hypoxia-induced HIF-1α levels in high-MP cells. High-MP cells uniquely demonstrated delayed shape changes after exposure to hypoxia-mimetic conditions, evolving into a more compact and rounded form, suggesting phenotypic plasticity instead of structural deterioration. Together, our findings provide insight into how oxygen deprivation impacts cell destiny and phenotypic plasticity in breast cancer models with varying metastatic risks.

Laser-driven accelerated particle beams have revolutionized the field of cancer treatment by enabling the delivery of extremely high dose-rates of radiation to solid tumors in a matter of femto- to picoseconds. In this study, radiobiological effectiveness of ultrashort pulsed electron beams generated by Advanced Research Electron Accelerator Laboratory (AREAL) accelerator and conventional X-rays were compared on two non-small cell lung cancer (NSCLC) cell lines, A549 (wild-type p53) and H1299 (p53-deficient). NSCLC cells were irradiated at using a AREAL accelerator (a peak dose rate of 1.6 × 1010 Gy/s, pulse duration of 4.5 × 10–13 s, repetition rate of 20 Hz) or X-ray unit at an absorbed dose rate of 0.85 Gy/min. Clonogenic survival analysis, γH2AX foci enumeration and genome-wide transcriptome analysis were performed. Clonogenic survival curves showed increased ra-diosensitivity of A549 cells after AREAL exposure compared to X-rays (RBEs=1.2), while H1299 radiosensitivity has not changed. AREAL exposure caused higher linear dose–dependent increase in the number of residual γH2AX foci in both A549 and H1299 cells compared to X-rays. The DNA double-strand breaks in H1299 cells were mainly repaired through homologous recombination following AREAL exposure. However, the gene ex-pression analysis of pathway activation levels revealed a down-regulation of the same pathways in A549 cells. This resulted in activation of Integrin-linked kinase-dependent apoptosis, G2 and proliferation arrest of A549 cells.

Metastasis is the leading cause of cancer-related mortality, highlighting the need for reliable diagnostic markers and molecular targets that distinguish primary and metastatic tumors. Genes involved in actin cytoskeleton organization and cell mechanics regulate cell migration and invasion and may represent both markers and functional targets of metastatic progression. This study investigated whether copy number alterations (CNAs) in an eleven-gene mechanobiology-related panel (FSCN1, GSN, ACTN4, EZR, CFL1, MYH9, PFN1, TAGLN, ECM1, ANXA2, ANXA6) can distinguish primary from metastatic tumors across breast, pancreatic, and gastric cancers. Publicly available cBioPortal datasets were analyzed using supervised machine-learning algorithms trained on gene-level CNA data. Models trained on combined multicancer datasets showed improved classification performance, evaluated on a held-out breast cancer test set, compared with breast cancer-only training, with Random Forest achieving the highest accuracy (0.851) and F1-score (0.844). Feature importance analysis identified PFN1, ECM1, and FSCN1 as the strongest contributors to metastatic classification, with ANXA6 also showing consistent relevance; permutation importance confirmed PFN1, ANXA6, and FSCN1 in both models. These findings suggest that copy number alterations in mechanobiology-related genes capture genomic patterns associated with metastatic status and may serve as candidate diagnostic biomarkers, warranting further functional and expression-level validation to assess their potential as therapeutic targets.

High-frequency proton therapy shows promise for breast cancer (BC) treatment. We previously showed that BC cells’ metastatic potential (MP) correlates with their nanoparticle (NP) uptake efficiency. MP is known to be associated with microenvironment stiffness and radiosensitivity. Here, proton beam-irradiated MCF-7 and MDA-MB-231 cells were assessed for NP uptake efficiency under stiff (plastic) or soft (fibrin gel) conditions. In a stiff microenvironment, control MDA-MB-231 cells internalized 1.35-fold more NPs than MCF-7 (p < 0.0017), with comparably low uptake in soft conditions. After proton beam irradiation at a dose of 6 Gy, in stiff conditions, MDA-MB-231 cells showed a 1.6-fold increase in NP internalization compared to non-treated MDA-MB-231 (p < 0.0001), while MCF-7 cells showed no change, leading to an overall 1.86-fold difference between proton-treated MDA-MB-231 and MCF-7 cells (p < 0.0001). In soft conditions, irradiated MDA-MB-231 retained a 1.47-fold higher uptake of NPs than MCF-7 cells (p < 0.0172), but this value was 1.7-fold lower (p < 0.0001) compared to non-irradiated MDA-MB-231 cells on stiff plastic. Hence, therapeutic strategies combining proton irradiation with targeting tumor microenvironment softening may reduce post-irradiation metastasis risk.