Immediate early response gene-3 (IER3/IEX-1) encodes a radiation-inducible stress-response adaptor protein that integrates pro-survival signaling pathways, yet the upstream mechanisms regulating its expression after irradiation remain incompletely understood. Here we investigated whether lysophosphatidic acid receptor (LPAR) signaling regulates IEX-1 expression during the radiation response and examined the radiobiological consequences of IEX-1 deficiency. LPAR activation enhanced IEX-1 expression in irradiated mouse whole blood, bone marrow, intestinal mucosa, and intestinal enteroids. In addition, IEX-1-deficient mice exhibited increased susceptibility to lethal radiation injury after total-body or partial body irradiation, while IEX-1-deficient enteroids displayed delayed growth and altered ERK phosphorylation kinetics. Together, these findings identify IEX-1 as a previously unrecognized downstream target of LPAR signaling after irradiation.

With the increasing use of computed tomography, the biological effects of low-dose ionizing radiation (LDIR) have gained attention. However, its effects on B lymphocytes remain understudied. We investigated LDIR-induced oxidative stress in lymphocytes to establish a theoretical basis for clinical radiation safety. First, we demonstrated that LDIR induces a rapid oxidative response in the human B-lymphoblastoid cell line IM-9. Reactive oxygen species (ROS) levels increased at 0.25 h and decreased at 1 h after LDIR. In parallel, the number and intensity of γ-H2A.X and 53BP1 DNA damage foci significantly increased in the irradiated group. Transcriptome analysis identified Forkhead box protein O1 (FOXO1) as a key gene. Western blotting analysis showed that Sirtuin-1 (SIRT1) activity decreased at 0.25 h after irradiation, while the activity of FOXO1 and acetylated FOXO1 increased. Superoxide dismutase 2 (SOD2) activity decreased initially and then increased at 1 h. A co-immunoprecipitation (Co-IP) assay confirmed the interaction between FOXO1 and SIRT1, supporting their coordinated role in oxidative regulation. LDIR induced oxidative stress via the SIRT1/FOXO1 axis. These results demonstrate that early ROS accumulation suppresses SIRT1 activity, impedes FOXO1 deacetylation, and transiently reduces SOD2 activity, followed by a compensatory induction of SOD2 to restore redox balance.

Radiation-induced liver damage (RILD) significantly limits the clinical application of radiotherapy for upper abdominal malignancies. Radiation can induce metabolic disorder in liver tissues. However, there is no systematic research on the effects of radiation on liver metabolism. In this study, we collected time-series liver tissue samples from irradiated rats at multiple time points, from 3 days to 4 weeks after exposure, to reveal dynamic alterations in metabolic profiles throughout RILD progression. Acetylcholic acid, p-hydroxyphenyl-lactic acid, ascorbic acid, daidzein, and glutamine (Gln) were identified as potential biomarkers, demonstrating good diagnostic sensitivity and specificity. As a metabolomic biomarker, Gln emerged as a potential metabolic target based on pathway enrichment analysis, which could modulate the radiosensitivity of both mouse (JS-1) and human (LX-2) hepatic stellate cells. Transcriptomics, co-immunoprecipitation (Co-IP) and functional rescue experiments demonstrated that the regulation of radiosensitivity by Gln is mediated through the Col1α2/ITGB1/AKT signaling axis. In the mouse RILD model, Gln prevented irradiation-induced body weight loss, preserved liver structure, and reduced TGF-β and TNF-α expression as well as collagen deposition. L-alanyl-glutamine (Ala-Gln) and liposomes were introduced to further enhance the radioprotective effects of Gln. This study provides the necessary theoretical and experimental basis for diagnosis and intervention of RILD.

