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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Mortality among workers at the Los Alamos National Laboratory, 1943–2017

During World War II (WWII), the Manhattan Engineering District established a secret laboratory in the mountains of northern New Mexico. The mission was to design, construct and test the first atomic weapon, nicknamed ‘The Gadget’ that was detonated at the TRINITY site in Alamogordo, NM. After WWII, nuclear weapons research continued, and the laboratory became the Los Alamos National Laboratory (LANL). The mortality experience of 26,328 workers first employed between 1943 and 1980 at LANL was determined through 2017. Included were 6157 contract workers employed by the ZIA Company. Organ dose estimates for each worker considered all sources of exposure, notably photons, neutrons, tritium, 238Pu and 239Pu. Vital status determination included searches within the National Death Index, Social Security Administration and New Mexico State Mortality Files. Standardized Mortality Ratios (SMR) and Cox regression models were used in the analyses. Most workers (55%) were hired before 1960, 38% had a college degree, 25% were female, 81% white, 13% Hispanic and 60% had died. Vital status was complete, with only 0.1% lost to follow-up. The mean dose to the lung for the 17,053 workers monitored for radiation was 28.6 weighted-mGy (maximum 16.8 weighted-Gy) assuming a Dose Weighting Factor of 20 for alpha particle dose to lung. The Excess Relative Risk (ERR) at 100 weighted-mGy was 0.01 (95%CI -0.02, 0.03; n = 839) for lung cancer. The ERR at 100 mGy was -0.43 (95%CI -1.11, 0.24; n = 160) for leukemia other than chronic lymphocytic leukemia (CLL), -0.06 (95%CI -0.16, 0.04; n = 3043) for ischemic heart disease (IHD), and 0.29 (95%CI 0.02, 0.55; n = 106) for esophageal cancer. Among the 6499 workers with measurable intakes of plutonium, an increase in bone cancer (SMR 2.44; 95%CI 0.98, 5.03; n = 7) was related to dose. The SMR for berylliosis was significantly high, based on 4 deaths. SMRs for Hispanic workers were significantly high for cancers of the stomach and liver, cirrhosis of the liver, nonmalignant kidney disease and diabetes, but the excesses were not related to radiation dose. There was little evidence that radiation increased the risk of lung cancer or leukemia. Esophageal cancer was associated with radiation, and plutonium intakes were linked to an increase of bone cancer. IHD was not associated with radiation dose. More precise evaluations will await the pooled analysis of workers with similar exposures such as at Rocky Flats, Savannah River and Hanford.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2021

Pre- and post-treatment image-based dosimetry in 90 Y-microsphere radioembolization using the TOPAS Monte Carlo toolkit

Objective.To evaluate the pre-treatment and post-treatment imaging-based dosimetry of patients treated with 90Y-microspheres, including accurate estimations of dose to tumor, healthy liver and lung. To do so, the Monte Carlo (MC) TOPAS platform is in this work extended towards its utilization in radionuclide therapy. Approach. In this study, five patients treated at the Massachusetts General Hospital were selected for this study. All patients had data for both pre-treatment SPECT-CT imaging using 99mTc-MAA as a surrogate of the 90Y-microspheres treatment and SPECT-CT imaging immediately after the 90Y activity administration. Pre- and post-treatment doses were computed with TOPAS using the SPECT images to localize the source positions and the CT images to account for tissue inhomoegeneities. We compared our results with analytical calculations following the voxel-based MIRD scheme. Main results. TOPAS results largely agreed with the MIRD-based calculations in soft tissue regions: the average difference in mean dose to the liver was 0.14 Gy GBq -1 (2.6%). However, dose distributions in the lung differed considerably: absolute differences in mean doses to the lung ranged from 1.2 to 6.3 Gy GBq -1 and relative differences from 153% to 231%. We also found large differences in the intra-hepatic dose distributions between pre- and post-treatment imaging, but only limited differences in the pulmonary dose. Significance. Doses to lung were found to be higher using TOPAS with respect to analytical calculations which may significantly underestimate dose to the lung, suggesting the use of MC methods for 90Y dosimetry. According to our results, pre-treatment imaging may still be representative of dose to lung in these treatments.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2022

