Keisuke S. Iwamoto

Exposure to ionizing radiation disrupts metabolic pathways and causes oxidative stress, which can lead to organ damage. In this study, urinary metabolites from mice exposed to high-dose and low-dose whole-body irradiation (WBI HDR, WBI LDR) or partial-body irradiation (PBI BM2.5) were analyzed using targeted and untargeted metabolomics approaches. Significant metabolic changes particularly in oxidative stress pathways were observed on Day 2 post-radiation. By Day 30, the WBI HDR group showed persistent metabolic dysregulation, while the WBI LDR and PBI BM2.5 groups were similar to control mice. Machine learning models identified metabolites that were predictive of the type of radiation exposure with high accuracy, highlighting their potential use as biomarkers for radiation damage and oxidative stress.

Image-guided mouse irradiation is essential to understand interventions involving radiation prior to human studies. Our objective is to employ Swin UNEt TRansformers (Swin UNETR) to segment native micro-CT and contrast-enhanced micro-CT scans and benchmark the results against 3D no-new-Net (nnU-Net). Swin UNETR reformulates mouse organ segmentation as a sequence-to-sequence prediction task using a hierarchical Swin Transformer encoder to extract features at five resolution levels, and it connects to a Fully Convolutional Neural Network (FCNN)-based decoder via skip connections. The models were trained and evaluated on open datasets, with data separation based on individual mice. Further evaluation on an external mouse dataset acquired on a different micro-CT with lower kVp and higher imaging noise was also employed to assess model robustness and generalizability. The results indicate that Swin UNETR consistently outperforms nnU-Net and AIMOS in terms of the average dice similarity coefficient (DSC) and the Hausdorff distance (HD95p), except in two mice for intestine contouring. This superior performance is especially evident in the external dataset, confirming the model’s robustness to variations in imaging conditions, including noise and quality, and thereby positioning Swin UNETR as a highly generalizable and efficient tool for automated contouring in pre-clinical workflows.

Nuclear factor erythroid 2-related factor 2 (NRF2) is recognized as a master transcription factor that regulates expression of numerous detoxifying and antioxidant cytoprotective genes. In fact, models of NRF2 deficiency indicate roles not only in redox regulation, but also in metabolism, inflammatory/autoimmune disease, cancer, and radioresistancy. Since ionizing radiation (IR) generates reactive oxygen species (ROS), it is not surprising it activates NRF2 pathways. However, unexpectedly, activation is often delayed for many days after the initial ROS burst. Here, we demonstrate that, as assayed by γ-H2AX staining, rapid DNA double strand break (DSB) formation by IR in primary mouse Nrf2–/– MEFs was not affected by loss of NRF2, and neither was DSB repair to any great extent. In spite of this, basal and IR-induced transformation was greatly enhanced, suggesting that NRF2 protects against late IR-induced genomic instability, at least in murine MEFs. Another possible IR- and NRF2-related event that could be altered is inflammation and NRF2 deficiency increased IR-induced NF-κB pro-inflammatory responses mostly late after exposure. The proclivity of NRF2 to restrain inflammation is also reflected in the reprogramming of tumor antigen-specific lymphocyte responses in mice where Nrf2 k.o. switches Th2 responses to Th1 polarity. Delayed NRF2 responses to IR may be critical for the immune transition from prooxidant inflammation to antioxidant healing as well as in driving cellular radioresistance and survival. Targeting NRF2 to reprogram immunity could be of considerable therapeutic benefit in radiation and immunotherapy.

Although there has been extensive research done on the biological response to doses of ionizing radiation relevant to radiodiagnostic procedures, very few studies have examined radiation schemes similar to those frequently utilized in CT exams. Instead of a single exposure, CT exams are often made up of a series of scans separated on the order of minutes. DNA damage dose-response kinetics after radiation doses and schemes similar to CT protocols were established in both cultured (ESW-WT3) and whole blood lymphocytes and compared to higher dose exposures. Both the kinetics and extent of H2AX phosphorylation were found to be dose dependent. Damage induction and detection showed a clear dose response, albeit different, at all time points and differences in the DNA repair kinetics of ESW-WT3 and whole blood lymphocytes were characterized. Moreover, using a modified split-dose in vitro experiment, we show that phosphorylation of H2AX is significantly reduced after exposure to CT doses fractionated over a few minutes compared to the same total dose delivered as a single exposure. Because the split-dose exposures investigated here are more similar to those experienced during a CT examination, it is essential to understand why and how these differences occur. This work provides compelling evidence supporting differential biological responses not only between high and low doses, but also between single and multiple exposures to low doses of ionizing radiation.

Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

Background: Many biological factors of 2-[¹⁸ F]fluoro-2-deoxy-d-glucose (¹⁸ F-FDG) in blood can affect ¹⁸ F-FDG uptake in tumors. In this study, longitudinal ¹⁸ F-FDG positron emission tomography (PET) studies were performed on tumor-bearing mice to investigate the effect of blood glucose level and tumor size on ¹⁸ F-FDG uptake in tumors. Methods: Six- to eight-week-old severe combined immunodeficiency mice were implanted with glioblastoma U87 (n = 8) or adenocarcinoma MDA-MB-231 (MDA) (n = 11) in the shoulder. When the tumor diameter was approximately 2.5 mm, a 60-min dynamic ¹⁸ F-FDG PET scan was performed weekly until the tumor diameter reached 10 mm. Regions of interests were defined in major organs and tumor. A plasma curve was derived based on a modeling method that utilizes the early heart time-activity curve and a late-time blood sample. The ¹⁸ F-FDG uptake constant K _i was calculated using Patlak analysis on the tumors without an apparent necrotic center shown in the PET images. For each tumor type, the measured K _i was corrected for partial volume (PV), and multivariate regression analysis was performed to examine the effects of blood glucose level ([Glc]) and tumor growth. Corrected Akaike's information criterion was used to determine the best model. Results: The regression model that best fit the PV-corrected K _i for U87 data was K _i /RC = (1/[Glc]) × (0.27 ± 0.027) mL/min/mL (where [Glc] is in mmol/L), and for MDA, it was K _i /RC = (0.04 ± 0.005) mL/min/mL, where K _i /RC denotes the PV-corrected K _i using an individual recovery coefficient (RC). The results indicated that ¹⁸ F-FDG K _i /RC for U87 was inversely related to [Glc], while [Glc] had no effect on ¹⁸ F-FDG K _i /RC of MDA. After the effects of PV and [Glc] were accounted for, the data did not support any increase of ¹⁸ F-FDG K _i as the tumor (of either type) grew larger in size. Conclusions: The effect of [Glc] on the tumor ¹⁸ F-FDG K _i was tumor-dependent. PV- and [Glc]-corrected ¹⁸ F-FDG K _i did not show significant increase as the tumor of either type grew in size.

Purpose: It is widely believed that the anticancer drug 5- fluorouracil (5-FU) must be administered chronically and in low doses to maximize radiosensitization during chemoradiotherapy. The rationale is based upon cell experiments that assumed identical mechanisms of 5-FU action between low-dose chronic (LDC) and high-dose pulsed (HDP) exposures. Here we challenge the paradigm and demonstrate the effectiveness of HDP 5-FU as a radiosensitizer and the wide range of dose/time schedules that can be used to synergize with radiation as compared to the relatively restrictive protocols prescribed for current LDC administrations. Materials and methods: Clonogenic survival of human glioblastoma and colon cancer cell lines, U87MG-VIII and HCT-116, respectively, was used to assess temporal and dose effects of 5-FU on radiosensitivity and in split-dose experiments to characterize changes in sublethal damage repair. Results: We show that HDP 5-FU administration does indeed radiosensitize both the highly radioresistant U87MG-VIII and HCT-116. Additionally, we show that this radiosensitization lasts for at least 24 h if cells are pre-irradiated with 2 Gy immediately after HDP 5-FU exposure as a result of a decrease in sublethal damage repair capacity for subsequent irradiations, suggesting the ideal combination of 5-FU bolus injection with fractionation radiotherapy schemes. Conclusions: 5-FU bolus administration protocols combined with radiation would not only help improve treatment outcomes and reduce development of 5-FU resistance, but it would greatly benefit patients by shortening clinical stays and lowering overall therapeutic costs.