International Journal of Radiation Oncology*Biology*Physics

Purpose: Elective nodal (EN) irradiation (ENI) during radiotherapy for locally advanced head-and-neck squamous cell carcinoma (LA-HNSCC) influences hematotoxicity, anti-tumor immunity, and synergy with immunotherapy. We evaluated whether EN-sparing upfront boosts affect DNA damage, systemic immune signaling in peripheral blood lymphocytes (PBLs), and radiation-induced lymphopenia (RIL). Methods and Materials: Twenty-eight patients with LA-HNSCC were randomized to either adjuvant or definitive chemoradiotherapy with standard ENI or EN-sparing upfront boost (adjuvant: 2x2 Gy; definitive: 5x2 Gy). Blood was collected pre-radiotherapy, 15 min, and 24 h after the first fraction, and before the sixth fraction. DNA damage in PBLs was assessed via {gamma}H2AX and 53BP1 foci and dicentric chromosome (DIC) assay. RNA sequencing was performed in two patients per group (definitive setting) at pre-CRT, before the sixth fraction, and at therapy end. Absolute lymphocyte counts (ALCs) were monitored weekly to assess RIL. Results: DNA damage in PBLs correlated with planning target volume and whole-body dose, both of which were reduced by EN-sparing by 9.9-fold and 4.4-fold, respectively (p < 0.001 each). Correspondingly, EN-sparing significantly reduced radiation-induced foci and DIC levels in PBLs (3-4-fold, p < 0.001) and lowered the fraction of radiation-damaged PBLs per fraction (11% vs. 23% with ENI, p < 0.001). EN-sparing preserved baseline ALCs during week 1 of chemoradiotherapy and delayed RIL, whereas ENI caused an immediate ALC decline and RIL. Lymphocyte counts after week 1 negatively correlated with planning target volume, whole-body dose, and DNA damage in PBLs (p < 0.01). Transcriptomics showed metabolic and interferon signaling associated with EN-sparing, versus sterile inflammatory and damage-associated patterns with ENI. Conclusions: EN-sparing by an upfront boost significantly reduced PBL damage and early RIL with distinct immune responses associated with lymphocyte viability and immune maturation. These findings support upfront EN-sparing strategies to mitigate RIL and improve radiotherapy-immunotherapy synergy in HNSCC.

Background: Reirradiation (reRT) is increasingly offered following progression in diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG), though optimal patient selection remains a challenge. This study evaluated clinical outcomes after reRT in a contemporary cohort of patients with DIPG/DMG. Methods: Patients <26 years old with DMG/DIPG treated with radiation therapy between 2011-2025 were retrospectively reviewed. Primary endpoints included overall survival (OS2) and progression-free survival (PFS2), measured from first progression, and change in neurologic symptoms after reRT. Survival was estimated using Kaplan Meier methods, with Cox proportional hazards modeling for prognostic factors. Results: Fifty eight patients were included; 37 (63.8%) underwent reRT. Tumors were predominantly pontine (74.1%). ReRT was associated with improvement in motor function (51.4% vs. 9.5%, p=0.002), cranial nerve function (29.7% vs. 4.8%, p=0.044), and gait ataxia (35.1% vs. 9.5%, p=0.059). Median OS2 and PFS2 were improved with reRT (OS2: 9.67 vs. 2.57 months, p<0.001; PFS2: 5.63 vs. 1.57 months, p<0.001). OS2 was independently associated with reRT (HR 0.27, p<0.0001), pontine location (HR 2.94, p=0.004), and steroid use at progression (HR 4.12, p=0.001). PFS2 was independently associated with reRT (HR 0.23, p < .0001) and distant pattern of failure (HR 2.83, p=.037). Among reRT patients, non-pontine location was associated with improved OS2 (p=0.02), and local failure was associated with improved PFS2 (p=0.003). Conclusion: ReRT was associated with neurologic improvement and prolonged survival. Patients with non-pontine tumors or local-only failure might derive the greatest benefit. Prospective studies are warranted to define optimal dose/fractionation and refine patient selection.

