Multi-assay profiling of blood lymphocytes radiosensitivity under protons or photons

BACKGROUND: Proton therapy is clinically associated with reduced radiation-induced lymphopenia (RIL), yet it remains unclear whether this effect arises solely from dose distribution advantages or also reflects differences in biological response. While Human Peripheral Blood Lymphocytes (HPBLs) are commonly used to identify and assess individual radiosensitivity, definitions of radiosensitivity remain assay-dependent and lack standardization. We aimed to compare the biological responses of HPBLs to low-to-moderate doses of protons and photons using a multi-assay approach. METHODS: HPBLs from five healthy donors were irradiated in vitro with increasing doses (0.3-4.0 Gy) of 60 MeV protons or 250 keV X-rays. Biological responses were assessed using apoptosis, necrosis, and caspase-3 activity to assess cell viability and death; the cytokinesis-block micronucleus (CBMN) assay and alkaline comet assay to evaluate DNA and nuclear/chromosomal damage; and premature chromosome condensation (PCC) to quantify chromosomal damage. Dose-response relationships were analyzed using linear, linear-quadratic, and saturation models. Correlation analyses and principal component analysis (PCA) were used to explore inter-individual radiation variability and the relationships among endpoints. RESULTS: Radiation-induced effects increased with dose for all assays. In the 1-4 Gy range, protons elicited significantly stronger cytotoxic responses than photons, particularly for apoptosis and necrosis. DNA and chromosomal damage endpoints showed linear dose-response patterns, while viability and apoptosis markers followed saturation kinetics. Across donors, protons consistently produced more pronounced biological effects than photons at equivalent doses. PCA revealed that apoptosis-based endpoints were most strongly correlated with overall radiation damage, suggesting they may serve as effective integrative biomarkers of lymphocyte radiosensitivity. CONCLUSIONS: Our findings indicate that HPBLs exhibit stronger cytotoxic responses to protons than to photons in vitro. These results support the interpretation that reduced RIL observed clinically with proton therapy likely results from volume-sparing effects rather than reduced intrinsic biological effectiveness. A multi-assay strategy remains essential to fully capture the complex nature of lymphocyte responses to radiation.