Abstract FLASH radiotherapy, delivered at ultra-high dose rates exceeding 100 Gy/s, spares normal tissues while maintaining tumor control, yet the molecular mechanisms underlying this differential response remain poorly understood. Here we employed spatial and bulk multi-omics to investigate lipid and protein remodeling in tongue tissue and Mouse oral carcinoma 2 (MOC2) tumors at two weeks post-irradiation with FLASH or conventional dose-rate (CONV) proton radiotherapy. Bulk lipidomics revealed that CONV irradiation induced marked triglyceride (TG) depletion in tongue tissue, whereas FLASH attenuated this depletion. Spatial lipidomics using MALDI-MSI demonstrated that this TG loss was spatially restricted to minor salivary glands, identifying these radiosensitive structures as focal points of radiation-induced lipid damage. Additionally, FLASH irradiation uniquely promoted increases in membrane phospholipids and their lysophospholipid intermediates, consistent with active phospholipid turnover rather than passive damage avoidance. Proteomics revealed divergent metabolic programs: CONV activated a destructive cascade characterized by Ces1d depletion, Acox1-mediated peroxisomal β-oxidation, and Acot7-driven fatty acid overflow, collectively defining a lipid droplet collapse; whereas FLASH engaged a protective program featuring Mgll suppression, Apoe-mediated triglyceride redistribution, and Lypla2-mediated lysophospholipid clearance. We propose a lipid metabolic reprogramming hypothesis in which FLASH not only minimizes acute oxidative damage but actively reprograms lipid metabolism to preserve lipid droplet stores and promote membrane remodeling. These findings provide a putative molecular foundation for the FLASH effect and support published data on the protection of normal tissues.

Head and neck cancer (HNC) is predominantly comprised of head and neck squamous cell carcinoma (HNSCC), ranking as the 6th most common cancer worldwide. There are roughly 450,000 documented annual fatalities with predictions of annual increases in incidence. Despite advances in these intensive anticancer techniques, patient survival remains low. The current standard of care for local recurrence of HNSCC tumors is salvage surgery (SS) or combined modality with adjuvant re-irradiation using (X-ray)-based therapy (XRT). However, due to critical structure dose limits of the head and neck region, adjuvant XRT can cause excess morbidity. Proton radiation (PT), although less researched, may be advantageous to minimize the risk of injury to highly critical structures and minimize the radiation dosage outside of the tumor volume due to its energy course of the Bragg’s Peak, which is thought to also have a higher relative biological effectiveness. While there may be high clinical relevancy of PT, there is currently a lack of translational and clinical data to confirm advantages of its use in HNSCC. Patients with HNSCC have impaired immune surveillance, and higher levels of inhibitory checkpoints such as PD-L1 permitting tumors to evade the immune system. Immunotherapy targeting PD-1, receptor to PD-L1, has greatly enhanced survival rates in these patients, but many patients fail to respond due to other immunosuppressive factors. Here we investigate the immune response effects of RT, with either XRT or PT combined with anti-PD1 antibody (αPD1) in vivo using an established murine oral cavity tumor model. Wild-type C57BL/6 mice were injected into the buccal region with 1.5 x 106 mouse oral cavity (MOC-1) cells. Once tumors reached 50-150mm3, mice were treated over one week with XRT (3 treatments of 5 Gy) or sham. The following week the mice received αPD1 or IgG isotype control antibody. Here we present effects of XRT vs PT on immune infiltration by flow cytometry and immunofluorescence in three independent studies. Growth curves in the studies indicated that proton therapy and αPD1 in combination may be advantageous over αPD1 or PT alone with increased CD8 T cell infiltration, suggesting that PT in combination with αPD1 may offer significant advantages in eliciting a strong anti-tumor immune response. Katelyn Jansen, Maria Lehn, Nicolas Nassar, Alexis Redmond, Mathieu Sertorio, Vinita Takiar, Dalia El-Gamal, Trisha Wise-Draper. Investigating the effects of radiation and anti-PD1 therapy on immune cell tumor infiltration in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4532.