PurposeGlioblastoma (GBM) is characterized by poor clinical outcomes and marked resistance to radiotherapy. Because effective radiosensitizing strategies for GBM remain limited, we investigated whether inhibition of KRAS/RAS signaling could enhance radiation response in GBM. In particular, we evaluated the radiosensitizing potential of RMC-6236, an RAS(ON) multiselective inhibitor that suppresses active RAS signaling across multiple RAS-dependent states. Experimental DesignHuman GBM cell lines (U251, LN-18, ACPK1, and OSU61) were treated with radiation, with or without genetic or pharmacological KRAS inhibition. KRAS signaling was suppressed by siRNA-mediated knockdown or RMC-6236 treatment. Radiation-induced KRAS activation and downstream MAPK signaling were assessed by Raf-RBD pull-down assays and immunoblotting. Radiosensitivity was evaluated using clonogenic survival assay. DNA damage persistence, cell cycle distribution, and mitotic catastrophe were analyzed by {gamma}H2AX immunofluorescence, flow cytometry, and nuclear morphology assessment, respectively. In vivo therapeutic efficacy was examined in an orthotopic U251 xenograft model. ResultsRadiation-induced transient activation and increased KRAS protein expression of KRAS, accompanied by activation of ERK, JNK, and p38 signaling in GBM cells. siKRAS suppressed radiation-induced KRAS and MAPK activation, and significantly enhanced radiosensitivity in all four GBM cell lines. Similarly, RMC-6236 inhibited radiation-induced KRAS activation and attenuated downstream MAPK signaling without reducing the total KRAS protein expression. RMC-6236 significantly increased the radiosensitivity across all GBM cell lines, with dose enhancement factors ranging from 1.33 1.46. Mechanistically, combined treatment with RMC-6236 and radiation increased persistent {gamma}H2AX foci and enhanced mitotic catastrophe without producing consistent redistribution of cells into radiosensitive cell cycle phases. In an orthotopic GBM model, the combination of RMC-6236 and radiation significantly prolonged survival compared to that of the control and radiation alone. ConclusionsThese findings indicate that radiation-induced KRAS signaling is a functionally important mediator of radioresistance in GBM and demonstrate that inhibition of KRAS/RAS signaling enhances the radiation response in vitro and in vivo. RMC-6236 may represent a promising radiosensitizing strategy for GBM by suppressing adaptive RAS/MAPK signaling and promoting persistent DNA damage and mitotic catastrophe following irradiation. However, clinical trials of this combination are warranted.

Patients with Fanconi anemia (FA) are particularly susceptible to developing squamous cell carcinoma of the head and neck due to impaired DNA repair pathways. However, their hypersensitivity to DNA damaging agents can limit effective treatment with standard radiotherapy due to severe side effects and complications. In pre-clinical models, ultra-rapid FLASH radiotherapy (FLASH) reduces radiation-induced toxicity in normal tissues while maintaining similar tumor control compared to conventional dose rate radiotherapy (CONV). Here, we investigated the safety of FLASH for treatment of the head and neck in a mouse model of FA. 129/Sv wild-type (WT) and Fanca-deficient (Fanca-/-) mice received single-dose oral cavity irradiation with electron beam FLASH or CONV to evaluate radiation-induced toxicity in non-tumor-bearing mice. Fanca WT and Fanca-/- mice were irradiated with 25 and 18 Gy, respectively, of FLASH (190 Gy/sec) or CONV (0.2 Gy/sec), with tongues harvested at 12 hours (hpi) and 10 days (dpi) post-irradiation. At 10 dpi, FLASH-irradiated tongues in both genetic backgrounds demonstrated reduced ulceration at the dorsal tongue surface compared to CONV-irradiated counterparts. Histopathological analysis of the tongue revealed lower mucositis severity scores with decreased epithelial thinning and ulceration in FLASH-irradiated tongues compared to CONV-irradiated ones. Analysis of {gamma}-H2AX foci formation at 12 hpi demonstrated fewer foci in WT mice treated with FLASH compared to CONV, with a similar trend observed in Fanca-/- mice. These findings suggest a potential normal tissue-sparing effect with FLASH and hold important clinical implications for the treatment of patients with Fanconi anemia and head and neck cancers.

Our study evaluated whether a deep learning auto segmentation model combined with machine learning triage can streamline radiotherapy clinical trial quality assurance (QA). We analyzed 107 stereotactic ablative radiotherapy (SABR) cases from a multi-institutional phase II clinical trial of neurovascular sparing prostate SABR, focusing on physician contours of the internal pudendal artery (IPA) as a novel organ-at-risk with substantial interobserver variability. Contours were scored by the trial principal investigator as Per-Protocol or Minor Deviation/Unacceptable. We applied a deep learning model for IPA auto-segmentation. Agreement between human and AI contours was then quantified using 14 overlap, distance, and surface metrics, and a supervised classifier was trained on these metrics to flag clinical trial protocol deviations. While AI segmentation achieved only modest geometric accuracy with mean Dice similarity coefficient of 0.446 and 95th percentile Hausdorff distance of 14.23, when incorporating all 14 metrics, a machine learning classifier yielded AUROC of 0.836, flagging all Minor Deviation/Unacceptable cases with 100% sensitivity on the 27 case hold-out set with 6 false positives and no false negatives. AI segmentation combined with metrics-based machine learning can triage protocol deviations within a multi-institution radiotherapy clinical trial, supporting prospective evaluation of AI-assisted trial QA.

