Enhancing the anticancer effect of androgen deprivation therapy by monocarboxylate transporter 1 inhibitor in prostate cancer cells.

BACKGROUND: Tumor initiation and progression necessitate a metabolic shift in cancer cells. Consequently, the progression of prostate cancer (PCa), a leading cause of cancer-related deaths in males globally, involves a shift from lipogenic to glycolytic metabolism. Androgen deprivation therapy (ADT) serves as the standard treatment for advanced-stage PCa. However, despite initial patient responses, castrate resistance emerges ultimately, necessitating novel therapeutic approaches. Therefore, in this study, we aimed to investigate the role of monocarboxylate transporters (MCTs) in PCa post-ADT and evaluate their potential as therapeutic targets. METHODS: PCa cells (LNCaP and C4-2 cell line), which has high prostate-specific membrane antigen (PSMA) and androgen receptor (AR) expression among PCa cell lines, was used in this study. We assessed the expression of MCT1 in PCa cells subjected to ADT using charcoal-stripped bovine serum (CSS)-containing medium or enzalutamide (ENZ). Furthermore, we evaluated the synergistic anticancer effects of combined treatment with ENZ and SR13800, an MCT1 inhibitor. RESULTS: Short-term ADT led to a significant upregulation in folate hydrolase 1 (FOLH1) and solute carrier family 16 member 1 (SLC16A1) gene levels, with elevated PSMA and MCT1 protein levels. Long-term ADT induced notable changes in cell morphology with further upregulation of FOLH1/PSMA and SLC16A1/MCT1 levels. Treatment with ENZ, a nonsteroidal anti-androgen, also increased PSMA and MCT1 expression. However, combined therapy with ENZ and SR13800 led to reduced PSMA level, decreased cell viability, and suppressed expression of cancer stem cell markers and migration indicators. Additionally, analysis of human PCa tissues revealed a positive correlation between PSMA and MCT1 expression in tumor regions. CONCLUSIONS: Our results demonstrate that ADT led to a significant upregulation in MCT1 levels. However, the combination of ENZ and SR13800 demonstrated a promising synergistic anticancer effect, highlighting a potential therapeutic significance for patients with PCa undergoing ADT.

Monocarboxylate transporter-1 (MCT-1) inhibitors screened from autodisplayed FV-antibody library.

Monocarboxylate transporter-1 (MCT-1) inhibitors were screened from the Fv-antibody library, which contained complementary determining region 3 with randomized amino acid sequences (11 residues) through site-directed mutagenesis. Fv-antibodies against MCT-1 were screened from the autodisplayed Fv-antibody library. Two clones were screened, and the binding affinity (KD) against MCT-1 was estimated using flow cytometry. The screened Fv-antibodies were expressed as soluble fusion proteins (Fv-1 and Fv-2) and the KD for MCT-1 was estimated using the SPR biosensor. The inhibitory activity of the expressed Fv-antibodies was observed in HEK293T and Jurkat cell lines by measuring intracellular pH and lactate accumulation. The level of cell viability in HEK293T and Jurkat cell lines was decreased by the inhibitory activity of the expressed Fv-antibodies. The binding properties of the Fv-antibodies to MCT-1 were analyzed using molecular docking simulations. Overall, the results showed that the screened Fv-antibodies against MCT-1 from the Fv-antibody library had high binding affinity and inhibitory activity against MCT-1, which could be used as potential therapeutic drug candidates for the MCT-1 inhibitor.

Acetyl-CoA synthetase 2 contributes to a better prognosis for liver cancer by switching acetate-glucose metabolism.