Adrenal Stress Hormone Regulation of Hepatic Homeostatic Function After an Acute Ozone Exposure in Wistar-Kyoto Male Rats

Ozone-induced lung injury, inflammation, and pulmonary/hypothalamus gene expression changes are diminished in adrenalectomized (AD) rats. Acute ozone exposure induces metabolic alterations concomitant with increases in epinephrine and corticosterone. We hypothesized that adrenal hormones are responsible for observed hepatic ozone effects, and in AD rats, these changes would be diminished. In total, 5–7 days after sham (SH) or AD surgeries, male Wistar-Kyoto rats were exposed to air or 0.8-ppm ozone for 4 h. Serum samples were analyzed for metabolites and liver for transcriptional changes immediately post-exposure. Ozone increased circulating triglycerides, cholesterol, free fatty-acids, and leptin in SH but not AD rats. Ozone-induced inhibition of glucose-mediated insulin release was absent in AD rats. Unlike diminution of ozone-induced hypothalamus and lung mRNA expression changes, AD in air-exposed rats (AD-air/SH-air) caused differential hepatic expression of ~1000 genes. Likewise, ozone in AD rats caused differential expression of ~1000 genes (AD-ozone/AD-air). Ozone-induced hepatic changes in SH rats reflected enrichment for pathways involving metabolic processes, including acetyl-CoA biosynthesis, TCA cycle, and sirtuins. Upstream predictor analysis identified similarity to responses produced by glucocorticoids and pathways involving forskolin. These changes were absent in AD rats exposed to ozone. However, ozone caused unique changes in AD liver mRNA reflecting activation of synaptogenesis, neurovascular coupling, neuroinflammation, and insulin signaling with inhibition of senescence pathways. In these rats, upstream predictor analysis identified numerous microRNAs likely involved in glucocorticoid insufficiency. These data demonstrate the critical role of adrenal stress hormones in ozone-induced hepatic homeostasis and necessitate further research elucidating their role in propagating environmentally driven diseases.

Toxicology↗
DOE OSTI2020

Development of a New Chelation Model: Bioassay Data Interpretation and Dose Assessment after Plutonium Intake via Wound and Treatment with DTPA

The administration of chelation therapy to treat significant intakes of actinides, such as plutonium, affects the actinide’s normal biokinetics. In particular, it enhances the actinide’s rate of excretion, such that the standard biokinetic models cannot be applied directly to the chelation-affected bioassay data in order to estimate the intake and assess the radiation dose. Here we propose a new chelation model that can be applied to the chelation-affected bioassay data after plutonium intake via wound and treatment with DTPA. In the proposed model, chelation is assumed to occur in the blood, liver, and parts of the skeleton. Ten datasets, consisting of measurements of 14 C-DTPA, 238 Pu, and 239 Pu involving humans given radiolabeled DTPA and humans occupationally exposed to plutonium via wound and treated with chelation therapy, were used for model development. The combined dataset consisted of daily and cumulative excretion (urine and feces), wound counts, measurements of excised tissue, blood, and post-mortem tissue analyses of liver and skeleton. The combined data were simultaneously fit using the chelation model linked with a plutonium systemic model, which was linked to an ad hoc wound model. The proposed chelation model was used for dose assessment of the wound cases used in this study.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Black-box optimization of CT acquisition and reconstruction parameters: a reinforcement learning approach

Protocol optimization is critical in Computed Tomography (CT) for achieving desired diagnostic image quality while minimizing radiation dose. Due to the inter-effect of influencing CT parameters, traditional optimization methods rely on the testing of exhaustive combinations of these parameters. This poses a notable limitation due to the impracticality of exhaustive parameter testing. This study introduces a novel methodology leveraging Virtual Imaging Trials (VITs) and reinforcement learning to more efficiently optimize CT protocols. Computational phantoms with liver lesions were imaged using a validated CT simulator and reconstructed with a novel CT reconstruction Toolkit. The optimization parameter space included tube voltage, tube current, reconstruction kernel, slice thickness, and pixel size. The optimization process was done using a Proximal Policy Optimization (PPO) agent which was trained to maximize the Detectability Index (d’) of the liver lesion for each reconstructed image. Results showed that our reinforcement learning approach found the absolute maximum d’ across the test cases while requiring 79.7% fewer steps compared to an exhaustive search, demonstrating both accuracy and computational efficiency, offering a efficient and robust framework for CT protocol optimization. The flexibility of the proposed technique allows for use of varying image quality metrics as the objective metric to maximize for. Our findings highlight the advantages of combining VIT and reinforcement learning for CT protocol management.