BackgroundATR activation following DNA damage from cancer treatments such as radiation can mitigate anticancer efficacy, making ATR inhibitors (ATRi) an attractive therapeutic. In vivo and in vitro studies have shown enhanced tumor cell radiosensitivity with the ATRi ceralasertib, elimusertib, and berzosertib, however, the potentiating effect of ATRi on ionizing radiation (IR) through immune-based mechanisms has only been studied with ceralasertib. MethodsWe aimed to determine if antitumor immune responses observed with ceralasertib in combination with IR extend to the other ATRi class members in the preclinical CT26 mouse model. We also examined the relationship between exposure and immune stimulation, efficacy and survival outcomes of each ATRi when combined with IR. ResultsCeralasertib and elimusertib, not berzosertib, synergized with IR in a dose and schedule-dependent manner to modify tumor antigen-specific CD8+ T cell populations in the draining lymph node. Transient ATRi therapy, combined with IR, enhances antitumor efficacy, promoted tumor shrinkage, and increased survival. ATRi elicited differential inflammatory gene induction and dose-dependent unique cytotoxicity profiles in vitro. ConclusionThe immune mediated antitumor effect of ATRi combined with radiation is dose and schedule dependent, and while likely a class effect, may differ between ATRi compounds.

The purpose of this work is to analyze the 2-year overall survival (OS2y) of limited-stage small cell lung cancer (LS-SCLC) treated with chemoradiotherapy (CRT), aiming at characterizing the response of LS-SCLC, and in particular the /{beta} value and proliferation parameters. Through a systematic analysis of the literature, we collated a dataset containing 57 entries (3363 patients) of response of LS-SCLC treated with CRT. Radiotherapy schedules ranged from hyper- to hypofractionation. Four radiobiological models to describe the OS2y were investigated, with progressive levels of complexity including the effect of radiotherapy, chemotherapy, treatment year and toxicity. The Akaike Information Criterion (AIC) was used to compare models, and the profile likelihood methodology to compute confidence intervals. Model 4, which includes the effect of radiotherapy, chemotherapy, treatment year and dose-dependent toxicity, provided the best fits of the experimental data (lowest AIC value). While being the best model, model 4 still fails to provide a good prediction of the OS2y, in particular failing to predict the survival of the schedules achieving the lower/higher survivals. The radiobiological analysis of the dose-response of LS-SCLC to CRT does not allow to narrowly constrain the value of response parameters. We attribute this limitation to the large heterogeneity of this disease. Nonetheless, our analysis shows a large /{beta} value (>9 Gy, 95% CI), which implies a low fractionation effect in the radiotherapy of LS-SCLC. and an accelerated proliferation of tumor cells, {lambda}' > 1.6 Gy/day (95% CI), after a kick-off time of ~4-5 weeks, which supports the use of accelerated protocols to avoid the effect of tumor proliferation on the clinical outcome.

Background: Prostate-specific membrane antigen (PSMA) PET/CT is central to prostate cancer staging and theranostic workflows. To our knowledge, no direct within-patient comparison of [18F]FC303 ([18F]Florastamin) and [68Ga]Ga-PSMA-11 has been reported. We performed a preliminary paired method-comparison study under non-harmonized acquisition protocols. Patients and Methods: Twenty patients with histologically confirmed prostate cancer underwent [68Ga]Ga-PSMA-11 PET/CT (185 +/- 37 MBq, 60 +/- 10 min) followed by [18F]FC303 PET/CT (370 +/- 37 MBq, 105 +/- 15 min) on the same PET/CT system within each patient (median interval, 29.5 days). Index targets were anatomically matched to the biopsied or surgically sampled lesion or target region. The primary malignant set included 18 histologically malignant targets; two histology-negative or indeterminate targets were included only in sensitivity analysis. Fixed [68Ga]Ga-PSMA-11-first scan order and the 45-min uptake-time difference were central interpretive constraints. Results: Across five predefined reference organs, [18F]FC303 showed lower SUVmean than [68Ga]Ga-PSMA-11 (all Benjamini-Hochberg-adjusted p < 0.001; [68Ga]/[18F]FC303 geometric mean ratio [GMR], 1.29-3.89). In the primary malignant set, [18F]FC303 lesion SUVmax was lower than [68Ga]Ga-PSMA-11 (median, 11.3 vs 18.1; paired median difference, -5.50; 95% CI, -6.85 to -2.90; Wilcoxon p = 8.4 x 10-4), with strong rank correlation (Spearman {rho} = 0.90). Passing-Bablok regression yielded {beta} = 1.13 (95% CI, 1.04-1.45), and log-Bland-Altman GMR (FC303/[68Ga]) was 0.75, consistent with proportional non-interchangeability. Tumor-to-liver and tumor-to-mediastinum ratios did not differ significantly (GMR, 1.17 [95% CI, 0.94-1.45] and 0.96 [0.80-1.15], respectively); the study was not powered for equivalence. The n = 20 sensitivity analysis showed consistent directionality. Conclusions: Under non-harmonized acquisition conditions, [18F]FC303 showed lower physiologic reference-organ SUVmean and malignant target-region SUVmax than [68Ga]Ga-PSMA-11, whereas tumor-to-liver and tumor-to-mediastinum ratios were not significantly different. Absolute SUVs were not interchangeable; [68Ga]Ga-PSMA-11-derived SUV thresholds should not be directly transferred to [18F]FC303 without tracer-specific calibration.

