Radiotherapy and Oncology

Background and purpose: Radiation-induced lymphopenia is associated with adverse outcomes in thoracic malignancies. FLASH radiotherapy delivers radiation over a timescale of hundreds of milliseconds, potentially reducing the fraction of irradiated circulating lymphocytes. In this study, we investigated whether FLASH mitigates lymphopenia after thoracic irradiation delivered with protons or photons. Materials and methods: C57BL/6 mice received three 13.5-Gy whole-heart fractions at 48-hour intervals using FLASH or standard dose-rate proton irradiation at the University of Pennsylvania (n=15), with photon validation at Queen's University Belfast (n=72). Leukocytes and CD4 T cells, CD8 T cells, B cells, and NK cells were quantified by hemocytometer and flow cytometry. A continuous-time Markov model simulated lymphocyte trafficking, dose accumulation, and post-irradiation recovery. Results: FLASH attenuated leukocyte depletion across both proton and photon irradiation modalities. In the proton cohort, white blood cell counts were significantly higher after FLASH at D1, D3, D7, and D14; CD4 T cells and NK cells were preserved through D14, while CD8 T cell sparing persisted through D21. Photon FLASH preserved CD45 leukocytes at D1, D3, D7, and D21, with sustained CD8 sparing at D21. Modeling showed that FLASH shifted the lymphocyte dose distribution toward lower exposures, increasing the proportion of lymphocytes receiving <1 Gy from 2.4% to 16.4%, and reduced the proportion of lymphocytes repeatedly irradiated across all three fractions from 36.3% at standard dose rate to 9.18%, despite similar median cumulative doses. The spleen contributed substantially to cumulative lymphocyte dose, and marrow-entering lymphocytes displayed a more high-dose-enriched distribution after FLASH irradiation. Conclusion: FLASH consistently mitigated radiation-induced lymphopenia for proton and photon modalities, with durable CD8 T cell preservation. These findings support a kinetic mechanism and provide a rationale for combining FLASH radiotherapy with immune-sparing planning and immunotherapy.

Ultra-high dose rate radiotherapy with electrons and protons has shown potential for cancer treatment by effectively targeting tumors while sparing healthy tissues (FLASH effect). This study aimed to investigate the potential FLASH sparing effect of ultra-high-dose rate helium ion irradiation, focusing on acute brain injury and subcutaneous tumor response in a preclinical in vivo setting. Raster-scanned helium ion beams were used to compare the effects of standard dose rate (SDR at 0.2 Gy/s) and FLASH (at 141 Gy/s) radiotherapy on healthy brain tissue. Irradiation-induced brain injury was studied in C57BL/6 mice via DNA damage response, using nuclear {gamma}H2AX as a marker for double-strand breaks (DSB). The integrity of neurovascular and immune compartments was assessed through CD31+ microvascular density and activation of microglia/macrophages. Iba1+ ramified and CD68+ phagocytic microglia/macrophages were quantified, along with the expression of inducible nitric oxide synthetase (iNOS). Tumor response to SDR (0.2 Gy/s) and FLASH (250 Gy/s) radiotherapy was evaluated in A549 carcinoma model, using tumor volume and Kaplan-Meier survival as endpoints. The results showed that helium FLASH radiotherapy significantly reduced acute brain tissue injury compared to SDR, evidenced by lower levels of DSB and preserved neurovascular endothelium. Additionally, FLASH radiotherapy reduced neuroinflammatory signals compared to SDR, as indicated by fewer CD68+ iNOS+ microglia/macrophages. FLASH radiotherapy achieved tumor control comparable to that of SDR radiotherapy. This study is the first to report the FLASH sparing effect of raster scanning helium ion radiotherapy in vivo, highlighting its potential for neuroprotection and effective tumor control.