Acetyl-CoA synthetase 2 (ACSS2)-dependent acetate usage has generally been associated with tumorigenesis and increased malignancy in cancers under nutrient-depleted conditions. However, the nutrient usage and metabolic characteristics of the liver differ from those of other organs; therefore, the mechanism of ACSS2-mediated acetate metabolism may also differ in liver cancer. To elucidate the underlying mechanisms of ACSS2 in liver cancer and acetate metabolism, the relationships between patient acetate uptake and metabolic characteristics and between ACSS2 and tumor malignancies were comprehensively studied in vitro, in vivo and in humans. Clinically, we initially found that ACSS2 expression was decreased in liver cancer patients. Moreover, PET-CT imaging confirmed that lower-grade cancer cells take up more 11C-acetate but less 18F-fluorodeoxyglucose (18F-FDG); however, this trend was reversed in higher-grade cancer. Among liver cancer cells, those with high ACSS2 expression avidly absorbed acetate even in a glucose-sufficient environment, whereas those with low ACSS2 expression did not, thereby showing correlations with their respective ACSS2 expression. Metabolomic isotope tracing in vitro and in vivo revealed greater acetate incorporation, greater lipid anabolic metabolism, and less malignancy in high-ACSS2 tumors. Notably, ACSS2 downregulation in liver cancer cells was associated with increased tumor occurrence in vivo. In human patient cohorts, patients in the low-ACSS2 subgroup exhibited reduced anabolism, increased glycolysis/hypoxia, and poorer prognosis. We demonstrated that acetate uptake by ACSS2 in liver cancer is independent of glucose depletion and contributes to lipid anabolic metabolism and reduced malignancy, thereby leading to a better prognosis for liver cancer patients.

Covalent coupling of functionalized outer membrane vesicles (OMVs) to gold nanoparticles.

Outer membrane vesicle-functionalized nanoparticles (OMV-NPs) have attracted significant interest, especially regarding drug delivery applications and vaccines. Here, we report on novel OMV-NPs by applying bioorthogonal click reaction for encapsulating gold nanoparticles (NPs) within outer membrane vesicles (OMVs) by covalent coupling. For this purpose, outer membrane protein A (OmpA), abundant in large numbers (due to 100,000 copies/cell [1]) in OMVs, was modified via the incorporation of the unnatural amino acid p-azidophenylalanine. The azide group was covalently coupled to alkyne-functionalized NPs after incorporation into OmpA. A simplified procedure using low-speed centrifugation (1,000 x g) was developed for preparing OMV-NPs. The OMV-NPs were characterized by zeta potential, Laurdan-based lipid membrane dynamics studies, and the enzymatic activity of functionalized OMVs with surface-displayed nicotinamide adenine dinucleotide oxidase (Nox). In addition, OMVs from attenuated bacteria (ClearColiTM BL21(DE3), E. coli F470) with surface-displayed Nox or antibody fragments were prepared and successfully coupled to AuNPs. Finally, OMV-NPs displaying single-chain variable fragments from a monoclonal antibody directed against epidermal growth factor receptor were applied to demonstrate the feasibility of OMV-NPs for tumor cell targeting.

Two-dimensional segmentation fusion tool: an extensible, free-to-use, user-friendly tool for combining different bidimensional segmentations.

Introduction: In several fields, the process of fusing multiple two-dimensional (2D) closed lines is an important step. For instance, this is fundamental in histology and oncology in general. The treatment of a tumor consists of numerous steps and activities. Among them, segmenting the cancer area, that is, the correct identification of its spatial location by the segmentation technique, is one of the most important and at the same time complex and delicate steps. The difficulty in deriving reliable segmentations stems from the lack of a standard for identifying the edges and surrounding tissues of the tumor area. For this reason, the entire process is affected by considerable subjectivity. Given a tumor image, different practitioners can associate different segmentations with it, and the diagnoses produced may differ. Moreover, experimental data show that the analysis of the same area by the same physician at two separate timepoints may result in different lines being produced. Accordingly, it is challenging to establish which contour line is the ground truth. Methods: Starting from multiple segmentations related to the same tumor, statistical metrics and computational procedures could be exploited to combine them for determining the most reliable contour line. In particular, numerous algorithms have been developed over time for this procedure, but none of them is validated yet. Accordingly, in this field, there is no ground truth, and research is still active. Results: In this work, we developed the Two-Dimensional Segmentation Fusion Tool (TDSFT), a user-friendly tool distributed as a free-to-use standalone application for MAC, Linux, and Windows, which offers a simple and extensible interface where numerous algorithms are proposed to "compute the mean" (i.e., the process to fuse, combine, and "average") multiple 2D lines. Conclusions: The TDSFT can support medical specialists, but it can also be used in other fields where it is required to combine 2D close lines. In addition, the TDSFT is designed to be easily extended with new algorithms thanks to a dedicated graphical interface for configuring new parameters. The TDSFT can be downloaded from the following link: https://sourceforge.net/p/tdsft.