BackgroundRadiation therapy is integral to the curative treatment of childhood brain tumors but contributes to late neurocognitive impairment in survivors. FLASH (ultra-high dose rate, >40Gy/s) reduces normal-tissue toxicity in preclinical models, and proton-FLASH is currently the only modality capable of delivering ultra-high dose rates to the deep targets, such as pediatric brain tumors. However, two questions remain unresolved before clinical translation: (1) whether the FLASH effect can be achieved on synchrotron-based proton systems, which deliver protons in discrete spills that may be insufficient to cover a clinical target within a single delivery, and (2) which dose-rate metric, among the multiple definitions currently used in the field, best predicts the biological FLASH effect. MethodsC57BL/6 mice (7-8 weeks) received 10 Gy whole-brain RT via a clinical Hitachi ProBEAT synchrotron with CBCT-guided delivery, using three transmission-beam techniques: single-spill pencil beam scanning (SS PBS), multi-spill PBS with [~]2-second inter-spot delay (MS PBS), and passive scatter (PS), compared to conventional (CONV) delivery and unirradiated controls (n=24-28/group, equal sex distribution). Dose rate was quantified using three frameworks: field dose rate (FDR), PBS dose rate (PBSDR), and dose-averaged dose rate (DADR). Recognition memory was assessed by novel object recognition (NOR) at 6 weeks post-RT, and cognitive flexibility was assessed via touchscreen visual discrimination and reversal learning at 14 weeks. Hippocampal neuroinflammation was evaluated by immunofluorescence and immunohistochemistry for Iba1, NeuN, and GFAP. ResultsFLASH conditions were met by SS PBS and PS under all three dose-rate definitions, but MS PBS qualified as FLASH only by DADR. Despite this, neuroprotection was preserved across all three FLASH techniques: discrimination index was significantly higher for SS PBS (P=0.021), MS PBS (P=0.008), and PS (P<0.001) versus CONV, with no significant difference between FLASH techniques. On touchscreen testing, FLASH-treated females demonstrated preserved cognitive flexibility (P=0.047 vs. CONV on reversal learning correct trials). Iba1+ microglia were reduced in FLASH compared to CONV mice, with morphology suggestive of preserved homeostatic state. ConclusionsSynchrotron-based proton FLASH preserves neurocognitive function across all delivery techniques, including under multi-spill delivery essential for treating clinical-scale pediatric brain tumors. Critically, this neuroprotection was observed even for deliveries that qualified as FLASH only by DADR, identifying DADR as the dose-rate metric most relevant to the biological FLASH effect with direct implications for clinical trial design and dose-rate reporting standards.

BackgroundBispecific T-cell Engagers (TCEs) targeting B7H3 (CD276) show promise for solid tumors but are limited by systemic toxicities and poor tumor penetration. Intratumoral (IT) delivery is proposed as a solution, but the safety and spatial pharmacodynamics (PD) remain poorly defined in these malignancies. Spontaneous canine tumors serve as a highly translatable model for human therapeutic development due to its clinical, genetic, and immunological similarities to human patients. This study evaluates the feasibility of an IT-delivered B7H3:CD3 TCE in a trial that enrolls companion dogs with solid tumors. MethodsWe engineered a canine B7H3:CD3 TCE and validated its ability to induce T-cell activation and T-cell mediated cytotoxicity in vitro on several B7H3-expressing canine tumor cell lines. Two STS canine patients received intratumoral columnar injections of the TCE and saline (internal control) at fixed distance of 1.5cm using a custom-engineered multi-needle assembly. Safety was evaluated by physical examinations and hematological and biochemical changes in peripheral blood. PD response was analyzed by H&E and immunohistochemistry. ResultsIn vitro assays validated the cytotoxicity of the B7H3:CD3 TCE on B7H3+ canine tumor cell lines. TCE IT administration (7.83 g / 148.2 pmol) was well tolerated with no adverse events greater than Grade 1 and no evidence of systemic cytokine release or organ toxicity. Immunohistochemistry of tumors collected 7 days after TCE administration revealed a significant five-fold increase in CD3+ T-cell density at the TCE injection site (within 0.5 cm radius) compared to internal saline controls. ConclusionsThis study demonstrated the feasibility of evaluating pharmacodynamic response to IT delivery of B7H3:CD3 TCE, namely local T-cell accumulation. T-cell localization around the TCE injection site supports our hypothesis that effective IT immunotherapy might require enhanced volumetric coverage using multi-needle injections and/or co-stimulatory strategies to convert T-cell localization into a robust, sustained anti-tumor response.