Fenwick, David [Duke University Medical Center]↗
DOE OSTI2020

Comparative RNA-Seq transcriptome analyses reveal dynamic time-dependent effects of 56 Fe, 16 O, and 28 Si irradiation on the induction of murine hepatocellular carcinoma

One of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z > 13), which can lead to the induction of hepatocellular carcinoma (HCC). However, little is known on the molecular mechanisms of HZE irradiation-induced HCC. We performed comparative RNA-Seq transcriptomic analyses to assess the carcinogenic effects of 600 MeV/n 56 Fe (0.2 Gy), 1 GeV/n 16 O (0.2 Gy), and 350 MeV/n 28 Si (0.2 Gy) ions in a mouse model for irradiation-induced HCC. C3H/HeNCrl mice were subjected to total body irradiation to simulate space environment HZE-irradiation, and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the biological effects of these HZE ions, particularly immunological, such as Acute Phase Response Signaling, B Cell Receptor Signaling, IL-8 Signaling, and ROS Production in Macrophages. Also seen in this study were novel unannotated transcripts that were significantly affected by HZE. To investigate the biological functions of these novel transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples (45 HZE-irradiated, 15 non-irradiated control) from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes (e.g., Acute Phase Response Signaling, B Cell Receptor Signaling, IL-3 Signaling), and RNA Transcription/Expression. A large number of transcripts were found differentially expressed post-HZE irradiation. These results provide valuable information for uncovering the differences in molecular mechanisms underlying HZE specific induced HCC carcinogenesis. Additionally, a handful of novel differentially expressed unannotated transcripts were discovered for each HZE ion. Taken together, these findings may provide a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation and may also have important implications for the discovery of potential countermeasures against and identification of biomarkers for HZE-induced HCC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The endocannabinoid system in bovine tissues: characterization of transcript abundance in the growing Holstein steer

Abstract Background The endocannabinoid system (ECS) is highly integrated with seemingly all physiological and pathophysiological processes in the body. There is increasing interest in utilizing bioactive plant compounds, for promoting health and improving production in livestock. Given the established interaction between phytochemicals and the ECS, there are many opportunities for identification and development of therapies to address a range of diseases and disorders. However, the ECS has not been thoroughly characterized in cattle, especially in the gastrointestinal tract. The objective of this study was to characterize the distribution and transcriptional abundance of genes associated with the endocannabinoid system in bovine tissues. Methods Tissues including brain, spleen, thyroid, lung, liver, kidney, mesenteric vein, tongue, sublingual mucosa, rumen, omasum, duodenum, jejunum, ileum and colon were collected from 10-mo old Holstein steers (n = 6). Total RNA was extracted and gene expression was measured using absolute quantification real time qPCR. Gene expression of endocannabinoid receptorsCNR1andCNR2, synthesis enzymesDAGLA,DAGLBandNAPEPLD, degradation enzymesMGLLandFAAH, and transient receptor potential vanilloidsTRPV3andTRPV6was measured. Data were analyzed in R using a Kruskal-Wallis followed by a Wilcoxon rank-sum test. Results are reported as the median copy number/20 ng of equivalent cDNA (CN) with interquartile range (IQR). Results The greatest expression ofCNR1andCNR2was in the brain and spleen, respectively. Expression of either receptor was not detected in any gastrointestinal tissues, however there was a tendency (P = 0.095) forCNR2to be expressed above background in rumen. Expression of endocannabinoid synthesis and degradation enzymes varied greatly across tissues. Brain tissue had the greatestDAGLAexpression at 641 CN (IQR 52;P ≤ 0.05).DAGLBwas detected in all tissues, with brain and spleen having the greatest expression (P ≤ 0.05). Expression ofNAPEPLDin the gastrointestinal tract was lowest in tongue and sublingual mucosal. There was no difference in expression ofNAPEPLDbetween hindgut tissues, however these tissues collectively had 592% greater expression than rumen and omasum (P ≤ 0.05). WhileMGLLwas found to be expressed in all tissues, expression ofFAAHwas only above the limit of detection in brain, liver, kidney, jejunum and ileum.TRPV3was expressed above background in tongue, rumen, omasum and colon. Although not different from each other, thyroid and duodenum had the greatest expression ofTRPV6, with 285 (IQR 164) and 563 (IQR 467) CN compared to all other tissues (P < 0.05). Conclusions These data demonstrate the complex distribution and variation of the ECS in bovine tissues. Expression patterns suggest that regulatory functions of this system are tissue dependent, providing initial insight into potential target tissues for manipulation of the ECS.