The gut microbiome plays a critical role in the pathophysiology of acute myocardial infarction (MI). MI events significantly impact intestinal integrity which results in leakage of bacterial products into the systemic circulation. We demonstrate that MI not only compromises intestinal integrity, leading to systemic leakage of bacterial products like LPS, but also results in the translocation and colonization of live, intact gut bacteria in the MI heart - a novel aspect of the heart-gut axis. Our initial findings with natural murine gut microbiome were substantiated using orally administered E. coli Nissle 1917 (EcN), as a tracer bacterium. Furthermore, we engineered EcN to express the microbial anti-inflammatory molecule (MAM) derived from the probiotic Faecalibacterium prausnitzii . Treatment with this engineered strain, EcN-MAM, led to significantly improved survival and cardiac function in MI mice. This was attributed to enhanced gut barrier integrity, resulting in reduced systemic bacterial permeation and subsequent inflammation. These findings shed light on a previously unrecognized dimension of the heart-gut axis and highlight the potential of microbiome-based interventions in MI management.

Cholesterol, an essential membrane component and a precursor for steroid hormones and bile acids, plays a vital role in various cellular processes. Cancer cells, in particular, exhibit a heightened demand for cholesterol to support their proliferation. This increased cholesterol requirement can be attributed to the upregulation of cholesterol biosynthesis or enhanced cholesterol uptake. Metabolic reprogramming in cancer cells allows them to sustain the energy demands associated with their aberrant growth characteristics. In normal cells, cholesterol uptake and synthesis are tightly regulated through various mechanisms within the cholesterol metabolism pathway. SREBP2 (Sterol Regulatory Element Binding Protein 2) is a critical master regulator of cholesterol homeostasis in normal cells. Dysregulation of cholesterol metabolism is intricately linked with the development of malignant phenotypes. Furthermore, emerging evidence highlights the crosstalk between SREBP2 and aberrant signaling pathways, such as PI3K/AKT/mTORC1, p53, TGF-β, c-Myc, Hippo, and FoxM1, which promote tumorigenesis. Understanding these molecular interactions between SREBP2 and signaling pathways is crucial for unraveling the mechanisms underlying cancer development. Identifying combinatorial treatment strategies targeting cholesterol metabolism holds great promise in deciphering mechanistic insights into metabolic vulnerabilities in cancer cells. Such strategies have the potential to enhance the efficacy of standard chemo/radiotherapy approaches for highly resistant cancer types. This review explores the regulation of SREBP2 in cancer and elucidates its role in dysregulated cholesterol metabolism. A detailed discussion on the implications of targeting cholesterol metabolism as a therapeutic approach for cancer treatment has also been elucidated.

Brain cancer is a complex, heterogeneous disease with a poor prognosis, reflected in a 5-year survival rate of approximately 33%. Current treatment includes surgery, radiotherapy, and chemotherapy. However, conventional whole-brain photon radiotherapy causes cognitive impairment due to collateral damage to non-tumoral neural tissue. Proton Conventional radiotherapy (pCONV-RT) has emerged as a more targeted alternative delivering precise tumor irradiation with reduced off-target toxicity. A novel form of this therapy is Proton FLASH radiotherapy (pFLASH-RT), which delivers an ultra-high dose rate within milliseconds. Preclinical studies suggest that pFLASH-RT may offer neuroprotective effects; nonetheless, its impact on healthy, particularly developing brain tissue, remains poorly understood. To address this knowledge gap, we developed a preclinical organotypic brain slice model from neonatal mice, which preserves 3D tissue architecture, reduces animal use, and supports diverse cellular and molecular analyses. Using this system, we hypothesized that pFLASH-RT induces less DNA damage and inflammation in healthy tissue compared to pCONV-RT. Coronal brain slices (250μm thick) from 7-day-old C57BL/6 mice (both sexes) were cultured for one week before irradiation with 10Gy pFLASH-RT (~50Gy/min) or pCONV-RT (0.1Gy/s). Tissues were harvested 24h post-irradiation for immunofluorescence and gene expression analyses. Our results showed that both radiation modalities increased nuclear γH2AX foci, indicating DNA damage. Cell proliferation decreased significantly in irradiated samples compared to unirradiated controls (SHAM), as assessed by Ki67 gene and protein expression. Inflammatory genes Il-1β and Il-6 were upregulated by both radiotherapies. Importantly, this inflammatory response was sex-dependent, with a significantly higher effect in females exposed to pCONV-RT, underscoring potential age- and sex-specific susceptibilities in the developing brain. Overall, our organotypic model represents a robust platform to investigate the mechanisms and safety profiles of emerging radiotherapy strategies. These findings support the potential of pFLASH-RT for pediatric brain cancer and emphasize the need for further research into sex- and age-dependent radiotoxicity.