BackgroundRadiotherapy efficacy is constrained by an immunosuppressive tumor microenvironment (TME) enriched in extracellular adenosine and suppressive myeloid populations that attenuate cytotoxic T-cell responses. The CD73-adenosine-A2a/A2b receptor axis represents a key metabolic immune checkpoint; however, the relative contributions of tumor cell-intrinsic versus host-derived adenosine signaling to radiotherapy response remain incompletely defined. MethodsUsing orthotopic murine breast carcinoma models, we interrogated radiation-induced adenosine dynamics and downstream immune remodeling through quantitative adenosine measurements, bulk RNA sequencing, and multiparameter flow cytometry. Genetically engineered models were employed to dissect the roles of tumor-derived CD73 and host A2a/A2b receptors in regulating radiosensitivity. Therapeutic studies evaluated combinatorial targeting of CD73 and A2a/A2b receptors with radiotherapy and anti-PD-1, followed by comprehensive immune profiling in breast carcinomas. ResultsTumor cell-intrinsic CD73 and host A2A receptor signaling cooperatively drive radioresistance and tumor progression. Radiotherapy induces a rapid surge in intratumoral adenosine, triggering transcriptional and cellular programs consistent with myeloid-mediated immunosuppression and lymphocyte dysfunction. Although T-cell infiltration increases at later time point post-irradiation, effector function remains constrained. Pharmacologic inhibition of CD73 and A2a/A2b receptors partially restores T-cell functionality but is insufficient for durable tumor control as monotherapy. In contrast, concurrent blockade of adenosine signaling during radiotherapy, followed by adjuvant PD-1 inhibition, amplifies adaptive antitumor immunity and significantly enhances tumor control. ConclusionsThese findings define a mechanistic link between radiation-induced adenosine signaling and immune dysfunction in the TME. Targeting the CD73-A2a/A2b axis in combination with radiotherapy and checkpoint blockade represents a rational strategy to overcome radioresistance and improve antitumor immunity. STATEMENT OF SIGNIFICANCEThe tumor and immune cell contributions to adenosine signaling play a central role in shaping the therapeutic outcomes of tumor irradiation. Therapeutic targeting of the adenosine signaling axis improves radiosensitivity and efficacy of checkpoint blockade.

Computed tomography is the largest contributor to population radiation dose from medical imaging, yet no diagnostic reference levels (DRLs) have been published from Kosovo or the Western Balkans. This retrospective audit analyzed all CT examinations performed on a 128- slice scanner at the University Clinical Centre of Kosovo between January and March 2026. After exclusions, 1,535 acquisitions from 1,092 patients across nine examination categories were analyzed. Local DRLs were defined as the 75th percentile and compared against German (BfS 2022) and Turkish (Kahraman et al., 2024) reference values. Head CT (n = 590) demonstrated CTDIvol 4.7% below the BfS DRL yet scan length 98.5% above the orientation value (median 25.8 vs 13 cm). Abdomen-pelvis CTDIvol matched the BfS reference while scan length exceeded it by 28%. Coronary CTA showed CTDIvol +377%, consistent with retrospective ECG gating. Excess scan length, not CTDIvol, is the major driver of elevated dose at this institution. The identified excesses are correctable through technologist landmarking training, protocol review, and enabling iterative reconstruction.