Combination of [18F]FDG and [18F]PSMA-1007 PET/CT predicts tumour aggressiveness at staging and biochemical failure postoperatively in patients with prostate cancer.

PURPOSE: [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT) has limitations in prostate cancer (PCa) detection owing to low glycolysis in the primary tumour. Recently, prostate-specific membrane antigen (PSMA) PET/CT has been useful for biochemical failure detection and radioligand therapy (RLT) guidance. However, few studies have evaluated its use in primary prostate tumours using PSMA and [18F]FDG PET/CT. This study aimed to evaluate [18F]PSMA-1007 and [18F]FDG PET/CT for primary tumour detection and understand the association of metabolic heterogeneity with clinicopathological characteristics at staging and postoperatively. METHOD: This prospective study included 42 index tumours (27 acinar and 15 ductal-dominant) in 42 patients who underwent [18F]PSMA-1007 and [18F]FDG PET/CT and subsequent radical prostatectomy. All patients were followed for a median of 26 mo, and serum prostate-specific antigen levels were measured every 3 mo to evaluate biochemical failure. One-way analysis of variance, Tukey's multiple comparison test, and Fisher's exact test were performed. RESULTS: All 42 index tumours were detected on [18F]PSMA-1007 PET/CT, whereas only 15 were detected on [18F]FDG PET/CT (62.3% vs. 37.7%, p < 0.0001). A high SUVmax for [18F]PSMA-1007 was observed in tumours with high Gleason scores (GS 6-7 vs. GS 8-10; 12.1 vs. 20.1, p < 0.05). Tumours with [18F]FDG uptake were mostly ductal dominant (acinar-dominant 4/27; ductal-dominant; 11/15, p < 0.001), with lower [18F]PSMA-1007 uptake than tumours without [18F]FDG uptake (SUVmax 16.58 vs. 11.19, p < 0.001). There were 16.6% (7/42) of patients with pStage IV in whom the primary tumours were [18F]FDG positive. Biochemical failure was observed in 14.8% (4/27) of patients with [18F]FDG negative tumours but in 53.3% (8/15) of patients with [18F]FDG positive tumours (p = 0.013). CONCLUSIONS: [18F]PSMA-1007 PET/CT was superior to [18F]FDG PET/CT in detecting primary PCa. In contrast, tumours with [18F]FDG uptake are associated with larger size, a ductal-dominant type, and likely to undergo metastasis at staging and biochemical failure postoperatively.

Monoamine Oxidase-A (MAO-A) Inhibitors Screened from the Autodisplayed Fv-Antibody Library.

Serotonin-like mimotopes were screened from the Fv-antibody library to be used as inhibitors against monoamine oxidase A (MAO-A). The Fv-antibody [corresponding to the VH region of immunoglobulin G (IgG)] consists of three complementarity-determining regions and four frame regions. The Fv-antibody library was prepared by site-directed mutagenesis of CDR3, which consists of 11 amino acid residues. Three target clones were screened from the Fv-antibody library, and the binding affinity of the screened clones to the monoclonal anti-serotonin antibody was analyzed using fluorescence-activated cell sorting. The screened Fv-antibodies were expressed as soluble proteins fused with green fluorescence protein. Additionally, the screened CDR3 regions (11 residues) of the selected Fv-antibodies were synthesized as peptides with linking amino acid residues. The binding constants (KD) of the three serotonin-like mimotopes (Fv-antibodies and peptides) were estimated using a surface plasmon resonance biosensor. The inhibitory activity (IC50) of the serotonin-like mimotopes (Fv-antibodies and peptides) was estimated separately for MAO-A and MAO-B enzymes and compared with that of conventional inhibitors. Finally, the screened serotonin-like mimotopes were used to treat a cell line (SH-SY5Y, ATCC code: CRL-2266) expressing serotonin receptors. This was done to confirm the following two aspects: (1) the binding of mimotopes to the serotonin receptors on the cell surface and (2) the inhibitory activity of mimotopes against MAO-A enzymes in the cell lysates.