Radiological nuclear scenarios remain a persistent threat due to civil use and military possession of radiation sources. Therefore, it is crucial to be prepared for events involving a large number of patients exposed to radiation. In such cases, it is important to quickly and simply predict whether potential patients will develop severe, mild, or no Acute Radiation Syndrome (ARS). To address this challenge, the Bundeswehr Institute of Radiobiology developed the H-module, a prediction module that estimates the severity of ARS based on peripheral blood cell counts in the context of an exposure event. For accurate predictions, correct blood counts, especially lymphocyte and granulocyte counts, are essential. Mobile hematology analyzers (HA) are a possible portable and easy-to-learn solution for assessing patient blood counts. These analyzers use impedance measurements to identify cell populations, with cell volume primarily responsible for providing differential blood cell counts. We investigated the influence of alterations in leukocyte size after irradiation on the performance of this method. Whole blood samples from healthy donors were irradiated with different doses (0, 1, and 5 Gy). The samples were analyzed after 0, 1, 6, and 24 h post-irradiation. At each time point, a blood count was performed using impedance measurements via HA, and in parallel, a differential blood count of white blood cells (WBC) was performed using imaging-based flow cytometry. Lymphocytes and granulocytes were differentiated using CD15, CD3, and CD19 markers, by their different side scatter, and by their size in brightfield mode. In addition, peripheral blood mononuclear cells were monitored using a live-cell imaging system equipped with image acquisition and cell size measurement. The HA indicated a discrete increase in the lymphocyte fraction when blood counts were performed 24 h after irradiation, whereas at earlier time points, all WBC fractions remained comparable between both methods. These findings demonstrate that morphological changes in WBC do not impair the ability of the device to distinguish granulocytes from lymphocytes, provided that measurements are performed promptly. However, after 24 h, a reduction in cell size, due to delayed handling of the samples, resulted in an artificial increase in the lymphocyte fraction, potentially masking the lymphocyte depletion characteristic of H-ARS. Thus, timely sample processing when using a HA is essential to ensure diagnostic accuracy.

The aim of this study is to assess the predictive efficacy of a radiomics model utilizing magnetic resonance imaging (MRI) for predicting locally advanced cervical squamous cell carcinoma response to concurrent chemoradiotherapy. We enrolled a cohort of 139 patients diagnosed with stage IIB to IV cervical squamous cell carcinoma, based on the 2018 FIGO classification, who underwent concurrent chemoradiotherapy and pre-/post-treatment MRI examinations. These patients were divided into complete response and partial response groups. Prior to the initiation of treatment, the areas of interest within the lesion were delineated on T2-weighted imaging (T2WI), diffusion-weighted imaging (DWI), and enhanced T1-weighted imaging (T1WI) sequences, from which radiomic features were extracted. The group was randomly divided into training (n = 111) and validation (n = 28) sets (8:2 ratio) to identify the optimal features. Logistic regression models were constructed to predict treatment response, with distinct models based on the following imaging modalities: enhanced T1WI, T2WI, DWI, a combination of T2WI and enhanced T1WI (joint model 1), and a combination of T2WI, DWI, and enhanced T1WI (joint model 2). Model fitness and predictive performance were assessed using receiver operating characteristic curves, while the clinical applicability of the models was analyzed using decision curve analysis. A cumulative count of 2,264 radiomic features was derived from the region of interest in each imaging sequence. Subsequently, 18, 16, 15, 16, and 13 optimal features were selectively identified from the five models. These selected features were employed to formulate a radiomics model designed for the prediction of treatment response. These selected features were used to construct individual radiomics models aimed at predicting treatment response. Subsequently, all models achieved AUCs > 0.8 in the validation set, with Joint Model 2 demonstrating the highest performance (AUC = 0.939, 95% CI: 0.826-1; sensitivity = 0.773, specificity = 0.833). No significant differences were observed between Joint Model 2 and other models (P > 0.05). The MRI-based radiomics model has high potential in effectively predicting the efficacy of concurrent chemoradiotherapy for locally advanced cervical squamous cell carcinoma.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is known for high levels of disease recurrence and resistance to conventional fractionated radiation therapy. In an analysis of primary patient HPV-negative HNSCC biopsies from our institutional tumor bank, we observed circulating and tumor-resident monocytic myeloid-derived suppressor cells (M-MDSCs) were enriched in patient samples from recurrent HPV-negative HNSCCs relative to treatment-naïve samples. To determine if radiation exposure alters myeloid chemotaxis to the tumor site, we measured myeloid chemotactic factor transcript expression in eight cancer-associated fibroblast (CAF) and EpCAM-expressing patient-derived malignant cell lines. In malignant cell lines, radiation exposure increased transcriptional expression of CCL5 and C-X-C ligand receptor 2 (CXCR2) ligands CXCL2 and CXCL8 (IL-8). Based on this finding, we evaluated whether an antagonist of CXCR2 (SB225002) in combination with radiation blocked murine MDSC chemotaxis in vivo and in vitro. Antagonism of CXCR2 signaling with SB225002, in combination with radiation, suppressed tumor growth, improved survival, and reduced intra-tumoral macrophages in a murine MOC1 model. Additionally, radiation-induced MDSC chemotaxis was inhibited by SB225002 in a transwell assay. Together, these data suggest CXCR2 inhibition in combination with radiation has therapeutic potential for treatment of HPV-negative HNSCC.