BackgroundRealistic PET/CT phantoms are essential for system evaluation, protocol optimization, and validation of advanced reconstruction methods. However, existing phantoms are often limited by simplified geometries, spatially uniform activity patterns, and complex preparation procedures. PurposeTo develop and evaluate PixelPrintPET, a 3D printing-based method for fabricating anatomically realistic PET/CT phantoms with spatially heterogeneous radiotracer distributions and a single-solution filling workflow that avoids physical compartmentalization. MethodsPixelPrintPET generates voxel-based printing instructions that encode spatially varying infill, which is realized during printing through modulation of filament extrusion, enabling heterogeneous activity distributions without compartmentalization of radioactivity at different activity concentrations. Calibration phantoms and anatomically structured phantoms were designed and printed using high-flow polylactic acid (PLA), with anatomical inputs derived from either digital atlas-based models or patient imaging data. The printed phantoms were subsequently filled by immersion in a radioactive solution, allowing activity distribution to be controlled by the internal porous structure. A bottom-up filling procedure with reduced surface tension was developed to ensure uniform infiltration and minimize air entrapment. Phantoms were imaged on the PennPET Explorer PET/CT system, and quantitative performance was evaluated using contrast recovery coefficient (CRC), target-to-background ratio (TBR), and comparisons with simulated or patient-derived reference data. ResultsA strong linear relationship between infill ratio and normalized signal (R2 = 0.998) was demonstrated by the calibration phantom, enabling reliable mapping between structure and activity. Additionally, air entrapment was minimized to less than 1% of the total phantom volume. In the contrast recovery phantom, CRC values were consistent with measurements using traditional phantoms. The brain phantom reproduced atlas-derived contrast patterns, with gray-to-white matter differences within 5% after accounting for resolution and other system effects. The patient-based thorax phantom showed high reproducibility across repeated scans, with differences within 3%, and closely matched the input patient image with regional differences within 10% in all regions except the lung. ConclusionsPixelPrintPET enables the fabrication of realistic, reproducible, and versatile PET/CT phantoms with a voxel-level control of the activity distribution. This approach provides a practical solution for generating patient-specific and application-specific phantoms, with the potential to accelerate system validation, protocol development, and clinical translation of advanced PET/CT technologies.

Human salivary stem/progenitor cell (hS/PC)-loaded hyaluronic acid (HA)-based hydrogels, termed 3D-salivary tissue constructs (3D-ST), hold great promise for restoring salivary gland function post-radiation injury. Here, we developed a next-generation 3D-ST using heparin-modified HA and bioactive peptide-modified hydrogels. This new formulation enables controlled pre-loading and localized presentation of heparin-binding growth factors prior to surgical implantation, providing opportunities to enhance in vivo hS/PC bioactivity. To model clinically relevant radiation injury, we established an athymic rat model subjected to computed tomography (CT)-guided fractionated radiation, resulting in hallmark features of radiation-induced salivary dysfunction. Over 60-days post-irradiation, glands exhibited progressive loss of acini, increased fibrosis, and disruption of endothelial, neuronal, and myoepithelial compartments. Within this injured environment, a surgical pocket was created to precisely implant 3D-STs to assess graft performance. Fluorescent labeling of the 3D-STs enabled longitudinal tracking post-implantation. Over 14 days, implanted 3D-STs remained structurally stable within irradiated glands, and hS/PCs remained viable without evidence of local inflammatory responses. Compared to non-injured glands, the irradiated microenvironment suppressed hS/PC proliferation and phenotype, indicating alterations in the irradiated local tissue negatively impact hS/PC bioactivity. In addition, host neurovascular migration into the 3D-ST was majorly restricted in irradiated glands, providing new opportunities to enhance biointegration. Overall, this work establishes a reproducible preclinical framework for assessing hydrogel biocompatibility and stability, cell bioactivity, and host-graft biointegration prior to scale up into preclinical large animal models. This study has successfully established a tractable approach for improving 3D-ST formulations to enhance hS/PC expansion, differentiation, and biointegration following implantation into radiation-injured beds.

BackgroundIonizing radiation (IR) accelerates atherosclerosis through induction of cellular senescence, DNA damage, defective efferocytosis, and dysregulation of clonal hematopoiesis (CH) drivers. Although low-dose colchicine reduces ischemic cardiovascular events in coronary artery disease, the precise molecular mechanisms underlying its vasculoprotective effects remain incompletely defined, and whether it mitigates radiation-associated vascular injury is unknown. MethodsBone marrow-derived macrophages (BMDMs) were pretreated with low-dose colchicine and exposed to 2 Gy IR. Molecular effects were assessed by RNA-seq, immunoblotting, and molecular docking. In vivo effects were tested in a partial carotid ligation (PLCL) model using spatial proteomics. Human monocyte-derived macrophages (HMDMs) from thoracic malignancy patients were analyzed before and after radiation therapy (RT). ResultsLow-dose colchicine suppressed IR-induced macrophage senescence signaling while preserving NRF2 activity. In a cell-free assay, colchicine directly activated aldehyde dehydrogenase 2 (ALDH2) in a dose-dependent manner (EC50 1-5 nM), identifying ALDH2 as a direct molecular target of colchicine. Following irradiation, colchicine restored ALDH2, reduced mitochondrial (mt)ROS-dependent p90 ribosomal S6 kinase (p90RSK) activation and lipid peroxidation, preserved TET2 and DNMT3A expression, and rescued impaired efferocytosis while preventing nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) depletion. These protective effects were ALDH2-dependent, as they were lost with ALDH2 inhibition or depletion and were mimicked by pharmacologic ALDH2 activation. In vivo, colchicine attenuated radiation-induced atherosclerosis and macrophage senescence-associated stemness (SAS). Consistently, macrophages from patients after RT showed reduced ALDH2 with increased mtROS, lipid peroxidation, and senescence. ConclusionThese findings identify ALDH2 as a previously unrecognized molecular target of colchicine that links mitochondrial redox control to suppression of radiation-induced macrophage senescence and atherosclerosis and may contribute to the efficacy of low-dose colchicine in cardiovascular disease.