Veterinary Sciences↗
DOE OSTI2023

Supranutrition of microalgal docosahexaenoic acid and calcidiol improved growth performance, tissue lipid profiles, and tibia characteristics of broiler chickens

Abstract Background Docosahexaenoic acid (DHA) and calcidiol could be enriched in chicken for improving public nutrition and health. It remains unclear if supranutritional levels of DHA and calcidiol impair growth performance or metabolism of broiler chickens. This study was to determine singular and combined effects of high levels of supplemental DHA-rich microalgal biomass or oil and calcidiol on growth performance, concentrations of triglycerides, cholesterol, and nonesterfied fatty acids in plasma, liver, breast, and thigh, and biophysical properties of tibia. Methods In Exp. 1, 144 day-old Cornish chicks were divided into 4 groups (6 cages/treatment, 6 birds/cage), and were fed a corn-soybean meal basal diet (BD), BD + 10,000 IU calcidiol/kg (BD + Cal), BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), and BD + Cal + DHA for 6 weeks. In Exp. 2, 180 day-old chicks were divided into 5 groups, and were fed: BD, BD + DHA (0.33% to 0.66% oil, 1.5 to 3.0 g DHA/kg), BD + DHA + EPA (1.9% to 3.8% eicosapentaenoic acid-rich Nannochloropsis sp. CO18, 0.3 to 0.6 g EPA/kg), BD + DHA + calcidiol (6000 to 12,000 IU/kg diet), and BD + DHA + EPA + Cal for 6 weeks. Results Birds fed BD + Cal diet in Exp. 1 and BD + DHA + EPA diet in Exp. 2 had higher ( P < 0.05) body weight gain (10%–11%) and gain:feed ratio (7%), and lower ( P < 0.05) total cholesterol and triglyceride concentrations in plasma (18%–54%), liver (8%–26%), breast (19%–26%), and thigh (10%–19%), respectively, over the controls. The two diets also improved ( P < 0.05) tibial breaking strength (8%–24%), total bone volume (2%–13%), and (or) bone mineral density (3%–19%) of chickens. Conclusion Supranutrition of dietary calcidiol and DHA alone or together did not produce adverse effects, but led to moderate improvements of growth performance, lipid profiles of plasma and muscle, and bone properties of broiler chickens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Data from Improved Annotation of Untargeted Metabolomics Data through Buffer Modifications That Shift Adduct Mass and Intensity

Annotation of untargeted high-resolution full-scan LC-MS metabolomics data remains challenging due to individual metabolites generating multiple LC-MS peaks arising from isotopes, adducts, and fragments. Adduct annotation is a particular challenge, as the same mass difference between peaks can arise from adduct formation, fragmentation, or different biological species. To address this, here we describe a buffer modification workflow (BMW) in which the same sample is run by LC-MS in both liquid chromatography solvent with 14NH3–acetate buffer and in solvent with the buffer modified with 15NH3–formate. Buffer switching results in characteristic mass and signal intensity changes for adduct peaks, facilitating their annotation. This relatively simple and convenient chromatography modification annotated yeast metabolomics data with similar effectiveness to growing the yeast in isotope-labeled media. Application to mouse liver data annotated both known metabolite and known adduct peaks with 95% accuracy. Overall, it identified 26% of ∼27 000 liver LC-MS features as putative metabolites, of which ∼2600 showed HMDB or KEGG database formula match. This workflow is well suited to biological samples that cannot be readily isotope labeled, including plants, mammalian tissues, and tumors.