Diffuse Midline Gliomas (DMGs) are universally fatal pediatric brain tumors. The current standard of care is radiation therapy, which provides only a transient benefit. The genetic landscape of DMG has revealed mutual exclusivity between TP53-mutant and PPM1D-mutant tumors, likely due to functional redundancy. While both TP53 and PPM1D mutations participate in DMG formation, different mechanisms of p53 pathway suppression may present unique therapeutic opportunities. We hypothesize that PPM1D-mutant tumors retain a suppressed yet functional p53 which can be reactivated under therapy stress. Targeting other complementary pathways and PPM1D-specific targets could help attain better overall outcomes. Moreover, PPM1D-mutant tumors frequently co-occur with sub-clonal PI3K pathway activating mutations leading to our hypothesis that they help accelerate tumorigenesis and confer therapy resistance. To investigate this, we have generated isogenic DMG IUE mouse models of Trp53-mutant, PPM1D-mutant, and PIK3CA-mutant (in the context of both Trp53- and PPM1D-mutant) to uncover shared and unique functions of these mutations. Ongoing studies are aimed to elucidate the differential response to radiation therapy using a combination of in vitro assays and transcriptomic profiling in each condition. We aim to test whether Trp53- and PPM1D-mutant DMG models display differences in response to CHK1 inhibition, which is a direct target of PPM1D phosphatase activity and regulator of DNA damage response and cell cycle progression. Similarly, experiments are focused on the use of PI3K/AKT/mTOR inhibitors, examining their in vitro and in vivo ability to improve response to DNA damaging agents in PPM1D-mutant DMGs. We expect to reveal PPM1D and PIK3CA specific targets that can be pursued to improve the outcomes for PPM1D-mutant DMG patients. This will help strategize specific targeted therapeutic approaches in combination with radiation to better treat PPM1D-mutant DMGs.

Abstract Proton therapy (PT) is a promising new alternative to conventional X-ray-based photon therapy (XRT) for patients with advanced head and neck squamous cell carcinomas (HNSCC) owing to its precise dosimetric characteristics and reduced toxicity. While tumor-derived exosomes contribute to immune suppression in HNSCC, the effects of PT on tumor-derived exosomes and anti-tumor immune responses are poorly understood. In this study, we generated primary HNSCC cell cultures from resected tumors and irradiated them with 5 Gy PT or XRT, and isolated exosomes from cell culture supernatants. We observed that compared to XRT, PT-exposed HNSCC cells produced 75% fewer exosomes. To assess the immunomodulatory effects of PT, we exposed peripheral blood mononuclear cells (PBMCs) to exosomes from PT- and XRT-irradiated HNSCC cells and conducted preliminary single-cell studies by transcriptome analysis (NanoString), secretome profiling (Isoplexis) and immune function (IFN-g release). Transcriptomic studies revealed that exosomes from PT- and XRT-irradiated cells comparably reduced cytotoxic and Th1 cell abundance, and inhibited immune responses associated with cytotoxicity, NK, and T cell functions. Exosomes from PT- and XRT-irradiated cells equally suppressed IFN-g release and reduced the polyfunctionality (the ability to secrete multiple cytokines from a single cell) of CD8+ T cells. Our results suggest that PT limits the immune suppressive effect of exosomes by reducing their production.