Impact of glucose metabolism on PD-L1 expression in sorafenib-resistant hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer-related mortality worldwide. Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that binds to programmed cell death-1 (PD-1), which is expressed in activated T cells and other immune cells and has been employed in cancer therapy, including HCC. Recently, PD-L1 overexpression has been documented in treatment-resistant cancer cells. Sorafenib is a multikinase inhibitor and the only FDA-approved treatment for advanced HCC. However, several patients exhibit resistance to sorafenib during treatment. This study aimed to assess the effect of glucose deprivation on PD-L1 expression in HCC cells. We used PD-L1-overexpressing HepG2 cells and IFN-γ-treated SK-Hep1 cells to explore the impact of glycolysis on PD-L1 expression. To validate the correlation between PD-L1 expression and glycolysis, we analyzed data from The Cancer Genome Atlas (TCGA) and used immunostaining for HCC tissue analysis. Furthermore, to modulate PD-L1 expression, we treated HepG2, SK-Hep1, and sorafenib-resistant SK-Hep1R cells with rapamycin. Here, we found that glucose deprivation reduced PD-L1 expression in HCC cells. Additionally, TCGA data and immunostaining analyses confirmed a positive correlation between the expression of hexokinase II (HK2), which plays a key role in glucose metabolism, and PD-L1. Notably, rapamycin treatment  decreased the expression of PD-L1 and HK2 in both high PD-L1-expressing HCC cells and sorafenib-resistant cells. Our results suggest that the modulation of PD-L1 expression by glucose deprivation may represent a strategy to overcome PD-L1 upregulation in patients with sorafenib-resistant HCC.

Screening of Pancreatic Ribonuclease A Inhibitors from an Autodisplayed Fv-Antibody Library.

Pancreatic ribonuclease A (RNase A) inhibitors were screened from an autodisplayed Fv-antibody library, which was prepared by randomizing amino acid sequences of the third complementary-determining region (CDR3) within the heavy chain variable region (VH region) of immunoglobulin G (called "Fv-antibody" comprising three CDRs and four frame regions (FRs)) through site-directed mutagenesis. The library was autodisplayed on the outer membrane of Escherichia coli. Target Fv-variants (clones) with specific binding affinity for RNase A were screened using fluorescein-labeled RNase A and flow cytometry. Three Fv variants (clones) were screened, and CDR3 amino acid sequences were analyzed. The screened Fv-antibodies were expressed as soluble proteins, and CDR3 was synthesized into peptides (11 residues). The binding affinity constants (KD) of the expressed Fv-antibodies and synthesized peptides to RNase A were estimated using surface plasmon resonance. Fitting analysis based on the adsorption model showed that KD values of the three expressed Fv-antibodies were estimated to be 17.5 ± 4.1, 28.8 ± 9.7, and 33.9 ± 8.9 nM (n = 3), and those of the three synthesized peptides were 1.3 ± 0.1, 1.3 ± 0.3, and 3.7 ± 1.3 µM (n = 3). From the RNase activity assay with an RNA probe labeled with fluorophore and quencher, inhibition constants (IC50) of the three expressed Fv-antibodies were estimated to be 90.2, 65.3, and 98.8 nM (n = 3), and those of the three synthesized peptides were 8.1, 3.6, and 0.4 µM (n = 3). The activity of RNase inhibitors constituting the expressed Fv-antibodies and synthesized peptides was demonstrated via an RNA cleavage test using the total RNA from HeLa cells.

Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment.

BACKGROUND: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explores 11C-acetate PET as an imaging technique for detecting reactive astrogliosis. METHODS: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and MCT1 expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis. RESULTS: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with worse prognosis. CONCLUSION: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.

Functionalized Parylene Films for Enhancement of Antibody Production by Hybridoma Cells.

In this study, the influence of microenvironments on antibody production of hybridoma cells was analyzed using six types of functionalized parylene films, parylene-N and parylene-C (before and after UV radiation), parylene-AM, and parylene-H, and using polystyrene as a negative control. Hybridoma cells were cultured on modified parylene films that produced a monoclonal antibody against the well-known fungal toxin ochratoxin-A. Surface properties were analyzed for each parylene film, such as roughness, chemical functional groups, and hydrophilicity. The proliferation rate of the hybridoma cells was observed for each parylene film by counting the number of adherent cells, and the total amount of produced antibodies from different parylene films was estimated using indirect ELISA. In comparison with the polystyrene, the antibody-production by parylene-H and parylene-AM was estimated to be observed to be as high as 210-244% after the culture of 24 h. These results indicate that the chemical functional groups of the culture plate could influence antibody production. To analyze the influence of the microenvironments of the modified parylene films, we performed cell cycle analysis to estimate the ratio of the G0/G1, S, and G2/M phases of the hybridoma cells on each parylene film. From the normalized proportion of phases of the cell cycle, the difference in antibody production from different surfaces was considered to result from the difference in the proliferation rate of hybridoma cells, which occurred from the different physical and chemical properties of the parylene films. Finally, protein expression was analyzed using an mRNA array to determine the effect of parylene films on protein expression in hybridoma cells. The expression of three antibody production-related genes (CD40, Sox4, and RelB) was analyzed in hybridoma cells cultured on modified parylene films.