Conversion↗
DOE OSTI2025

Prediction of cccDNA dynamics in hepatitis B patients by a combination of serum surrogate markers

Quantification of intrahepatic covalently closed circular DNA (cccDNA) is a key for evaluating an elimination of hepatitis B virus (HBV) in infected patients. However, quantifying cccDNA requires invasive methods such as a liver biopsy, which makes it impractical to access the dynamics of cccDNA in patients. Although HBV RNA and HBV core-related antigens (HBcrAg) have been proposed as surrogate markers for evaluating cccDNA activity, they do not necessarily estimate the amount of cccDNA. Here, we employed a recently developed multiscale mathematical model describing intra- and intercellular viral propagation and applied it in HBV-infected patients under treatment. We developed a model that can predict intracellular HBV dynamics by use of extracellular viral markers, including HBsAg, HBV DNA, and HBcrAg in peripheral blood. Importantly, the model prediction of the amount of cccDNA in patients over time was confirmed to be well correlated with the data for quantified cccDNA by paired liver biopsy. Thus, our method combining classic and emerging surrogate markers enables us to predict the decay dynamics of cccDNA in patients undergoing treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

225 Ac-MACROPATATE: A Novel α-Particle Peptide Receptor Radionuclide Therapy for Neuroendocrine Tumors

Neuroendocrine tumors (NETs) express somatostatin receptors (SSTRs) 2 and 5. Modified variants of somatostatin, the cognate ligand for SSTR2 and SSTR5, are used in treatment for metastatic and locoregional disease. Peptide receptor radionuclide therapy with 177 Lu-DOTATATE (DOTA-octreotate), a β-particle–emitting somatostatin derivative, has demonstrated survival benefit in patients with SSTR-positive NETs. Despite excellent results, a subset of patients has tumors that are resistant to treatment, and alternative agents are needed. Targeted α-particle therapy has been shown to kill tumors that are resistant to targeted β-particle therapy, suggesting that targeted α-particle therapy may offer a promising treatment option for patients with 177 Lu-DOTATATE–resistant disease. Although DOTATATE can chelate the clinically relevant α-particle–emitting radionuclide 225 Ac, the labeling reaction requires high temperatures, and the resulting radioconjugate has suboptimal stability. Methods: We designed and synthesized MACROPATATE (MACROPA-octreotate), a novel radioconjugate capable of chelating 225 Ac at room temperature, and assessed its in vitro and in vivo performance. Results: MACROPATATE demonstrated comparable affinity to DOTATATE (dissociation constant, 21 nM) in U2-OS-SSTR2, a SSTR2-positive transfected cell line. 225 Ac-MACROPATATE demonstrated superior serum stability at 37°C over time compared with 225 Ac-DOTATATE. Biodistribution studies demonstrated higher tumor uptake of 225 Ac-MACROPATATE than of 225 Ac-DOTATATE in mice engrafted with subcutaneous H69 NETs. Therapy studies showed that 225 Ac-MACROPATATE exhibits significant antitumor and survival benefit compared with saline control in mice engrafted with SSTR-positive tumors. However, the increased accumulation of 225 Ac-MACROPATATE in liver and kidneys and subsequent toxicity to these organs decreased its therapeutic index compared with 225 Ac-DOTATATE. Conclusion: 225 Ac-MACROPATATE and 225 Ac-DOTATATE exhibit favorable therapeutic efficacy in animal models. Because of elevated liver and kidney accumulation and lower administered activity for dose-limiting toxicity of 225 Ac-MACROPATATE, 225 Ac-DOTATATE was deemed the superior agent for targeted α-particle peptide receptor radionuclide therapy.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2023

A perfused multi-well bioreactor platform to assess tumor organoid response to a chemotherapeutic gradient