PurposeLow-dose radiation-induced adaptive response (LDRIAR) is well documented, but its role in early DNA damage signalling remains unclear. This study aimed to investigate whether adaptive response influences initial DNA double-strand break (DSB) recognition, as reflected by {gamma}H2AX foci formation, and to evaluate its time-dependent expression in human lymphocytes. Materials and MethodsPeripheral blood lymphocytes from three healthy donors were exposed to a priming dose followed by a challenging dose at defined time intervals. DNA damage was assessed using {gamma}H2AX foci analysis, comparing acute and split-dose exposures in both PHA-stimulated (large) and non-stimulated (small) lymphocytes. ResultsA clear time-dependent adaptive response was observed. No significant reduction in {gamma}H2AX foci was detected at 1 h (p > 0.05). At 2 h, a significant decrease was observed ([~]7-8% in large and [~]13% in small lymphocytes; p < 0.01), which increased at 4 h ([~]12% and [~]22%, respectively; p < 0.001). The maximal response occurred at 15 h, with reductions of [~]40- 43% in large and [~]27% in small lymphocytes (p < 0.001). Small lymphocytes exhibited an earlier response, while large lymphocytes showed a greater magnitude at later time points. The temporal trend was consistent across donors, with minor variability at later intervals. ConclusionsThe findings demonstrate that LDRIAR is reflected at the level of DNA damage signalling and follows a defined temporal pattern with cell-type specificity. This suggests that adaptive response may influence early DSB-associated processes, contributing to a better understanding of radiation response mechanisms in radiobiology.

Nano- and microplastics (NMPs), by-products of the fragmentation and degradation of plastic products, are ubiquitous environmental contaminants, yet their burden in pediatric immune tissues and functional consequences for developing immunity remain unknown. Here we report the first comprehensive characterization of NMPs in surgically excised pediatric tonsils (n = 30) using pyrolysis gas chromatography- mass spectrometry (Py-GC/MS), Nile Red fluorescence microscopy, and optical photothermal infrared (O-PTIR) spectroscopy. NMPs were detected in all specimens, with polystyrene, polyethylene, polyethylene terephthalate, and acrylonitrile butadiene styrene present in >90% of samples. To bridge clinical exposure data with mechanistic insight, we formulated a cryo-milled multi-polymer mixture reflecting the patient-derived polymer profile and challenged human lymphoid aggregate culture (HLAC) tonsil organoids at environmentally relevant concentrations. Multiplexed cytokine profiling of culture supernatants revealed a robust early inflammatory response at day 3, with significant upregulation of IL-6 (p = 0.011) and MIP-1{beta}/CCL4 (p = 0.011), followed by convergence toward control levels by day 14. Functional cytokine modules spanning immune, metabolic, structural, and growth factor pathways showed coordinated deviation from controls at day 3 post-exposure with subsequent normalization. Fluorescence-guided depth profiling demonstrated time-dependent penetration of 100 nm particles into organoid aggregates (70% tissue depth at day 3 versus 95% at day 14), and transmission electron microscopy revealed intracellular polyethylene within lymphocyte lysosomes. These findings establish pediatric tonsils as a sentinel tissue for NMP bioaccumulation and demonstrate that environmentally relevant polymer mixtures elicit transient but significant immunomodulatory responses in human lymphoid tissue, with implications for mucosal and systemic immune health in children. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/728317v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@19395f4org.highwire.dtl.DTLVardef@5a0380org.highwire.dtl.DTLVardef@19c0741org.highwire.dtl.DTLVardef@a052c5_HPS_FORMAT_FIGEXP M_FIG Structure: Translational pipeline from clinical tissue characterization to patient-informed preclinical modeling of nano-microplastic (NMP) exposure in pediatric lymphoid tissue. Pediatric tonsil tissue collected from clinically indicated tonsillectomies underwent tissue digestion for NMP characterization to identify NMP type and size distributions. In parallel, tonsil tissue was used to generate human lymphoid aggregate culture (HLAC) organoids that recapitulate the cellular complexity of the native tissue. These patient-derived organoids were then exposed to environmentally relevant compositions and concentrations of NMPs over time-course experiments, with longitudinal assessment of immunomodulatory responses including cytokine profiling and functional readouts. This bedside-to-bench approach establishes a physiologically relevant human system for investigating NMP-immune interactions, bridging clinical tissue analysis with mechanistic preclinical modeling to inform understanding of pediatric environmental exposures and their potential health impacts. C_FIG