Extracellular Citrate Treatment Induces HIF1α Degradation and Inhibits the Growth of Low-Glycolytic Hepatocellular Carcinoma under Hypoxia.

HCC is well known for low glycolysis in the tumors, whereas hypoxia induces glycolytic phenotype and tumor progression. This study was conducted to evaluate the expression of SLCs in human HCCs and investigated whether extracellular nutrient administration related to SLCs in low-glycolytic HCC can prevent hypoxic tumor progression. SLCs expression was screened according to the level of glycolysis in HCCs. Then, whether extracellular nutrient treatment can affect hypoxic tumor progression, as well as the mechanisms, were evaluated in an in vitro cell line and an in vivo animal model. Low-glycolytic HCCs showed high SLC13A5/NaCT and SLC16A1/MCT1 but low SLC2A1/GLUT1 and HIF1α/HIF1α expression. Especially, high SLC13A5 expression was significantly associated with good overall survival in the Cancer Genome Atlas (TCGA) database. In HepG2 cells with the highest NaCT expression, extracellular citrate treatment upon hypoxia induced HIF1α degradation, which led to reduced glycolysis and cellular proliferation. Finally, in HepG2-animal models, the citrate-treated group showed smaller tumor with less hypoxic areas than the vehicle-treated group. In patients with HCC, SLC13A5/NaCT is an important SLC, which is associated with low glycolysis and good prognosis. Extracellular citrate treatment induced the failure of metabolic adaptation to hypoxia and tumor growth inhibition, which can be a potential therapeutic strategy in HCCs.

11 C-Acetate PET/CT Detects Reactive Astrogliosis Helping Glioma Classification.

PURPOSE: 11 C-acetate ( 11 C-ACE) uptake on PET/CT was recently discovered to represent reactive astrocytes in the tumor microenvironment. This study aimed at evaluating the role of 11 C-ACE PET/CT as an imaging biomarker of reactive astrogliosis in characterizing different types of gliomas. METHODS: In this prospective study, a total of 182 patients underwent 11 C-ACE PET/CT before surgery. The ratio of SUV max of a glioma to the SUV mean of the contralateral choroid plexus ( 11 C-ACE TCR) on PET/CT was calculated. 11 C-ACE TCRs were compared with the World Health Organization grades and isocitrate dehydrogenase 1 ( IDH1 ) mutation status. Grade 2 was considered low-grade tumor, and grades 3 and 4 were considered high-grade tumors. RESULTS: The median 11 C-ACE TCR was significantly higher in IDH1 wild-type (wt) tumors (n = 91) than in IDH1 -mutant (mt) tumors (n = 91) (2.38 vs 1.30, P < 0.001). Of the 91 IDH1 -mt tumors, there were no differences in the median 11 C-ACE TCRs between oligodendrogliomas (ODs) and astrocytic tumors (1.40 vs 1.20, P > 0.05). In grading low- versus high-grade gliomas, the receiver operating characteristic curve analyses showed a higher area under the curve (0.951) in IDH1 -wt tumors than in IDH1 -mt tumors (0.783, P = 0.002). Grade 2 ODs were well differentiated from high-grade gliomas. The 11 C-ACE TCR of grade 3 ODs was significantly lower than that of IDH1 -wt glioblastomas. CONCLUSIONS: High 11 C-ACE uptake is associated with high-grade IDH1 -wt tumors, thus facilitating differentiation from high-grade IDH1-mt and low-grade gliomas. In particular, low 11 C-ACE uptake in ODs is advantageous in overcoming the limitation of radiolabeled amino acid tracers.