In addition to immediate cell lethality, radiation exposure has long-term effects on the immune system, particularly T lymphocytes, causing persistent functional abnormalities post-exposure. Uncertainties remain regarding the long-lasting consequences on T cell immunity in survivors of the acute radiation syndrome and the underlying mechanisms of those consequences. Here, we investigated delayed effects of acute radiation exposure on CD8+ T cell immunity using the Listeria monocytogenes infection model. Impaired CD8+ T cell activation, reduced terminal effector formation upon infection, and exacerbated listeriosis were found in mice at 4 weeks postirradiation with sublethal doses. To elucidate how radiation affects the functionality of CD8+ T cells at various differentiation stages, we performed single-cell transcriptomic profiling. Radiation altered cluster distribution pre-infection and impaired terminal effector and memory precursor effector formation post-infection. Differential gene analysis highlighted radiation-induced gene expression changes, including cytokines and mitochondrial proteins, in a cluster-specific manner post-infection. The study demonstrates dysfunction of newly replenished naïve T cells, which results in impaired CD8+ T cell immunity after irradiation.

Laryngeal squamous cell carcinoma (SQCC), in both early and advanced stages, is often treated non-surgically with definitive-intent radiotherapy or chemoradiotherapy. In advanced-stage disease, uninvolved lymphatic structures of the neck are electively irradiated, whereas in early-stage disease the larynx alone is irradiated. The neutrophil-to-lymphocyte ratio (NLR) has been implicated as a prognostic marker in head and neck SQCC. As lymphocytes are extremely radiosensitive, the absolute lymphocyte count (ALC) may fluctuate during and after radiotherapy, and consequently, the NLR may vary across these time points. This study aimed to evaluate dynamic changes in NLR, ALC, and absolute neutrophil count (ANC) during and after radiotherapy and to assess their association with oncologic outcomes in patients with laryngeal SQCC. We retrospectively reviewed the charts of patients treated with definitive-intent radiotherapy or chemoradiotherapy for laryngeal carcinoma at our institution between 2015 and 2021. NLR, ANC, and ALC values were recorded at three time points: treatment initiation, treatment completion, and three to six months after treatment completion. Oncologic outcomes were also recorded. A total of 174 patients treated for laryngeal SQCC were included. The mean NLR level was significantly higher at treatment completion than at treatment initiation (6.23 ± 4.61 vs. 3.12 ± 3.32, P < 0.001) and was significantly lower at 3-6 months after treatment than at treatment completion (3.75 ± 2.15 vs. 6.23 ± 4.61, P < 0.001). An NLR level above 4 at treatment completion was associated with worse overall survival (log-rank P = 0.005). NLR above 4 at treatment completion and 3-6 months afterward was associated with worse disease-free survival (log-rank P = 0.047 and P = 0.011, respectively). Mean ALC declined from 1.91 ± 0.70 × 103/µL at baseline to 0.90 ± 0.51 × 103/µL at treatment completion and partially recovered to 1.36 ± 0.52 × 103/µL at follow-up, whereas ANC remained relatively stable (4.95 ± 1.70, 4.38 ± 1.99, and 4.38 ± 1.44 × 103/µL, respectively). NLR significantly increases during oncologic treatment. Our study demonstrates a novel association between NLR at the end of treatment for laryngeal SQCC and survival. Patients whose NLR remains high 3-6 months after treatment experience worse disease-free survival. The transient rise in NLR appears to be largely driven by radiation-induced lymphocyte depletion. These findings support routine monitoring of both NLR and ALC in patients undergoing radiotherapy for laryngeal cancer.