Background: Traditional bone scintigraphy for detecting malignant bone metastases is limited by suboptimal accuracy and radiation exposure. Whole-body magnetic resonance imaging (WB-MRI), while an alternative, requires lengthy scan times and high patient compliance. Purpose: To develop a novel, rapid whole body bone screening (WB-RBS) MRI protocol and evaluate its diagnostic performance for bone metastasis detection. Materials and Methods: Patients with pathologically confirmed malignancies and healthy controls were prospectively enrolled. All participants underwent WB-RBS (acquisition time: about 10 min); patients additionally underwent WB-MRI (about 70 min). Three radiologists, blinded to clinical data, independently evaluated the images for bone metastases. A consensus expert diagnosis served as the reference standard to calculate the diagnostic performance of WB-RBS. Specificity was further assessed in the healthy control group. Results: Seventy patients and 19 healthy controls were included. WB-RBS demonstrated excellent inter-reader agreement at the patient level. Compared with the reference standard, WB-RBS achieved an accuracy of 77.1%-91.4% at the patient level and a slightly lower accuracy (70.6%-82.5%) at the lesion level. At diagnostic confidence thresholds 1-3, the correlations between WB-RBS ratings and the reference standard were statistically significant for both patient- and lesion-level analyses. Conclusion: WB-RBS showed favorable inter-reader agreement and high accuracy for bone metastasis screening at the patient level, while substantially reducing scan time and cost. Its rapid, radiation-free nature and high accessibility offer distinct clinical advantages, supporting its potential as an alternative screening tool to conventional bone scintigraphy.

Ionizing radiation induces molecular responses that may be used to estimate exposure when physical dosimeters are unavailable. Here we present two large-scale proteomics datasets generated from mouse dorsal skin punch samples collected following controlled X-ray exposures spanning multiple doses, dose rates, and post-exposure time points. Experiment 1 comprised 96 samples (including 16 reference samples) collected 6 days after exposure to 0-75 cGy delivered at either 30 or 300 cGy/min. Experiment 2 comprised 936 samples (including 236 reference samples) exposed to 0-100 cGy at either 3 or 28 cGy/min dose rates and harvested between 7 and 150 days post-exposure. All samples were processed using a standardized workflow involving automated bead-based digestion and data-independent acquisition mass spectrometry. The datasets include multiple pooled reference sample types, process controls, and system suitability standards ensuring high quality data. All data presented are available via ProteomeXchange at several levels of processing, from raw files through normalized peptide- and protein-level abundance matrices suitable for biomarker discovery and machine learning applications. This dataset will facilitate generation of new insights into the biological changes and molecular signatures resulting from X-ray exposure in mice and may also help inform future studies in humans.

Treatment of advanced head and neck squamous cell carcinoma (HNSCC) often involves radiotherapy combined with chemotherapy, targeted therapy, or immunotherapy. However, due to its anatomical and molecular heterogeneity, identifying the most effective treatment for each patient remains a major clinical challenge. To address this need, we developed a high-throughput organoid-based drug screening platform that uses patient-derived organoids to assess candidate treatment regimens. We validated the platform by establishing bioprinted 3D organoids of human HNSCC cell lines and exposing them to X-ray radiation in combination with various small-molecule drugs and biologics. We quantified viability using ATP release assays and assessed extracellular matrix (ECM) invasion with a machine learning-based brightfield image analysis pipeline. Proof-of-concept experiments with HPV-negative HNSCC lines (HN30 and HN31, established from primary and metastatic disease from the same patient) and HPV-positive HNSCC cells (SCC154) revealed different therapy agents that can radiosensitize each cell line. Image analysis showed that copanlisib, afatinib, and ibrutinib could limit ECM invasion of HN31, while the AKT inhibitor ipatasertib promotes invasion of HN30 cells, consistent with previous studies. Application of the platform to patient-derived HPV+ oropharyngeal tumor organoids showed that they shared sensitivity to several agents while also exhibiting differences against certain therapies. Cetuximab, sorafenib, and nedisertib significantly radiosensitized organoids from two clinical samples. This work demonstrates the feasibility of performing sensitivity screening by integrating bioprinting, conventional viability assays, and advanced image analysis techniques. This platform has the potential to enable a personalized therapeutic pipeline for patients with advanced HNSCC, optimizing responses to radiotherapy and targeted agents to improve clinical outcomes while avoiding modulators that may promote tumor invasion.