Proton therapy (PT) is emerging as an effective and less toxic alternative to conventional X-ray-based photon therapy (XRT) for patients with advanced head and neck squamous cell carcinomas (HNSCCs) owing to its clustered dose deposition dosimetric characteristics. For optimal efficacy, cancer therapies, including PT, must elicit a robust anti-tumor response by effector and cytotoxic immune cells in the tumor microenvironment (TME). While tumor-derived exosomes contribute to immune cell suppression in the TME, information on the effects of PT on exosomes and anti-tumor immune responses in HNSCC is not known. In this study, we generated primary HNSCC cells from tumors resected from HNSCC patients, irradiated them with 5 Gy PT or XRT, and isolated exosomes from cell culture supernatants. HNSCC cells exposed to PT produced 75% fewer exosomes than XRT- and non-irradiated HNSCC cells. This effect persisted in proton-irradiated cells for up to five days. Furthermore, we observed that exosomes from proton-irradiated cells were identical in morphology and immunosuppressive effects (suppression of IFN-γ release by peripheral blood mononuclear cells) to those of photon-irradiated cells. Our results suggest that PT limits the suppressive effect of exosomes on cancer immune surveillance by reducing the production of exosomes that can inhibit immune cell function.

Abstract Background FLASH therapy is a treatment technique in which radiation is delivered at ultra-high dose rates (≥ 40 Gy/s). The first-in-human FAST-01 clinical trial demonstrated the clinical feasibility of proton FLASH in the treatment of extremity bone metastases. The objectives of this investigation are to assess the toxicities of treatment and pain relief in study participants with painful thoracic bone metastases treated with FLASH radiotherapy, as well as workflow metrics in a clinical setting. Methods This single-arm clinical trial is being conducted under an FDA investigational device exemption (IDE) approved for 10 patients with 1–3 painful bone metastases in the thorax, excluding bone metastases in the spine. Treatment will be 8 Gy in a single fraction administered at ≥ 40 Gy/s on a FLASH-enabled proton therapy system delivering a single transmission proton beam. Primary study endpoints are efficacy (pain relief) and safety. Patient questionnaires evaluating pain flare at the treatment site will be completed for 10 consecutive days post-RT. Pain response and adverse events (AEs) will be evaluated on the day of treatment and on day 7, day 15, months 1, 2, 3, 6, 9, and 12, and every 6 months thereafter. The outcomes for clinical workflow feasibility are the occurrence of any device issues as well as time on the treatment table. Discussion This prospective clinical trial will provide clinical data for evaluating the efficacy and safety of proton FLASH for palliation of bony metastases in the thorax. Positive findings will support the further exploration of FLASH radiation for other clinical indications including patient populations treated with curative intent. Registration ClinicalTrials.gov NCT05524064.

Targeting cholesterol metabolism in combination with RT could be beneficial for treating HNSCC. In vivo validation in immunocompetent mouse models and orthotopic patient-derived xenograft models is ongoing.