Pentaazamacrocyclic Mn (II)-containing (MnPAM) superoxide dismutase mimetics can selectively modulate the effects of radiotherapy in tumors and normal tissues by enhancing oxidative stress in tumors and mitigating it in normal tissues. However, radiosensitizing effects in tumors are only achieved with stereotactic body radiotherapy-type dosing (≥7 Gy per fraction). These effects are limited at lower fractional doses that are clinically relevant for many tumors, including locally advanced rectal cancer. Here, we show that combining the MnPAM dismutase mimetic, rucosopasem manganese (GC4711), with pharmacological ascorbate significantly enhances radiosensitization to fractionated radiation doses relevant in colorectal cancer treatment (2-5 Gy) compared to either agent alone in multiple tumor models in vitro and significantly prolongs tumor growth delay in vivo. Mechanistically, enhanced radiosensitization with combination therapy is mediated by increased oxygen consumption and hydrogen peroxide generation. Despite having no effect on oxygen consumption in tumor cell media on its own, GC4711 significantly increases pharmacological ascorbate-induced oxygen consumption. Inducible catalase expression, or administration of exogenous catalase, attenuates the anti-cancer activity when combined. As predicted, increased peroxide fluxes with combination therapy enhanced postirradiation DNA damage and G2/M cell cycle accumulation. Importantly, treatment with pharmacological ascorbate and/or GC4711 had no significant effect on postirradiation clonogenic survival or DNA damage in non-malignant FHs74 enterocytes. These results support the hypothesis that adding pharmacological ascorbate to MnPAM dismutase mimetics selectively induces more peroxide-mediated radiosensitization than either agent alone in tumors without increasing radiosensitivity in cells treated with clinically relevant radiation fraction sizes used for radiotherapy of colorectal cancers.

Several radiation medical countermeasures (MCMs) are currently approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). Continued development of additional candidates remains a priority to enhance force protection capabilities, combat readiness, and operational preparedness in nuclear/radiological threat environments. One such candidate under development is BBT-059, a long-acting PEGylated interleukin-11 (IL-11) analog with known hematopoietic-promoting and anti-apoptotic properties. BBT-059 is under advanced development as a potential radiation MCM for H-ARS and has been shown to improve survival in lethally irradiated murine models; however, its proteomic responses after irradiation have not been fully characterized in nonhuman primates (NHPs). In this study, NHPs exposed to 4 Gy total-body gamma radiation were subsequently treated with a single subcutaneous dose of either 37.5 or 75 µg/kg of BBT-059 at 24 h postirradiation, and longitudinal serum proteomic profiling was performed using a mass spectrometry (MS)-based approach. Proteomic changes were compared to baseline levels to comprehensively identify time-dependent changes induced by irradiation and BBT-059 administration. These analyses revealed changes in proteins related to inflammation, innate immune activation, and hematological and platelet functions, which peaked at days 2 and 4 and gradually decreased to near baseline levels by the end of the study. Pathway enrichment analysis identified consistent activation of neutrophil degranulation, platelet degranulation, insulin-like growth factor transport, and calcium signaling pathways.