Antibody-Mediated Screening of Peptide Inhibitors for Monoamine Oxidase-B (MAO-B) from an Autodisplayed FV Library.

Inhibitors for monoamine oxidase-B (MAO-B) were screened from an FV library with a randomized complementarity-determining region 3 (CDR3) region using a monoclonal antibody against dopamine. As the first step, the FV library was expressed on the outer membrane of E. coli by site-directed mutagenesis of the randomized CDR3 region. Among the FV library, variants with a binding affinity to monoclonal antibodies against dopamine were screened and cloned. From the comparison of the binding activity of the screened clones to a control clone with a modified FV antibody (only with CDR1 and CDR2), the CDR3 regions of screened clones were determined to directly interact with the monoclonal antibody against dopamine. These CDR3 sequences were then synthesized as mimotopes (mimicking peptides) of dopamine. The inhibitory activity of two mimotopes against MAO-B was analyzed using HeLa cells overexpressing MAO-B, as well as using activated human astrocytes; their inhibitory activity was compared to that of a commercial inhibitor of MAO-B, selegiline. The inhibition efficiency of the two mimotopes (in comparison with selegiline) was estimated to be 67.2% and 69.4% in the HeLa cells and 64.4% and 58.0% in the human astrocytes. The gene expression pattern in astrocytes after treatment with the two mimotopes was also analyzed and compared with that in the human astrocytes treated with selegiline. Finally, the interaction between two mimotopes and MAO-B was analyzed using docking simulation, and the candidate regions of MAO-B for the interaction with each mimotope were explored through the docking simulation.

Total Lesion Glycolysis on 18F-FDG PET/CT Is a Better Prognostic Factor Than Tumor Dose on 90Y PET/CT in Patients With Hepatocellular Carcinoma Treated With 90Y Transarterial Radioembolization.

PURPOSE: This study aimed to evaluate the prognostic value of metabolic parameters on 18F-FDG PET/CT and tumor dose (TD) on posttreatment 90Y PET/CT in patients with hepatocellular carcinoma (HCC) who underwent 90Y transarterial radioembolization (TARE). PATIENTS AND METHODS: Forty-seven HCC patients treated with 90Y TARE were retrospectively enrolled between January 2013 and October 2018. 18F-FDG PET/CT was performed before treatment. Maximum tumor SUV-to-mean normal liver SUV ratio (TLR), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) were measured for each patient. Voxel dosimetry was performed on 90Y PET/CT images to measure TD. The prognostic significance of metabolic parameters on 18F-FDG PET/CT, TD on 90Y PET/CT, and clinical factors for overall survival (OS) was evaluated. In addition, TD on 90Y PET/CT was analyzed in relation to the administered dose of 90Y-labeled microspheres and metabolic parameters on 18F-FDG PET/CT. RESULTS: The median patient age was 57 years, and 37 patients (78.7%) were men. During the follow-up period, 25 patients (53.2%) died. In univariable analysis, Barcelona Clinic Liver Cancer stage C, Child-Pugh score, TD on 90Y PET/CT, TLR, MTV, and TLG were significant prognostic factors affecting OS (P < 0.05). In multivariable analysis, Barcelona Clinic Liver Cancer stage C and high TLG on 18F-FDG PET/CT were independent prognostic factors for OS (P < 0.05). The 1-year OS rates were 72.9% in patients with low TLG and 33.3% in patients with high TLG (P < 0.05). We also found that TD on 90Y PET/CT was not correlated with the administered dose of 90Y-labeled microspheres, but negatively correlated with TLG on pretreatment 18F-FDG PET/CT (P < 0.05). CONCLUSIONS: TLG, a parameter incorporating both the degree of 18F-FDG uptake and amount of metabolically active tumor volume on pretreatment 18F-FDG PET/CT, is a better prognostic factor than TD on 90Y PET/CT for predicting OS in HCC patients treated with 90Y TARE.