Radiation therapy (RT) is a crucial treatment modality for central nervous system (CNS) tumors but toxicity to healthy CNS tissues remains a challenge. Additionally, environmental exposure to radiation during nuclear catastrophes or space travel presents a risk of CNS toxicity. However, the underlying mechanisms of radiation-induced CNS toxicity are not fully understood. Neural progenitor cells (NPCs) are highly radiosensitive, resulting in decreased neurogenesis in the hippocampus. This study aimed to characterize a novel platform utilizing rat NPCs cultured as 3D neurospheres (NSps) to screen the safety and efficacy of experimental drugs with and without radiation exposure. The effect of radiation on NSp growth and differentiation was assessed by measuring sphere volume and the expression of neuronal differentiation markers Nestin and GFAP and proliferation marker Ki67. Radiation exposure inhibited NSp growth, decreased proliferation, and increased GFAP expression, indicating astrocytic differentiation. RNA sequencing analysis supported these findings, showing upregulation of Notch, BMP2/4, S100b, and GFAP gene expression during astrogenesis. By recapitulating radiation-induced toxicity and astrocytic differentiation, this single-NSp culture system provides a high-throughput preclinical model for assessing the effects of various radiation modalities and evaluates the safety and efficacy of potential therapeutic interventions in combination with radiation.

BACKGROUND:In preclinical studies, FLASH therapy, in which radiation delivered at ultrahigh dose rates of ≥40 Gy per second, has been shown to cause less injury to normal tissues than radiotherapy delivered at conventional dose rates. This paper describes the protocol for the first-in-human clinical investigation of proton FLASH therapy.OBJECTIVE:FAST-01 is a prospective, single-center trial designed to assess the workflow feasibility, toxicity, and efficacy of FLASH therapy for the treatment of painful bone metastases in the extremities.METHODS:Following informed consent, 10 subjects aged ≥18 years with up to 3 painful bone metastases in the extremities (excluding the feet, hands, and wrists) will be enrolled. A treatment field selected from a predefined library of plans with fixed field sizes (from 7.5 cm × 7.5 cm up to 7.5 cm × 20 cm) will be used for treatment. Subjects will receive 8 Gy of radiation in a single fraction-a well-established palliative regimen evaluated in prior investigations using conventional dose rate photon radiotherapy. A FLASH-enabled Varian ProBeam proton therapy unit will be used to deliver treatment to the target volume at a dose rate of ≥40 Gy per second, using the plateau (transmission) portion of the proton beam. After treatment, subjects will be assessed for pain response as well as any adverse effects of FLASH radiation. The primary end points include assessing the workflow feasibility and toxicity of FLASH treatment. The secondary end point is pain response at the treated site(s), as measured by patient-reported pain scores, the use of pain medication, and any flare in bone pain after treatment. The results will be compared to those reported historically for conventional dose rate photon radiotherapy, using the same radiation dose and fractionation.RESULTS:FAST-01 opened to enrollment on November 3, 2020. Initial results are expected to be published in 2022.CONCLUSIONS:The results of this investigation will contribute to further developing and optimizing the FLASH-enabled ProBeam proton therapy system workflow. The pain response and toxicity data acquired in our study will provide a greater understanding of FLASH treatment effects on tumor responses and normal tissue toxicities, and they will inform future FLASH trial designs.TRIAL REGISTRATION:: ClinicalTrials.gov NCT04592887; http://clinicaltrials.gov/ct2/show/NCT04592887.INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID):DERR1-10.2196/41812.