Normal tissues exposed to radiation during radiotherapy or radiation accidents are susceptible to chronic inflammation, which poses a significant health concern. Here, we used the mouse mammary gland as a model to investigate radiation-induced damage to normal tissue. Five-week-old mice were chosen to capture the radiation-sensitive pubertal developmental window, and transcriptomic sequencing was used to profile gene expression changes at 1-5 months postirradiation. Single-cell RNA sequencing was performed to identify immune cell populations and characterize their transcriptional features at the subpopulation level. By combining transcriptomic data with morphological analysis of the irradiated mammary gland, we identified gene expression signatures of specific macrophage and T cell subtypes within the postirradiation microenvironment and highlighted their roles in modulating inflammation and maintaining cell junction homeostasis. Notably, dysregulation of adipocyte differentiation and lipid metabolism pathways was observed as early as 1 month postirradiation, and this metabolic disturbance was associated with persistently elevated oxidative stress in the tissue. Single-cell analysis showed that CD163+ macrophages were functionally enriched in lipid metabolism pathways, and immunofluorescence staining confirmed their significant accumulation in the irradiated mammary gland. Overall, these findings suggest that CD163+macrophages are key mediators of chronic tissue inflammation after irradiation. This study offers a comprehensive characterization of microenvironmental changes in the irradiated mammary gland and identifies potential targets for radioprotection strategies to modulate tissue microenvironment factors.

To clarify the mechanisms of FLASH radiotherapy using C ion beams (373 MeV/u and 90 MeV/u), we investigated the dose rate dependence of yields of water radiolysis species (OH radicals, hydrated electrons, and hydrogen peroxide). Additionally, we evaluated changes in the induction rate of DNA strand breaks as a function of dose rate using pBR322 plasmid DNA in solutions. Average dose rates were 0.2 Gy/s [conventional (CONV)] and 75 Gy/s [ultra-high dose rate (UHDR)] for 373 MeV/u, and 0.3 Gy/s (CONV) and 100 Gy/s (UHDR) for 90 MeV/u. The G values of water radiolysis species decreased with UHDR irradiation compared to CONV dose rate, except for hydrogen peroxide exposed to 90 MeV/u C ions, whose yield remained independent of dose rate. Furthermore, the induction rate of single-strand breaks (SSB) was unaffected by dose rate, while the induction of double-strand breaks (DSB) decreased after UHDR irradiation. These findings suggest that radical-radical reactions occur more efficiently with increasing dose rate, resulting in a reduced contribution of water radiolysis species to DNA strand breaks, especially DSBs, after irradiation with C ions. Therefore, changes in the contribution of water radiolysis species to DNA strand breaks could play a crucial role in the sparing effects observed after UHDR irradiation.

Experimental data on magnitudes of sparing effects of whole-abdominal pulse irradiation, whole-brain pulse irradiation, and bilateral thorax pulse/continuous irradiation at high doses and dose rates from conventional to FLASH irradiation rates (> 40 Gy/s) on normal intestinal, brain, and lung tissues in mice, which were reported in the literature, are analyzed by making use of recently developed mathematical models. These models are capable of predicting the fractions of surviving blood lymphocytes in mice after the exposures. Results of juxtaposing those experimental data with the respective modeling results suggest a possible relationship between them. Recent experimental findings confirming this relationship are presented. Finally, an algebraic formula to predict FLASH dose rate thresholds for the exposures is derived and verified, providing a tool for future experimental design.

Irradiation at ultra-high dose rates is gaining increased attention due to its ability to limit damage to surrounding healthy tissues, thereby preventing radiation-induced side effects. Although there are some exceptions, this protective effect (the FLASH effect) has been demonstrated in many preclinical models, primarily after low-LET irradiation and high-dose irradiation. Here, we report that both ultra-high dose rate (UHDR) and conventional dose rate (CONV) irradiations produce similar growth delays in 3D tumor spheroids (composed of HCT116 and T98G, colorectal carcinoma or glioblastoma cell lines), indicating comparable efficacy in tumor control. In normal cells (HIEC-6, a non-malignant epithelial cell line from the small intestine), UHDR and CONV irradiation induced comparable senescence, while a lower induction of apoptosis was observed after UHDR irradiation at clinically relevant doses and early time points postirradiation. Collectively, these findings highlight the potential of UHDR irradiation to modulate normal tissue responses while achieving comparable tumor control.