Acute Hypoxia Does Not Alter Tumor Sensitivity to FLASH Radiation Therapy.

PURPOSE: Tumor hypoxia is a major cause of treatment resistance, especially to radiation therapy at conventional dose rate (CONV), and we wanted to assess whether hypoxia does alter tumor sensitivity to FLASH. METHODS AND MATERIALS: We engrafted several tumor types (glioblastoma [GBM], head and neck cancer, and lung adenocarcinoma) subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20-Gy fraction at either CONV or FLASH, measured oxygen tension, monitored tumor growth, and sampled tumors for bulk RNAseq and pimonidazole analysis. Next, we inhibited glycolysis with trametinib in GBM tumors to enhance FLASH efficacy. RESULTS: Using various subcutaneous tumor models, and in contrast to CONV, FLASH retained antitumor efficacy under acute hypoxia. These findings show that in addition to normal tissue sparing, FLASH could overcome hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered a FLASH-specific profile in human GBM that involved cell-cycle arrest, decreased ribosomal biogenesis, and a switch from oxidative phosphorylation to glycolysis. Glycolysis inhibition by trametinib enhanced FLASH efficacy in both normal and clamped conditions. CONCLUSIONS: These data provide new and specific insights showing the efficacy of FLASH in a radiation-resistant context, proving an additional benefit of FLASH over CONV.

Infrared microspectroscopy to elucidate the underlying biomolecular mechanisms of FLASH radiotherapy.

BACKGROUND: FLASH-radiotherapy (FLASH-RT) is an emerging modality that uses ultra-high dose rates of radiation to enable curative doses to the tumor while preserving normal tissue. The biological studies showed the potential of FLASH-RT to revolutionize radiotherapy cancer treatments. However, the complex biological basis of FLASH-RT is not fully known yet. AIM: Within this context, our aim is to get deeper insights into the biomolecular mechanisms underlying FLASH-RT through Fourier Transform Infrared Microspectroscopy (FTIRM). METHODS: C57Bl/6J female mice were whole brain irradiated at 10 Gy with the eRT6-Oriatron system. 10 Gy FLASH-RT was delivered in 1 pulse of 1.8µs and conventional irradiations at 0.1 Gy/s. Brains were sampled and prepared for analysis 24 h post-RT. FTIRM was performed at the MIRAS beamline of ALBA Synchrotron. Infrared raster scanning maps of the whole mice brain sections were collected for each sample condition. Hyperspectral imaging and Principal Component Analysis (PCA) were performed in several regions of the brain. RESULTS: PCA results evidenced a clear separation between conventional and FLASH irradiations in the 1800-950 cm-1 region, with a significant overlap between FLASH and Control groups. An analysis of the loading plots revealed that most of the variance accounting for the separation between groups was associated to modifications in the protein backbone (Amide I). This protein degradation and/or conformational rearrangement was concomitant with nucleic acid fragmentation/condensation. Cluster separation between FLASH and conventional groups was also present in the 3000-2800 cm-1 region, being correlated with changes in the methylene and methyl group concentrations and in the lipid chain length. Specific vibrational features were detected as a function of the brain region. CONCLUSION: This work provided new insights into the biomolecular effects involved in FLASH-RT through FTIRM. Our results showed that beyond nucleic acid investigations, one should take into account other dose-rate responsive molecules such as proteins, as they might be key to understand FLASH effect.

In vivo measurements of change in tissue oxygen level during irradiation reveal novel dose rate dependence.