PurposeIn vitro experimental radiobiology is a fundamental tool for understanding the cellular and molecular mechanisms involved in the response to ionizing radiation. Conventional experimental designs require separate irradiations to achieve different absorbed doses, thereby introducing experimental variability between irradiation sessions due to inter-session variability in both culture conditions and irradiation geometry. Here, we developed a multidose irradiation system for multiwell cell culture plates that enables the simultaneous delivery of three distinct dose levels within a single plate, thereby reducing resource consumption and operating time MethodsThe system was designed using a clinical linear accelerator delivering 6 MV X-rays and a 3D conformal irradiation approach based on CT imaging. Dose calculations for 200, 400 and 600 cGy were performed using Monaco Version 5.11. The irradiation geometry was optimized to achieve distinct and well-separated dose regions while preserving dose uniformity within each dose level. Treatment planning showed good agreement between estimated and prescribed dose levels, with mean dose deviation ranging from 0.35-1.63%. Physical verification using TLDs and radiochromic films demonstrated high dosimetric accuracy, showing deviations of 0.5-1.0% and 0.57-3.0%, respectively, expressed as the deviation of the measured mean dose from the nominal administered dose levels. Biological assessment included clonogenic assays in human tumor cell lines and a metabolic assay. Clonogenic assays showed a high concordance between the multidose and single-dose irradiation, with comparable linear-quadratic model fits (extra sum-of-squares F-test, p = 0.0923), confirming preservation of the intrinsic radiobiological response under simultaneous irradiation. ConclusionsThese results support the reliability of the proposed multidose irradiation system for in vitro radiobiology. This robust and reproducible system enables efficient characterization of radiobiological parameters.

PurposeThis study systematically investigates the radiobiological effects of Very High Energy Electrons (VHEE) generated by a laser-plasma accelerator (LPA), in comparison with Conventional Intermediate Energy Electrons (CIEE) from a conventional linear accelerator (LINAC). Using in vitro, ex vivo, and in vivo models, we evaluate and compare their potential toxicity on healthy tissues. Methods and MaterialsCell survival, tissue response, and developmental toxicity were assessed across three biological models. In vitro, human fibroblasts (MRC5-hTERT) were used to generate post-irradiation survival curves. Ex vivo, precision-cut lung slices (PCLS) from mice were analyzed for radiation-induced inhibition of cell proliferation. In vivo, zebrafish embryos were used to evaluate developmental toxicity through body length and spinal curvature measurements. VHEE irradiations were performed using a broadband electron beam spanning 50-300 MeV, using the Salle Jaune LPA (Laboratoire dOptique Appliquee, France), while CIEE exposures were performed with a 7 MeV conventional LINAC (Institut Curie, France). ResultsIn vitro, MRC5-hTERT cells showed no significant difference in radiosensitivity between VHEE and CIEE, with comparable D10 values (p-value = 0.7). In the ex vivo model, both beams induced a dose-dependent decrease in cell division with no significant inter-beam differences at any dose level (p-value > 0.99). In vivo, zebrafish embryos exhibited dose-dependent body shortening and increased spinal curvature following both VHEE and CIEE exposure. No significant differences were observed between the two modalities at matched doses for any measured metric (p-value[&ge;] 0.5). ConclusionThis study presents the first comprehensive radiobiological evaluation of a laser-driven VHEE beam across multiple biological models. Under the investigated conditions, VHEE and CIEE irradiations exhibit similar biological toxicity. These findings support the feasibility and potential of VHEE generated with LPA for future clinical applications.

Herein, we assess the potential of low-dose stereotactic radiosurgery (SRS) to modulate neuronal network states without apparent damage to cellular integrity. Using a small animal radiation research platform (SARRP), a 1 mm3 focal target in the mouse visual cortex was irradiated with doses of 5, 20, and 40 Gy. One-month later a significant dose-dependent increase in excitatory synapse numbers was observed, notably limited to the treated visual cortex and not the adjacent somatosensory cortex. Six months post-irradiation, cortical neuronal microcircuit activity was monitored in awake mice using high sensitivity two-photon calcium imaging. A single 5 Gy dose resulted in a significant microcircuit-wide increase of spontaneous neuronal activity, consistent with a lasting shift in the functional architecture of the irradiated nodal network. At higher SRS doses (40 Gy) this neuromodulatory window appears to close. In aggregate, these data suggest that low-dose radiation could, in some circumstances, be exploited by selected high precision SRS technologies to durably modulate neuronal circuit disorders. Some, or even all the clinical benefits reported in the companion article by Zhao et al. are likely attributable to the biological properties we sought to characterize in our research. One Sentence SummaryLow-dose stereotactic radiosurgery effectively and durably modulates neuronal excitability via synaptic re-organization and could open new clinical possibilities for neuromodulation.