There is an urgent need to develop new therapies for colorectal cancer that has metastasized to the liver and, more fundamentally, to develop improved preclinical platforms of colorectal cancer liver metastases (CRCLM) to screen therapies for efficacy. To this end, we developed a multi-well perfusable bioreactor capable of monitoring CRCLM patient-derived organoid response to a chemotherapeutic gradient. CRCLM patient-derived organoids were cultured in the multi-well bioreactor for 7 days and the subsequently established gradient in 5-fluorouracil (5-FU) concentration resulted in a lower IC 50 in the region near the perfusion channel versus the region far from the channel. We compared behaviour of organoids in this platform to two commonly used PDO culture models: organoids in media and organoids in a static (no perfusion) hydrogel. The bioreactor IC 50 values were significantly higher than IC 50 values for organoids cultured in media whereas only the IC 50 for organoids far from the channel were significantly different than organoids cultured in the static hydrogel condition. Using finite element simulations, we showed that the total dose delivered, calculated using area under the curve (AUC) was similar between platforms, however normalized viability was lower for the organoid in media condition than in the static gel and bioreactor. Our results highlight the utility of our multi-well bioreactor for studying organoid response to chemical gradients and demonstrate that comparing drug response across these different platforms is nontrivial.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Src-mediated Tyr353 phosphorylation of IP3R1 promotes its stability and causes apoptosis in palmitic acid-treated hepatocytes

Palmitic acid (PA)-induced hepatocyte apoptosis is critical for the progression of nonalcoholic fatty liver disease (NAFLD). Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is an intracellular Ca{sup 2+}-release channel and is involved in PA-induced hepatocyte apoptosis. While the expression of IP3R1 is elevated in patients with NAFLD and in hepatocytes treated with PA, it remains unclear how PA promotes the expression of IP3R1. In present study, our results showed that PA induced mitochondrial dysfunction and apoptosis, which is accompanied with the increase of the IP3R1 expression in hepatic cells. The inhibition of IP3R1 expression using siRNA ameliorated the PA-induced mitochondrial dysfunction. Furthermore, PA enhanced the stability of the IP3R1 protein instead of an increase in its mRNA levels. PA also promoted the phosphorylation of IP3R1 at the Tyr353 site and increased the phosphorylation of src in hepatic cells. Moreover, an inhibitor of src kinase (SU6656) significantly reduced the Tyr353 phosphorylation of IP3R1 and decreased its stability. In addition, SU6656 improved mitochondrial function and reduced apoptosis in hepatocytes. Conclusion: PA promotes the Tyr353 phosphorylation of IP3R1 by activating the src pathway and increasing the protein stability of IP3R1, which consequently results in mitochondrial Ca{sup 2+} overload and mitochondrial dysfunction in hepatic cells. Our results also suggested that inhibition of the src/IP3R1 pathway, such as by SU6656, may be a novel potential therapeutic approach for the treatment of NAFLD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Hepatocellular Metabolic Profile: Understanding Post-Thawing Metabolic Shift in Primary Hepatocytes In Vitro

Primary human hepatocytes (PHHs) are widely used as in vitro models for liver function and drug metabolism studies, yet their metabolic stability post-thawing remains an open question. To better characterize early metabolic changes, we conducted a time-course experiment using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze metabolic shifts in PHHs cultured in suspension. Unexposed and exposed (acetaminophen-treated) samples were evaluated, and TITAN analysis was applied to determine the time point of maximal metabolic change at both individual metabolite and global metabolic profile levels. Our results indicate that the majority of metabolic shifts occur within the first five hours post-thawing. In the early culture time points, substantial metabolic overlap was observed between unexposed and exposed cells, suggesting a conserved biological response likely related to cellular recovery. However, at later time points, metabolite profiles diverged, with acetaminophen treatment-specific metabolic changes emerging, potentially reflecting differences in homeostatic restoration versus hepatotoxic responses. Our study highlights the importance of considering early post-thawing metabolic dynamics in experimental design and offers insights for optimizing hepatocyte culture protocols to better replicate in vivo physiological conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Current Status of Radiopharmaceutical Therapy