BACKGROUND AND PURPOSE: This study aimed to investigate the radiochemical oxygen depletion (ROD) in vivo by directly measuring oxygen levels in various mouse tissues during ultra-high dose rate (UHDR) irradiation at clinically relevant doses and dose rates. MATERIALS AND METHODS: Mice bearing subcutaneous human glioblastoma (U-87 MG) tumors were used for tumor and normal tissue (skin, muscle, brain) measurements. An oxygen-sensitive phosphorescent probe (Oxyphor PtG4) was injected into the tissues, and oxygen levels were monitored using a fiberoptic phosphorometer during UHDR irradiation with a 6 MeV electron linear accelerator (LINAC). Dose escalation experiments (10-40 Gy) were performed at a dose rate of 1300 Gy/s, and dose rate escalation experiments were conducted at a fixed dose of 40 Gy with dose rates ranging from 2 to 101 Gy/s. RESULTS: Radiation-induced change in tissue oxygenation (ΔpO2) increased linearly with dose and correlated with baseline tissue oxygenation levels in the range of 0 - 30 mmHg. At higher baseline tissue oxygenation levels, such as those observed in muscle and brain, there was no corresponding increase in ΔpO2. When we modulated dose rate, ΔpO2 increased steeply up to âˆ¼ 20 Gy/s and plateaued thereafter. The relationship between ΔpO2 and dose rate showcases the interplay between ROD and reoxygenation. CONCLUSION: While UHDR irradiation induces measurable oxygen depletion in tissues, the observed changes in oxygenation levels do not support the hypothesis that ROD-induced radioresistance is responsible for the FLASH tissue-sparing effect at clinically relevant doses and dose rates. These findings highlight the need for further investigation into alternative mechanisms underlying the FLASH effect.

Antitumor Effect by Either FLASH or Conventional Dose Rate Irradiation Involves Equivalent Immune Responses.

PURPOSE: The capability of ultrahigh dose rate FLASH radiation therapy to generate the FLASH effect has opened the possibility to enhance the therapeutic index of radiation therapy. The contribution of the immune response has frequently been hypothesized to account for a certain fraction of the antitumor efficacy and tumor kill of FLASH but has yet to be rigorously evaluated. METHODS AND MATERIALS: To investigate the immune response as a potentially important mechanism of the antitumor effect of FLASH, various murine tumor models were grafted either subcutaneously or orthotopically into immunocompetent mice or in moderately and severely immunocompromised mice. Mice were locally irradiated with single dose (20 Gy) or hypofractionated regimens (3 × 8 or 2 × 6 Gy) using FLASH (≥2000 Gy/s) and conventional (CONV) dose rates (0.1 Gy/s), with/without anti-CTLA-4. Tumor growth was monitored over time and immune profiling performed. RESULTS: FLASH and CONV 20 Gy were isoeffective in delaying tumor growth in immunocompetent and moderately immunodeficient hosts and increased tumor doubling time to >14 days versus >7 days in control animals. Similar observations were obtained with a hypofractionated scheme, regardless of the microenvironment (subcutaneous flank vs ortho lungs). Interestingly, in profoundly immunocompromised mice, 20 Gy FLASH retained antitumor activity and significantly increased tumor doubling time to >14 days versus >8 days in control animals, suggesting a possible antitumor mechanism independent of the immune response. Analysis of the tumor microenvironment showed similar immune profiles after both irradiation modalities with significant decrease of lymphoid cells by ∼40% and a corresponding increase of myeloid cells. In addition, FLASH and CONV did not increase transforming growth factor-ß1 levels in tumors compared with unirradiated control animals. Furthermore, when a complete and long-lasting antitumor response was obtained (>140 days), both modalities of irradiation were able to generate a long-term immunologic memory response. CONCLUSIONS: The present results clearly document that the tumor responses across multiple immunocompetent and immunodeficient mouse models are largely dose rate independent and simultaneously contradict a major role of the immune response in the antitumor efficacy of FLASH. Therefore, our study indicates that FLASH is as potent as CONV in modulating antitumor immune response and can be used as an immunomodulatory agent.

More May Not be Better: Enhanced Spacecraft Shielding May Exacerbate Cognitive Decrements by Increasing Pion Exposures during Deep Space Exploration.

The pervasiveness of deep space radiation remains a confounding factor for the transit of humans through our solar system. Spacecraft shielding both protects astronauts but also contributes to absorbed dose through galactic cosmic ray interactions that produce secondary particles. The resultant biological effects drop to a minimum for aluminum shielding around 20 g/cm2 but increase with additional shielding. The present work evaluates for the first time, the impact of secondary pions on central nervous system functionality. The fractional pion dose emanating from thicker shielded spacecraft regions could contribute up to 10% of the total absorbed radiation dose. New results from the Paul Scherrer Institute have revealed that low dose exposures to 150 MeV positive and negative pions, akin to a Mars mission, result in significant, long-lasting cognitive impairments. These surprising findings emphasize the need to carefully evaluate shielding configurations to optimize safe exposure limits for astronauts during deep space travel.