BackgroundAfferent baroreflex failure (ABF) is an underrecognized but debilitating complication among head and neck cancer survivors, especially in oropharyngeal cancer (OPC), a malignancy with excellent prognosis. ABF is mainly caused by radiation therapy (RT), with neck surgery and some chemotherapies also contributing. It manifests as blood pressure lability, including severe hypertension or hypotension, syncope, and arrhythmias. ObjectivesTo determine the prevalence and predictors of ABF-associated manifestations among OPC survivors treated with modern RT. MethodsWe retrospectively studied OPC patients treated with RT at a tertiary cancer center between 2016-2019. Clinical data were collected from RT initiation to last follow-up or death. ABF-associated manifestations included new or worsening hypertension, hypotension requiring intervention, arrhythmias, and syncope. Secondary endpoints included new or worsened carotid artery atherosclerosis, stenosis, transient ischemic attack (TIA), stroke, and all-cause mortality. ResultsAmong 393 patients (88% men, 91% White, mean age 61{+/-}10 years), 9.4% developed hypertension, 5.3% hypotension, 3.8% syncope, and 3.3% arrhythmias over median 6.3-year follow-up. Overall, 19.1% developed at least one ABF-associated manifestations. New or worsened carotid atherosclerosis occurred in 38.9%, with 7.1% developing >50% stenosis and 2.3% experiencing TIA or stroke. Mortality was 21.4%. On cause-specific multivariable Cox analysis, older age (adjusted hazard ratio [aHR] 1.03; 95% confidence interval [CI] 1.01-1.06), valvular disease (aHR 2.85; CI 1.03-7.92), T4 cancer (aHR 1.90; CI 1.10-3.27), and platinum-taxane chemotherapy (aHR 1.86; CI 1.13-3.05) independently increased risk of ABF-associated manifestations. ConclusionsNearly 1 in 5 OPC survivors treated with RT develop ABF-associated manifestations, highlighting the need for early recognition and surveillance.

Background and purposeThe normal tissue sparing afforded by FLASH radiotherapy (RT) is being intensely investigated for potential clinical translation. Here, we studied the effects of FLASH proton RT (F-PRT) in the reirradiation setting, with or without hypofractionation. Chronic toxicities in three murine models of normal tissue toxicity including the intestine, skin, and bone were investigated. Materials and methodsIn studies of the intestine, single-dose irradiation was performed with 12 Gy of Standard proton RT (S-PRT), followed by a second dose of 12 Gy of F-PRT or S-PRT. Additionally, a hypofractionation scheme was applied in the reirradiation setting (3 x 6.4 Gy of F-PRT or S-PRT, given every 48 hrs). In studies of skin/bone of the murine leg, 15 Gy of S-PRT was followed by hypofractionated reirradiation with F-PRT or S-PRT (3 x 11 Gy). ResultsCompared to reirradiation with S-PRT, F-PRT reduced intestinal fibrosis and collagen deposition in the reirradiation setting and significantly increased survival rate, demonstrating its protective effects on intestinal tissues. In previously irradiated leg tissues, reirradiation with hypofractionated F-PRT created transient dermatitis that fully resolved in contrast to reirradiation with hypofractionated S-PRT. Lymphedema was also alleviated after a second course of radiation with F-PRT, along with significant reductions in the accumulation of fibrous connective tissue in the skin compared to mice reirradiated with S-PRT. The delivery of a second course of fractionated S-PRT induced tibial fractures in 83.3% of the mice, whereas only 20% of mice reirradiated with F-PRT presented with fractures. ConclusionThese studies provide the first evidence of the sparing effects of F-PRT, in the setting of hypofractionated reirradiation. The results support FLASH as highly relevant to the reirradiation regimen where it exhibits significant potential to minimize chronic complications for patients undergoing RT.