In radiopharmaceutical therapy (RPT), a radionuclide is systemically or locally delivered with the goal of targeting and delivering radiation to cancer cells while minimizing radiation exposure to untargeted cells. Examples of current RPTs include thyroid ablation with the administration of {sup 131}I, treatment of liver cancer with {sup 90}Y microspheres, the treatment of bony metastases with {sup 223}Ra, and the treatment of neuroendocrine tumors with {sup 177}Lu-DOTATATE. New RPTs are being developed where radionuclides are incorporated into systemic targeted therapies. To assure that RPT is appropriately implemented, advances in targeting need to be matched with advances in quantitative imaging and dosimetry methods. Currently, radiopharmaceutical therapy is administered by intravenous or locoregional injection, and the treatment planning has typically been implemented like chemotherapy, where the activity administered is either fixed or based on a patient’s body weight or body surface area. RPT pharmacokinetics are measurable by quantitative imaging and are known to vary across patients, both in tumors and normal tissues. Therefore, fixed or weight-based activity prescriptions are not currently optimized to deliver a cytotoxic dose to targets while remaining within the tolerance dose of organs at risk. Methods that provide dose estimates to individual patients rather than to reference geometries are needed to assess and adjust the injected RPT dose. Accurate doses to targets and organs at risk will benefit the individual patients and decrease uncertainties in clinical trials. Imaging can be used to measure activity distribution in vivo, and this information can be used to determine patient-specific treatment plans where the dose to the targets and organs at risk can be calculated. The development and adoption of imaging-based dosimetry methods is particularly beneficial in early clinical trials. In this work we discuss dosimetric accuracy needs in modern radiation oncology, uncertainties in the dosimetry in RPT, and best approaches for imaging and dosimetry of internal radionuclide therapy.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2021

MiR-205–5p suppresses angiogenesis in gastric cancer by downregulating the expression of VEGFA and FGF1

Highlights: • MiR-205-5p was commonly downregulated in GC tissues from TCGA database and our hospital, and these events were associated with a worse TNM stagedistant metastasis and poor survival. • MiR-205-5p and CD31 expressions were negatively inter-related within GC tissues from TCGA database and our hospital. • GC cell lines expressed lower level of miR-205-5p significantly as compared with the normal gastric cell line. • MiR-205-5p upregulation significantly impaired proliferation and colony formation of GC cells and neovascularization induced by GC cells in vitro and in vivo. • The angiogenesis and ERK-signaling were depressed by VEGFA and FGF1 downregulation induced by miR-205-5p overexpression. The miR-205-5p inhibitor promoted malignant phenotypes by enhancing VEGFA and FGF1 expressions, as well as the activation of ERK-signaling. Anti-angiogenic therapy represents one of the most promising treatment modalities for human cancers. However, the response to antiangiogenic therapy in gastric cancer (GC) remains dismal. To help identify new strategies for antiangiogenic therapy in GC, we evaluated miR-205–5p expression in GC tissues from TCGA database and our hospital, and its functions in angiogenesis were explored in vitro and in vivo. We investigated miR-205–5p expression and microvessel densities (MVDs) in GC tissues and liver metastases from patients. The function and mechanisms of miR-205–5p were examined in human cell lines and in xenograft mouse models. Associations between miR-205–5p expression and clinical characteristics were analyzed using either Pearson's χ{sup 2} test or Fisher's exact test. Differences in overall survival (OS) distributions were evaluated using the log-rank test. Differences in measurement data were compared using Student's t-test and one-way ANOVA. We found that miR-205–5p expression was downregulated in GC tissues and was negatively correlated with CD31 expression in both TCGA and our clinical samples. GC cell lines expressed low levels of miR-205–5p, and miR-205–5p upregulation significantly impaired the proliferation and angiogenesis of GC cells. Moreover, vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 1 (FGF1) expression and activation of extracellular-related kinase (ERK) signaling were suppressed by miR-205–5p. MiR-205–5p inhibition promoted malignant phenotypes by enhancing VEGFA and FGF1 expression, as well as the activation of ERK signaling. Angiogenesis and ERK signaling were decreased in response to VEGFA and FGF1 downregulation induced by miR-205–5p overexpression. The dual-luciferase reporter assay showed that VEGFA and FGF1 were direct targets of miR-205–5p. Xenograft mouse models revealed that miR-205–5p suppressed tumor growth by inhibiting neovascularization. Altogether, these results demonstrate that miR-205–5p suppresses angiogenesis in GC by attenuating the expression of VEGFA and FGF1, indicating that upregulation of miR-205–5p may represent as an antiangiogenic therapy for GC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Upregulation of a novel LncRNA AC104958.2 stabilized by PCBP2 promotes proliferation and microvascular invasion in hepatocellular carcinoma