FLASHlab@PITZ: New R&D platform with unique capabilities for electron FLASH and VHEE radiation therapy and radiation biology under preparation at PITZ.

At the Photo Injector Test facility at DESY in Zeuthen (PITZ), an R&D platform for electron FLASH and very high energy electron radiation therapy and radiation biology is being prepared (FLASHlab@PITZ). The beam parameters available at PITZ are worldwide unique. They are based on experiences from 20 + years of developing high brightness beam sources and an ultra-intensive THz light source demonstrator for ps scale electron bunches with up to 5 nC bunch charge at MHz repetition rate in bunch trains of up to 1 ms length, currently 22 MeV (upgrade to 250 MeV planned). Individual bunches can provide peak dose rates up to 1014 Gy/s, and 10 Gy can be delivered within picoseconds. Upon demand, each bunch of the bunch train can be guided to a different transverse location, so that either a "painting" with micro beams (comparable to pencil beam scanning in proton therapy) or a cumulative increase of absorbed dose, using a wide beam distribution, can be realized at the tumor. Full tumor treatment can hence be completed within 1 ms, mitigating organ movement issues. With extremely flexible beam manipulation capabilities, FLASHlab@PITZ will cover the current parameter range of successfully demonstrated FLASH effects and extend the parameter range towards yet unexploited short treatment times and high dose rates. A summary of the plans for FLASHlab@PITZ and the status of its realization will be presented.

Ultra-high-dose-rate FLASH and Conventional-Dose-Rate Irradiation Differentially Affect Human Acute Lymphoblastic Leukemia and Normal Hematopoiesis.

PURPOSE: Ultra-high-dose-rate FLASH radiation therapy has been shown to minimize side effects of irradiation in various organs while keeping antitumor efficacy. This property, called the FLASH effect, has caused enthusiasm in the radiation oncology community because it opens opportunities for safe dose escalation and improved radiation therapy outcome. Here, we investigated the impact of ultra-high-dose-rate FLASH versus conventional-dose-rate (CONV) total body irradiation (TBI) on humanized models of T-cell acute lymphoblastic leukemia (T-ALL) and normal human hematopoiesis. METHODS AND MATERIALS: We optimized the geometry of irradiation to ensure reproducible and homogeneous procedures using eRT6/Oriatron. Three T-ALL patient-derived xenografts and hematopoietic stem/progenitor cells (HSPCs) and CD34+ cells isolated from umbilical cord blood were transplanted into immunocompromised mice, together or separately. After reconstitution, mice received 4 Gy FLASH and CONV-TBI, and tumor growth and normal hematopoiesis were studied. A retrospective study of clinical and gene-profiling data previously obtained on the 3 T-ALL patient-derived xenografts was performed. RESULTS: FLASH-TBI was more efficient than CONV-TBI in controlling the propagation of 2 cases of T-ALL, whereas the third case of T-ALL was more responsive to CONV-TBI. The 2 FLASH-sensitive cases of T-ALL had similar genetic abnormalities, and a putative susceptibility imprint to FLASH-RT was found. In addition, FLASH-TBI was able to preserve some HSPC/CD34+ cell potential. Interestingly, when HSPC and T-ALL were present in the same animals, FLASH-TBI could control tumor development in most (3 of 4) of the secondary grafted animals, whereas among the mice receiving CONV-TBI, treated cells died with high leukemia infiltration. CONCLUSIONS: Compared with CONV-TBI, FLASH-TBI reduced functional damage to human blood stem cells and had a therapeutic effect on human T-ALL with a common genetic and genomic profile. The validity of the defined susceptibility imprint needs to be investigated further; however, to our knowledge, the present findings are the first to show benefits of FLASH-TBI on human hematopoiesis and leukemia treatment.