PurposeThis preclinical study aimed to verify the FLASH effect of compact single high-energy X-ray source (CHEXs) and to explore whether three irradiations with single-gantry rotation two 30 s pauses can generate FLASH effect in mice. Materials and methodsThe absolute dose and pulsed beam of the CHEXs were measured using an EBTXD radiochromic film and fast current transformer. Healthy C57BL/6J female mice and a subcutaneous tumor model were irradiated under different conditions: sham (control), FLASH-RT (FLASH1: delivering the total dose in 1 fraction; FLASH3: delivering the total dose with two 30 second pauses to simulate a three-field delivery where the gantry rotation is occurring within 30 seconds), and conventional dose rate radiotherapy (CONV-RT). Various total doses were administered to the corresponding normal tissues (whole thorax, 30 Gy; whole abdomen, 12 Gy; and skin, 36 Gy) and tumors (CT26, 16.5 Gy; and LLC, 18 Gy). Survival status, normal tissue damage, and tumor growth suppression were recorded in each group. ResultsThe average dose rate of the CHEXs exceeded 40 Gy/s. For whole-thorax and skin irradiation, both FLASH1 and FLASH3 demonstrated protective effects. For whole-abdomen irradiation, FLASH1 exhibited a superior protective effect. No significant differences in tumor growth responses were observed between the FLASH1, FLASH3, and CONV-RT groups (P>0.05). ConclusionThis study confirmed that the FLASH effect could be triggered using CHEXs FLASH radiotherapy, and demonstrated that three irradiations with single gantry rotation two 30 s pauses can trigger the FLASH effect, indicating the potential benefit of CHEXs 3D conformal radiotherapy. Our findings indicate that further clinical trials on CHEXs are warranted.

BackgroundConventional x-ray-based radiotherapy is a standard treatment for patients with brain tumors. However, is associated with systemic effects like lymphopenia that correlates with poor prognosis. Proton therapy has emerged as a new radiation strategy, given that the lower entry dose and absence of exit dose can be exploited to spare healthy brain tissues and reduce side-effects caused by systemic inflammation. We evaluated if brain irradiation with protons could spare circulating leukocytes along with other variables in rodent models. MethodsTumor-free C57BL/6 mice were irradiated with a total dose of 20Gy in 2.5Gy twice-daily sessions over four consecutive days with either x-rays or protons. Groups of mice were defined according to irradiation volume (whole-brain or hemisphere) and dose rate (1 or 2Gy/min). Blood was withdrawn at various time points and circulating lymphoid, with myeloid subpopulations analyzed using flow cytometry. Brain tissue histochemical analyses were performed late after irradiation. ResultsBlood sampling showed severe and acute radiation-induced lymphopenia after x-rays, with marked depletion of 50% CD4+ and CD8+, as well as B and NK cells. With protons, the decrease was 20% on average for whole-brain irradiations, suggesting a conservative effect on circulating lymphocytes. The data showed no effect in CD11b+ myeloid cells for both x-rays and protons. Histological analyses revealed a more intense expression level of CD68 and Iba1 immunostaining after x-ray irradiation. GFAP staining was well detected after both beams. ConclusionProton therapy for brain tumors differs from photon therapy in terms of its effects on circulating cells and tissues. Key pointsO_LIX-ray brain irradiation induced an acute severe lymphopenia, with a reduction of at least 50% lymphocytes. The whole-brain irradiation caused a more pronounced decrease in lymphocytes than hemisphere irradiation. Proton brain irradiation exhibited a conservative effect on circulating leukocytes. C_LIO_LIX-ray irradiation-induced lymphopenia is followed by a recovery of all lymphocyte subpopulations to control levels. However, this recovery is longer for CD3+ lymphocytes, and B and NK cells, depending on irradiation modalities. C_LIO_LILong-term brain tissue histochemical analyses demonstrated differences between the two beams, consisting of a macrophage/microglial activation seen mostly after x-rays while an astrocyte reaction was seen after brain exposure to the two beams. These differences may explain the disparities observed in leukocytes, thereby favoring a specific biological reaction between the brain and blood. C_LI Importance of the StudyOur study demonstrated that while whole-brain or hemispheric irradiation with x-rays resulted in lymphopenia, proton brain irradiation exhibited a conservative effect on circulating lymphocytes, which was paralleled by a less intense brain tissue reaction.