Importance:To our knowledge, there have been no clinical trials of ultra-high-dose-rate radiotherapy delivered at more than 40 Gy/sec, known as FLASH therapy, nor first-in-human use of proton FLASH.Objectives:To assess the clinical workflow feasibility and treatment-related toxic effects of FLASH and pain relief at the treatment sites.Design, Setting, and Participants:In the FAST-01 nonrandomized trial, participants treated at Cincinnati Children's/UC Health Proton Therapy Center underwent palliative FLASH radiotherapy to extremity bone metastases. Patients 18 years and older with 1 to 3 painful extremity bone metastases and life expectancies of 2 months or more were eligible. Patients were excluded if they had foot, hand, and wrist metastases; metastases locally treated in the 2 weeks prior; metal implants in the treatment field; known enhanced tissue radiosensitivity; and implanted devices at risk of malfunction with radiotherapy. One of 11 patients who consented was excluded based on eligibility. The end points were evaluated at 3 months posttreatment, and patients were followed up through death or loss to follow-up for toxic effects and pain assessments. Of the 10 included patients, 2 died after the 2-month follow-up but before the 3-month follow-up; 8 participants completed the 3-month evaluation. Data were collected from November 3, 2020, to January 28, 2022, and analyzed from January 28, 2022, to September 1, 2022.Interventions:Bone metastases were treated on a FLASH-enabled (≥40 Gy/sec) proton radiotherapy system using a single-transmission proton beam. This is consistent with standard of care using the same prescription (8 Gy in a single fraction) but on a conventional-dose-rate (approximately 0.03 Gy/sec) photon radiotherapy system.Main Outcome and Measures:Main outcomes included patient time on the treatment couch, device-related treatment delays, adverse events related to FLASH, patient-reported pain scores, and analgesic use.Results:A total of 10 patients (age range, 27-81 years [median age, 63 years]; 5 [50%] male) underwent FLASH radiotherapy at 12 metastatic sites. There were no FLASH-related technical issues or delays. The average (range) time on the treatment couch was 18.9 (11-33) minutes per patient and 15.8 (11-22) minutes per treatment site. Median (range) follow-up was 4.8 (2.3-13.0) months. Adverse events were mild and consistent with conventional radiotherapy. Transient pain flares occurred in 4 of the 12 treated sites (33%). In 8 of the 12 sites (67%) patients reported pain relief, and in 6 of the 12 sites (50%) patients reported a complete response (no pain).Conclusions and Relevance:In this nonrandomized trial, clinical workflow metrics, treatment efficacy, and safety data demonstrated that ultra-high-dose-rate proton FLASH radiotherapy was clinically feasible. The treatment efficacy and the profile of adverse events were comparable with those of standard-of-care radiotherapy. These findings support the further exploration of FLASH radiotherapy in patients with cancer.Trial Registration:ClinicalTrials.gov Identifier: NCT04592887.

Targeted radionuclide therapy (TRT) is an emerging therapeutic modality for the treatment of various solid cancers. Current approaches rely on the presence of cancer-specific epitopes and receptors against which a radiolabeled ligand is systemically administered to specifically deliver cytotoxic doses of α and β particles to tumors. In this proof-of-concept study, tumor-colonizing Escherichia coli Nissle 1917 (EcN) is utilized to deliver a bacteria-specific radiopharmaceutical to solid tumors in a cancer-epitope independent manner. In this microbe-based pretargeted approach, the siderophore-mediated metal uptake pathway is leveraged to selectively concentrate copper radioisotopes, 64 Cu and 67 Cu, complexed to yersiniabactin (YbT) in the genetically modified bacteria. 64 Cu-YbT facilitates positron emission tomography (PET) imaging of the intratumoral bacteria, whereas 67 Cu-YbT delivers a cytotoxic dose to the surrounding cancer cells. PET imaging with 64 Cu-YbT reveals persistence and sustained growth of the bioengineered microbes in the tumor microenvironment. Survival studies with 67 Cu-YbT reveals significant attenuation of tumor growth and extends survival of both MC38 and 4T1  tumor-bearing mice harboring the microbes. Tumor response to this pretargeted approach correlates with promising anti-tumor immunity, with noticeable CD8+ T:Treg cell ratio. Their strategy offers a pathway to target and ablate multiple solid tumors independent of their epitope and receptor phenotype.