Hypofractionated FLASH-RT as an Effective Treatment against Glioblastoma that Reduces Neurocognitive Side Effects in Mice.

PURPOSE: Recent data have shown that single-fraction irradiation delivered to the whole brain in less than tenths of a second using FLASH radiotherapy (FLASH-RT), does not elicit neurocognitive deficits in mice. This observation has important clinical implications for the management of invasive and treatment-resistant brain tumors that involves relatively large irradiation volumes with high cytotoxic doses. EXPERIMENTAL DESIGN: Therefore, we aimed at simultaneously investigating the antitumor efficacy and neuroprotective benefits of FLASH-RT 1-month after exposure, using a well-characterized murine orthotopic glioblastoma model. As fractionated regimens of radiotherapy are the standard of care for glioblastoma treatment, we incorporated dose fractionation to simultaneously validate the neuroprotective effects and optimized tumor treatments with FLASH-RT. RESULTS: The capability of FLASH-RT to minimize the induction of radiation-induced brain toxicities has been attributed to the reduction of reactive oxygen species, casting some concern that this might translate to a possible loss of antitumor efficacy. Our study shows that FLASH and CONV-RT are isoefficient in delaying glioblastoma growth for all tested regimens. Furthermore, only FLASH-RT was found to significantly spare radiation-induced cognitive deficits in learning and memory in tumor-bearing animals after the delivery of large neurotoxic single dose or hypofractionated regimens. CONCLUSIONS: The present results show that FLASH-RT delivered with hypofractionated regimens is able to spare the normal brain from radiation-induced toxicities without compromising tumor cure. This exciting capability provides an initial framework for future clinical applications of FLASH-RT.See related commentary by Huang and Mendonca, p. 662.

Extracellular Vesicle-Derived miR-124 Resolves Radiation-Induced Brain Injury.

Radiation-induced cognitive dysfunction (RICD) is a progressive and debilitating health issue facing patients following cranial radiotherapy to control central nervous system cancers. There has been some success treating RICD in rodents using human neural stem cell (hNSC) transplantation, but the procedure is invasive, requires immunosuppression, and could cause other complications such as teratoma formation. Extracellular vesicles (EV) are nanoscale membrane-bound structures that contain biological contents including mRNA, miRNA, proteins, and lipids that can be readily isolated from conditioned culture media. It has been previously shown that hNSC-derived EV resolves RICD following cranial irradiation using an immunocompromised rodent model. Here, we use immunocompetent wild-type mice to show that hNSC-derived EV treatment administered either intravenously via retro-orbital vein injection or via intracranial transplantation can ameliorate cognitive deficits following 9 Gy head-only irradiation. Cognitive function assessed on the novel place recognition, novel object recognition, and temporal order tasks was not only improved at early (5 weeks) but also at delayed (6 months) postirradiation times with just a single EV treatment. Improved behavioral outcomes were also associated with reduced neuroinflammation as measured by a reduction in activated microglia. To identify the mechanism of action, analysis of EV cargo implicated miRNA (miR-124) as a potential candidate in the mitigation of RICD. Furthermore, viral vector-mediated overexpression of miR-124 in the irradiated brain ameliorated RICD and reduced microglial activation. Our findings demonstrate for the first time that systemic administration of hNSC-derived EV abrogates RICD and neuroinflammation in cranially irradiated wild-type rodents through a mechanism involving miR-124. SIGNIFICANCE: Radiation-induced neurocognitive decrements in immunocompetent mice can be resolved by systemic delivery of hNSC-derived EVs involving a mechanism dependent on expression of miR-124.

Extracellular Vesicles for the Treatment of Radiation-Induced Normal Tissue Toxicity in the Lung.