Radiation induced gastrointestinal mucositis (GIM) is a severe complication of radiotherapy that compromises patient quality of life and treatment efficacy. This study assessed the therapeutic potential and molecular mechanisms of two forms of herbal extracts, Compound Kushen Powder (CKP) and Compound Kushen Injection (CKI) in a rat model of GIM. Administration of either CKP or CKI to irradiated animals significantly reduced the severity of GIM symptoms. Transcriptomic analysis of jejunum and colon mucosa identified candidate mechanisms as well as common pathways between CKP and CKI. CKP modulated innate immune activation and inflammatory signalling, affecting genes such as Socs3 and Tlr4, while CKI promoted tissue repair and oxidative stress resistance through genes including Osm, Epha2, and Stat3. However, both forms of herbal extract enhanced stress response and stimulus regulation in our GIM model. These findings reveal common mechanisms shared by CKP and CKI that suppress symptoms of radiation induced GIM, along with extract specific effects on distinct pathways, acting on different set of genes.

BackgroundThoracic radiation therapy (TRT) is commonly used for breast, lung, and lymphoid cancers. While its cardiotoxic effects, particularly coronary artery disease, are well recognized, less is known about its impact on arrhythmia-related hospitalizations. MethodsA retrospective cohort study using the National Inpatient Sample (2016-2022) was conducted. Admissions for atrial fibrillation or flutter were identified using ICD-10 codes, and prior TRT was determined from thoracic malignancy and radiation history codes. Propensity score matching and doubly robust multivariable regression were used to evaluate outcomes. The primary endpoint was in-hospital mortality; secondary endpoints included length of stay (LOS) and total charges. ResultsAmong 3,198,304 weighted admissions, 8,570 (0.27%) had prior TRT. After matching, TRT was associated with higher odds of in-hospital mortality (adjusted odds ratio [aOR] 1.97; 95% CI 1.17- 3.32; p=0.010) and longer LOS (+0.30 days; 95% CI 0.05-0.55; p=0.019) without increased costs (p=0.202). TRT patients also had higher odds of palliative consultation (aOR 2.60, p<0.001) and DNR status (aOR 1.97, p<0.001), but lower odds of acute kidney injury (aOR 0.66, p<0.001). ConclusionPrior TRT is linked to greater in-hospital mortality and resource utilization during atrial fibrillation or flutter admissions, likely reflecting cumulative cardiovascular injury. These findings support closer surveillance and early intervention for this high-risk population.

Purpose Radiation injury to growth plates commonly leads to skeletal late complications including short stature, limb length-discrepancy, and scoliosis/kyphosis in pediatric oncology patients. We aimed to understand the acute responses of direct growth plate irradiation that result in skeletal late complications. Materials and methods We first established an in vivo model of focal growth plate irradiation that recapitulates the clinical development of skeletal late complications and used it to explore the responses of growth plate chondrocytes within the first 72 hours of radiation exposure. To monitor acute effects of radiation exposure on human chondrocytes, rare human growth plate biopsies were exposed to ionizing radiation ex vivo. Using these approaches, we applied clonal genetic tracing and immunofluorescence to monitor changes at the cellular and molecular levels. Functional in vivo perturbations were conducted with clinically-relevant autophagy inhibitor, hydroxychloroquine. Results Growth plate irradiation disrupted the continuous production of chondrocytes required for bone elongation and was associated with DNA damage throughout the growth plate. Indicators of growth plate activity, SOX9 and the phosphorylated form of ribosomal protein S6, decreased during a 6- and 24-hour post-irradiation window but returned to normal levels 72 hours after irradiation. We identified a surge in autophagic flux throughout the growth plate during this window, based on temporal SQSTM1 and LAMP1 protein levels. The earliest stages of these response mechanisms are conserved between species and relevant to humans. Hydroxychloroquine treatment immediately after radiation injury in mice impaired growth plate regeneration, resulting in more severe late complications. Conclusion Our findings demonstrate that autophagy is an important acute response to irradiation in growth plate chondrocytes, revealing a novel potential therapeutic target for preventing radiation-induced skeletal late complications.