Highlights: • AC104958.2 is a novel lncRNA identified to be upregulated in HCC tissues and associated with MVI. • Knockout of AC104958.2 inhibited cell proliferation, invasion, migration and EMT. • AC104958.2 interacted with PCBP2. • PCBP2 was upregulated in HCC and enhanced the RNA stability of AC104958.2. Long non-coding RNAs (lncRNAs) were reported to be involved in tumorigenesis and progression of hepatocellular carcinoma (HCC). Microvascular invasion (MVI) is an independent predictor for early recurrence and overall survival in postoperative patients with HCC. However, the mechanisms how lncRNAs affect HCC and MVI remain elusive. By RNA sequencing (RNA-seq) in a series of 65 HCC samples and 30 paired adjacent non-tumor liver tissue, we identified a novel lncRNA AC104958.2 that was significantly upregulated in HCC tissues and associated with MVI. Overexpression of AC104958.2 obviously elevated cell viability, metastasis, invasion and epithelial-mesenchymal transition (EMT), while knockout of AC104958.2 mediated by CRISPR/Cas9 technique showed the opposite effects. In addition, the interaction between AC104958.2 and Poly (rC) binding protein 2 (PCBP2) was identified by RNA pull down and mass spectrometry (MS), which was further validated by RNA immunoprecipitation (RIP). PCBP2 was also upregulated in HCC and associated with MVI. High expression of both AC104958.2 and PCBP2 was correlated with tumor size, TNM stage and MVI in HCC. Overexpression of PCBP2 greatly increased the cell viability, metastasis, invasion and EMT. Moreover, actinomycin D assay showed that overexpression of PCBP2 enhanced the RNA stability of AC104958.2. In conclusion, our study showed that a novel lncRNA AC104958.2 exerted oncogenic roles in HCC and might be a promising biomarker and therapeutic target.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

NFATc1 promotes epithelial-mesenchymal transition and facilitates colorectal cancer metastasis by targeting SNAI1

Highlights: • Metastasis remains a major cause of colorectal cancer (CRC) mortality. • In this study, we examined the role of nuclear factor of activated T cells 1 (NFATc1), which showed increased expression in metastatic CRC tissues and positively correlated with CRC clinical stages. • Mechanistically, SNAI1 was transcriptionally activated by NFATc1 and interacted with SLUG to promote EMT and CRC metastasis. • Calcineurin-NFAT inhibitor FK506 could reverse these effects, offering novel therapeutic strategies for metastatic CRC. Metastatic recurrence remains a major cause of colorectal cancer (CRC) mortality. In this study, we investigated the mechanistic role of nuclear factor of activated T cells 1 (NFATc1) in CRC metastasis. First, we explored the potential role of NFATc1 in CRC using bioinformatics and hypothesized that NFATc1 might play different roles at different stages of CRC development. Then, we examined the relative expression of NFATc1 in 25 CRC tissues and adjacent normal tissues, and further analyzed the correlation between NFATc1 expression levels and clinical stages in 120 CRC patients. The role of NFATc1 in CRC metastasis and the molecular mechanisms were investigated in both in vitro and in vivo models. Our results showed that the expression of NFATc1 was increased in metastatic CRC tissues and positively associated with clinical stages (stage I vs. stage II, III or IV) of CRC. Overexpression of NFATc1 promoted CRC cell migration, invasion, and epithelial-mesenchymal transition (EMT). Moreover, SNAI1 was verified as the direct transcriptional target of NFATc1 and interacted with SLUG to promote EMT. Remarkably, our lung and liver metastasis mouse model demonstrated that NFATc1 overexpression accelerated CRC metastasis, and treatment with FK506, a calcineurin-NFAT pathway inhibitor, could suppress CRC metastasis in vivo. Taken together, our findings suggest that NFATc1 could transcriptionally activate SNAI1, which in turn interacts with SLUG to mediate EMT to promote CRC metastasis. Thus, making NFATc1 a promising therapeutic target in the treatment of metastatic CRC.

60 APPLIED LIFE SCIENCES↗