Human stem cell-derived extracellular vesicles (EV) provide many advantages over cell-based therapies for the treatment of functionally compromised tissue beds and organ sites. Here we sought to determine whether human embryonic stem cell (hESC)-derived EV could resolve in part, the adverse late normal tissue complications associated with exposure of the lung to ionizing radiation. The hESC-derived EV were systemically administered to the mice via the retro-orbital sinus to explore the potential therapeutic benefits following exposure to high thoracic doses of radiation (14 Gy). Data demonstrated that hESC-derived EV treatment significantly improved overall survival of the irradiated cohorts (P < 0.001). Increased survival was also associated with significant reductions in lung fibrosis as quantified by CBCT imaging (P < 0.01, 2 weeks post-irradiation). Qualitative histological analyses revealed reduced indications of radiation induced pulmonary injury in animals treated with EV. EV were then subjected to a rigorous proteomic analysis to ascertain the potential bioactive cargo that may prove beneficial in ameliorating radiation-induced normal tissue toxicities in the lung. Proteomics validated several consensus exosome markers (e.g., CD68) and identified major classes of proteins involved in nuclear pore complexes, epigenetics, cell cycle, growth and proliferation, DNA repair, antioxidant function, and cellular metabolism (TCA cycle and oxidative phosphorylation, OXYPHOS). Interestingly, EV were also found to contain mitochondrial components (mtDNA, OXYPHOS protein subunits), which may contribute to the metabolic reprograming and recovery of radiation-injured pulmonary tissue. To evaluate the safety of EV treatments in the context of the radiotherapeutic management of tumors, mice harboring TC1 tumor xenografts were subjected to the same EV treatments shown to forestall lung fibrosis. Data indicated that over the course of one month, no change in the growth of flank tumors between treated and control cohorts was observed. In conclusion, present findings demonstrate that systemic delivery of hESC-derived EV could ameliorate radiation-induced normal tissue complications in the lung, through a variety of potential mechanisms based on EV cargo analysis.

miRNA-based therapeutic potential of stem cell-derived extracellular vesicles: a safe cell-free treatment to ameliorate radiation-induced brain injury.

PURPOSE: This review compiles what is known about extracellular vesicles (EVs), their bioactive cargo, and how they might be used to treat radiation-induced brain injury. Radiotherapy (RT) is effective in cancer treatment, but can cause substantial damage to normal central nervous system tissue. Stem cell therapy has been shown to be effective in treating cognitive dysfunction arising from RT, but there remain safety concerns when grafting foreign stem cells into the brain (i.e. immunogenicity, teratoma). These limitations prompted the search for cell-free alternatives, and pointed to EVs that have been shown to have similar ameliorating effects in other tissues and injury models. CONCLUSIONS: EVs are nano-scale and lipid-bound vesicles that readily pass the blood-brain barrier. Arguably the most important bioactive cargo within EVs are RNAs, in particular microRNAs (miRNA). A single miRNA can modulate entire gene networks and signalling within the recipient cell. Determining functionally relevant miRNA could lead to therapeutic treatments where synthetically-derived EVs are used as delivery vectors for miRNA. Stem cell-derived EVs can be effective in treating brain injury including radiation-induced cognitive deficits. Of particular interest are systemic modes of administration which obviate the need for invasive procedures.

Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.

Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different "omics," clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental "virtual" liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal-fetal-placental immune interactions in preeclampsia. The description of these novel pathways in the "molecular phase" of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.

Ancestral perinatal obesogen exposure results in a transgenerational thrifty phenotype in mice.

Ancestral environmental exposures to non-mutagenic agents can exert effects in unexposed descendants. This transgenerational inheritance has significant implications for understanding disease etiology. Here we show that exposure of F0 mice to the obesogen tributyltin (TBT) throughout pregnancy and lactation predisposes unexposed F4 male descendants to obesity when dietary fat is increased. Analyses of body fat, plasma hormone levels, and visceral white adipose tissue DNA methylome and transcriptome collectively indicate that the F4 obesity is consistent with a leptin resistant, thrifty phenotype. Ancestral TBT exposure induces global changes in DNA methylation and altered expression of metabolism-relevant genes. Analysis of chromatin accessibility in F3 and F4 sperm reveals significant differences between control and TBT groups and significant similarities between F3 and F4 TBT groups that overlap with areas of differential methylation in F4 adipose tissue. Our data suggest that ancestral TBT exposure induces changes in chromatin organization transmissible through meiosis and mitosis.