Radiotherapy (RT) is constrained by the narrow therapeutic window between tumour control and normal-tissue toxicity. While FLASH RT has been reported to reduce normal-tissue toxicity at ultra-high dose rates (UHDR) with preserved tumour control in several preclinical models, the radiobiological potential of temporal regimes intermediate between conventional fractionation and FLASH remains largely unexplored. Here, we use a laser-plasma accelerator (LPA) to access this regime by delivering sub-picosecond very high-energy electron (VHEE, 50-300 MeV) pulses at ultra-high instantaneous dose rates (UHIDR; > 109 Gy s-1) with externally programmable inter-pulse intervals. We introduce Pulsed Fast Fractionation (PFF): ultrashort (fs-ps) pulses delivered under UHIDR conditions with second-scale inter-pulse intervals and implementation-dependent dose per pulse. In this work, we vary the inter-pulse interval from 1 to 10 s while keeping dose per pulse and total dose constant, and identify an interval of 1 s that enhances normal-tumour separation: human fibroblasts exhibit reduced toxicity, whereas colorectal carcinoma cells show increased cytotoxicity under matched dose conditions. In vivo, zebrafish embryos reproduce the interval-dependent normal-tissue protection, supporting relevance in a whole-organism context. These results establish second-scale pulse timing as a biologically active degree of freedom for VHEE delivery, positioning PFF as a temporally optimised approach that complements spatial dose modulation and extends the current fractionation-FLASH framework.

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.

Definitive radiotherapy (RT) for prostate cancer (PC) with dose intensification and/or focal boosting has excellent oncologic outcomes, but many patients experience adverse events. Dose escalation to the whole prostate improves outcomes at the expense of increased late adverse events. Intraprostatic recurrence after definitive RT typically occurs at the site of the primary tumor, suggesting that dose to the site of the dominant lesion is an important predictor of future failure. The efficacy and safety of tumor-focused RT compared to that of standard RT for definitive treatment of localized PC has not been assessed. RadTARGET (RAdiation Dose TAiloRing Guided by Enhanced Targeting) is a phase II randomized trial that aims to demonstrate superior safety of image-guided, tumor-focused RT compared to standard RT for acute genitourinary (GU) or gastrointestinal (GI) in the setting of definitive RT for intermediate- and high-risk PC. The study intervention is image-guided, tumor-focused RT with dose intensification of cancer visible on imaging and dose de-intensification to remaining prostate. Patients will be randomized to two arms: those who receive standard RT dose and those that receive tumor-focused RT. The study population will be patients with intermediate- or high-risk PC planning to undergo definitive RT with or without systemic therapy. The primary endpoint to compare between randomized arms is acute GU or GI grade [&ge;]2 adverse events. Participant and study duration are 5 years and 8 years, respectively. RadTARGET will compare the efficacy and safety of tumor-focused RT to that of standard RT for definitive treatment of localized PC. We hypothesize that the tumor-focused approach will substantially reduce adverse events after prostate RT while retaining high efficacy. If this hypothesis is confirmed, we will conclude that a phase III randomized control trial is warranted to formally establish oncologic non-inferiority compared to the current standard of whole-gland dose escalation.