Recent studies suggest that ultra-high dose rates of proton radiation (>40 Gy/s; FLASH) confer less toxicity to exposed healthy tissue and reduce cognitive decline compared with conventional radiation dose rates (~1 Gy/s), but further preclinical data are required to demonstrate this sparing effect. In this study, postnatal day 11 (P11) rats were treated with whole brain irradiation with protons at a total dose of 0, 5, or 8 Gy, comparing a conventional dose rate of 1 Gy/s vs. a FLASH dose rate of 100 Gy/s. Beginning on P64, rats were tested for locomotor activity, acoustic and tactile startle responses (ASR, TSR) with or without prepulses, novel object recognition (NOR; 4-object version), striatal dependent egocentric learning ([configuration A] Cincinnati water maze (CWM-A)), prefrontal dependent working memory (radial water maze (RWM)), hippocampal dependent spatial learning (Morris water maze (MWM)), amygdala dependent conditioned freezing, and the mirror image CWM [configuration B (CWM-B)]. All groups had deficits in the CWM-A procedure. Weight reductions, decreased center ambulation in the open-field, increased latency on day-1 of RWM, and deficits in CWM-B were observed in all irradiated groups, except the 5 Gy FLASH group. ASR and TSR were reduced in the 8 Gy FLASH group and day-2 latencies in the RWM were increased in the FLASH groups compared with controls. There were no effects on prepulse trials of ASR or TSR, NOR, MWM, or conditioned freezing. The results suggest striatal and prefrontal cortex are sensitive regions at P11 to proton irradiation, with reduced toxicity from FLASH at 5 Gy.

In vitro studies allow evaluation of normal or cancer cell responses to radiation, either alone or in combination with agents used to modify these biological responses. Ionizing radiation can be produced by a variety of particles and sources, with varying energy spectra, interaction probabilities, linear energy transfer, dose uniformity, dose rates, and delivery methods. Multiple radiation sources have been used to irradiate cells in the published literature. However, the equivalence of response in cell culture models across radiation sources has not been rigorously established. Moreover, current reporting of radiation source parameters lacks consistency and rigor which may impact the reproducibility of pre-clinical data between laboratories. Relevant choices of radiation source are also of high importance due to growing interest in comparing photon versus particle radiation effect on biological responses. Therefore, this study robustly evaluates the cellular response (cell survival, apoptosis, and DNA damage) of three distinct cell lines using four unique photon generating radiation sources. We hypothesize there may be subtle differences across the radiation sources, without an appreciable difference in cellular response. The four photon irradiation energies investigated, 662 keV, 100 kVp, 220 kVp, 6 MV, did produce subtle differences in DNA damage and cell survival when treating three distinct tumor cell lines. These variations in cellular response emphasize the need to carefully consider irradiation source, energy, and dose rate depending on study goal and endpoint.

PURPOSE:FLASH proton pencil beam scanning (p-PBS) showed a reduction in mouse skin toxicity and fibrosis when delivered as a single, uninterrupted, high-dose fraction. Clinical p-PBS treatment usually requires multiple beams to achieve good conformality, and these beams are separated by minutes to allow patient and equipment repositioning. We evaluate the impact of multibeam versus single-beam proton radiation on the FLASH sparing effect on skin toxicity. METHODS AND MATERIALS:The right hind leg of 10-week-old female C57Bl/6j mice was irradiated using a Varian ProBeam proton beam scanning gantry system at conventional (1 Gy/s) or FLASH (100 Gy/s) average field dose rate. We scored the skin toxicity after different doses for 7 weeks. The treatment was delivered as 1, 2, or 3 equal beams with an interruption of 2 minutes. For each beam delivery, the equipment remained in the same position so that there was a full overlap of beams administered. RESULTS:Single-beam delivery confirmed a benefit for p-PBS FLASH in this model at 30, 35, and 40 Gy. At 30 and 35 Gy, a single beam interruption of 2 minutes (2 × 15 Gy or 2 × 17.5 Gy) reduced the FLASH sparing effect, which remained significant (P < .001). However, 2 interruptions (3 × 10 Gy or 3 × 11.6 Gy) abrogated the normal tissue sparing effect. CONCLUSIONS:Our results indicate that the FLASH sparing effect in areas of beam overlap can be compromised by interruptions in delivery time. Time gap between overlapping beams and spatial arrangement of the delivered beams are important parameters for FLASH studies. The effect of multibeam needs to be studied on different organs of interest.