BackgroundRadiation therapy is a standard-of-care oncological treatment for central nervous system (CNS) malignancies. However, as survival outcomes improve, radiation-induced injury to normal brain tissue has increased in clinical significance. CNS radiation injury is a delayed, multifactorial process characterized by impaired neurogenesis, reactive gliosis, and persistent functional deficits. Mechanistic exploration and development of effective radiation mitigators have been limited by the lack of scalable, human-relevant models. MethodsMature human iPSC-derived cortical organoids were exposed to single-dose or clinically relevant fractionated radiation (5 x 2 Gy). DNA damage, apoptosis, and growth dynamics were assessed longitudinally. Structural organization, synaptic integrity, and neuroinflammatory responses were evaluated by immunofluorescence and real-time PCR. Transcriptomic profiling was performed at 72 hours and 2 weeks after fractionated radiation to capture acute and delayed effects. Two candidate radiation mitigators, NSPP and amisulpride, were tested for their therapeutic effects within the organoid system. ResultsCortical organoids exhibited partial recovery following single doses up to 4 Gy or fractioned irradiation. Transcriptomic analyses revealed that radiation not only reduced overall cell viability but also reshaped lineage trajectories, characterized by depletion of neural stem/progenitor populations, loss of neuronal identity, enhanced gliogenesis, increased inflammatory cytokines, and disrupted cortical layering and synaptic integrity. Treatment with NSPP or amisulpride attenuated injury-associated transcriptional and structural alterations. ConclusionHuman cortical organoids recapitulate key features of radiation-induced neural injury, recovery, and therapeutic modulation, providing a robust, scalable, and human-relevant platform for studying CNS radiation biology and preclinical screening of candidate radiation mitigators. Key pointsO_LIHuman iPSC-derived cortical organoids enable study of human CNS radiation responses. C_LIO_LIOrganoids recover after single-dose and fractionated radiation relevant to clinical exposure. C_LIO_LIThe platform supports scalable, human-relevant testing of radiation mitigation strategies. C_LI

Significance3D tumor spheroids are more physiologically representative of in vivo patient tumors compared to 2D monolayer culture. However, their 3D nature challenges the use of conventional biological techniques like proliferation assays, fluorescence microscopy, and the clonogenic assay, which is the gold standard method for assessing cell survival following radiation. However, clonogenic assay requires spheroid disaggregation. AimNon-invasive volumetric imaging with dynamic optical coherence tomography (dOCT) enables cellular activity to be visualized with spatial resolution within 3D tumor spheroids. Cellular activity observed via dOCT in irradiated prostate tumor spheroids was quantified for comparison with conventional biological techniques. ApproachA Varian TrueBeam linear accelerator was used to irradiate spheroid and monolayer cultures with a 6 MV beam. Cellular activity was estimated from dOCT images generated via frequency banding and compared to clonogenic assay, proliferation assay, fluorescence microscopy, and 3D cell simulation. ResultsProstate cancer cells cultured as spheroids demonstrated improved radio-resistance via clonogenic assay compared to monolayer culture. The dOCT method demonstrated quantitative and qualitative agreement with proliferation assay and fluorescence microscopy, respectively. ConclusionsA longer duration of repeated dOCT measurement in tumor spheroids following radiation treatment could offer a non-invasive alternative to the clonogenic assay.

ObjectiveThis study evaluated the hypothesis that baseline tissue oxygen (pO2) would modulate FLASH damage sparing in murine skin, comparing MeV electron ultra-high dose rate (UHDR) versus conventional dose rate (CDR) irradiations. ApproachMurine leg skin pO2 was systematically varied and measured during irradiation from a Mobetron 9 MeV linac at 25 Gy, comparing UHDR ({approx}240 Gy/s) to CDR ({approx}0.16 Gy/s), for skin damage outcomes. Radiolytic oxygen consumption, go2 (mmHg/Gy), was also quantified in vivo. Baseline tissue pO2 was systematically modulated in 5 different treatment cohorts, using known methods of altering the inhaled gas (room air, 100% oxygen, or carbogen) and through applied limb vascular compression (partial or full). Induced skin damage was scored daily per mouse. Main ResultsFLASH skin sparing was observed in groups with partial leg clamping (pO2{approx}7{+/-}4mmHg), inhaled air (pO2{approx}12{+/-}6mmHg) and oxygen (tissue pO2{approx}16{+/-}4mmHg), while reduction in ulceration progression was significant just in the air inhalation group. No FLASH effect was observed at zero oxygen, via complete blood flow occlusion (pO2{approx}0{+/-}1mmHg), or when modulated by inhaled carbogen (pO2{approx}21{+/-}7mmHg). In vivo measurements of radiolytic consumption, go2, correlated to initial pO2 when FLASH was present (pO2{approx}4-16mmHg) and saturated above pO2>16mmHg. Inspired carbogen induced the highest pO2 and maximum damage at 25 Gy. Reductions in dose to 20 and 15 Gy reduced skin damage with carbogen, but did not result in FLASH sparing. SignificanceThese findings indicate that tissue pO2 directly modulates in vivo FLASH skin tissue sparing, requiring roughly room air or 100% oxygen anesthetic gas levels for this effect to be present. The effect is diminished by the absence of oxygen or from carbogen induced oxygen values. Variations in dose do not appear to alter this observation at carbogen induced pO2 levels.

PurposeTumor cell networks formed by tumor microtubes (TMs) are thought to drive therapy resistance in glioblastoma (GB). X-ray irradiation enhances TM formation, thereby increasing radioresistance. We hypothesize that high linear energy transfer (LET) particle radiotherapy is less affected by TM-mediated resistance due to its reduced reliance on indirect DNA damage. This study explores the impact of LET-induced DNA damage on TMs formation and GB survival Material and MethodsFormation of TMs was investigated in the primary patient derived glioblastoma stem-like cell lines (S24 and T269) irradiated with different LET, ranging from 3 - 107 keV/{micro}m, across dose series (1, 2, 4, 6 Gy) of clinical proton, helium, and carbon ion beams. TM networks and DNA damage patterns, specifically {gamma}H2AX foci, were visualized using fluorescence microscopy. Cell survival was evaluated through clonogenic survival assays. ResultsThe formation of TMs, radiation-induced nuclear DNA damage repair foci, and GB cell survival were correlated with a gradual increase in LET. Consistent with conventional photon/X-rays, low-LET proton irradiation promoted TMs formation in a dose-dependent manner. In contrast, an anti-correlation between LET and TMs induction was found, i.e., a decreased network connectivity with gradual increase of LET and formation of complex DNA damage. Consequently, LET increase correlated with reduced cell survival, with the most pronounced cell killing observed after high-LET carbon irradiation. Moreover, the inverse correlation between LET and TMs density was further confirmed for a broad range of LET modulated within the carbon ion irradiation. ConclusionThis is the first report on the relevance of LET as a novel mean to overcome TMs network-mediated radioresistance in GB, with ramifications for the clinical translation of high-LET particle radiotherapy to further improve outcome in this still devastating disease.

Background and purposeCombining radiation therapy with immunotherapy may be beneficial in treatment of head and neck cancer (HNC), but the combined effect may depend on tumor characteristics and the type of radiation. The purpose was to compare responses for two syngeneic tumor models in mice following X-ray or proton irradiation with or without immune checkpoint inhibition (ICI). Materials and methodsMOC1 and MOC2 tumors were inoculated in the right hind leg of each mouse (C57BL/6J, n=159). Single-dose irradiation with X-rays or protons and administration of anti-PDL1 started when the tumors reached 200 mm3. Doses of 5-30 Gy were given. Time-dependent tumor volume data were analyzed with a regression model yielding the growth rate {gamma} without irradiation and the reduction in growth rate per dose {eta}. Relative biological effectiveness (RBE) was calculated as the ratio of {eta} for X-rays to that of protons. Synergy between radiation and ICI was estimated as the ratio of {eta}s. ResultsMOC2 tumors grew faster and were more radioresistant than MOC1 tumors. ICI reduced the growth rate for MOC1 with 20{+/-}2% compared to controls, while no reduction was seen for MOC2. RBE for MOC1 wo/w ICI was 0.89{+/-}0.04 and 0.93{+/-}0.06, respectively, while it was 1.15{+/-}0.12 and 1.60{+/-}0.17, respectively, for MOC2. Combination synergy for X-rays was 1.22{+/-}0.08 and 0.96{+/-}0.11 in MOC1 and MOC2, respectively, while was it 1.27{+/-}0.06 and 1.33 {+/-}0.13, respectively, for protons. ConclusionsRBE for protons was dependent on use of ICI and tumor type. A greater synergy may be achieved when combining protons with ICI compared to X-rays and ICI.