Impact of Exogenous Treatment with Histidine on Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Sorafenib, a multi-kinase inhibitor, is the first-line therapy for advanced HCC. However, long-term exposure to sorafenib often results in reduced sensitivity and the development of resistance. Although various amino acids have been shown to contribute to cancer initiation and progression, little is known about the effects of histidine, a dietary essential amino acid that is partially taken up via histidine/large neutral amino acid transporter (LAT1), on cancer cells. In this study, we evaluated the effects of histidine on HCC cells and sensitivity to sorafenib. Remarkably, we found that exogenous histidine treatment induced a reduction in the expression of tumor markers related to glycolysis (GLUT1 and HK2), inflammation (STAT3), angiogenesis (VEGFB and VEGFC), and stem cells (CD133). In addition, LAT1 expression was downregulated in HCC tumor regions with high expression of GLUT1, CD133, and pSTAT3, which are known to induce sorafenib resistance. Finally, we demonstrated that combined treatment with sorafenib and histidine could be a novel therapeutic strategy to enhance the sensitivity to sorafenib, thereby improving long-term survival in HCC.

One-step immunoassay for food allergens based on screened mimotopes from autodisplayed FV-antibody library.

One-step immunoassay detects a target analyte simply by mixing a sample with a reagent solution without any washing steps. Herein, we present a one-step immunoassay that uses a peptide mimicking a target analyte (mimotope). The key idea of this strategy is that the mimotopes are screened from an autodisplayed FV-antibody library using monoclonal antibodies against target analytes. The monoclonal antibodies are bound to fluorescence-labeled mimotopes, which are quantitatively released into the solution when the target analytes are bound to the monoclonal antibodies. Thus, the target analyte is detected without any washing steps. For the mimotope screening, an FV-antibody library was exhibited on the outer membrane of E. coli with a diversity of >106 clones/library using autodisplay technology. The targeted clones were screened from the autodisplayed FV-antibody library using magnetic beads with immobilized monoclonal antibodies against food allergens. The analysis of binding properties of a control strain with mutant FV -antibodies composed of only CDR1 and CDR2 demonstrated that the CDR3 regions of the screened FV-antibodies showed binding affinity to food allergens. The CDR3 regions were synthesized into peptides as mimotopes for the corresponding food allergens (mackerel, peanuts, and pig fat). One-step immunoassays for food allergens were demonstrated using mimotopes against mackerel, peanut, and pig fat without any washing steps in solution without immobilization of antibodies to a solid support.

Fluorescein and Rhodamine B-Binding Domains from Autodisplayed Fv-Antibody Library.

In this study, the binding domains for fluorescent dyes were presented that could be used as synthetic peptides or fusion proteins. Fv-antibodies against two fluorescent dyes (fluorescein and rhodamine B) were screened from the Fv-antibody library, which was prepared on the outer membrane of Escherichia coli using the autodisplay technology. Two clones with binding activities to each fluorescent dye were screened separately from the library using flow cytometry. The binding activity of the screened Fv-antibodies on the outer membrane was analyzed using fluorescent imaging with the corresponding fluorescent dyes. The CDR3 regions of the screened Fv-antibodies (11 amino acid residues) were synthesized into peptides, and each peptide was analyzed for its binding activity to each fluorescent dye using fluorescence resonance energy transfer (FRET) experiments. These CDR3 regions were demonstrated to have a binding activity to each fluorescent dye when the regions were co-expressed as a fusion protein with Z-domain.

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout.

To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG VH chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (Kd) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin-horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0-300 µg/mL.

Elevated miR-16-5p induces somatostatin receptor 2 expression in neuroendocrine tumor cells.

Somatostatin analogs, which are used to treat neuroendocrine tumors, inhibit hormone secretion or promote tumor shrinkage; however, their efficacy varies between patients, possibly because of differential expression of somatostatin receptors (SSTRs) in tumors. In this study, we evaluated the regulatory mechanism underlying the expression of SSTR2, the main octreotide target. Thirty miRNAs were found to be dysregulated in neuroendocrine cells (INS-1 cells) incubated with octreotide compared to that in placebo-treated cells. Among the upregulated miRNAs, miR-16-5p was elevated after short-term octreotide treatment. We conducted in vitro experiments to determine whether the expression of miR-16-5p was associated with the regulation of SSTR2 expression and affected octreotide sensitivity in INS-1 cells. Overexpression of miR-16-5p by transfected mimics induced upregulation of SSTR2 expression. Additionally, the expression of miR-16-5p further enhanced octreotide-induced reduction in cell proliferation in both two- and three-dimensional culture of INS-1 cells. Thus, our results reveal the mechanism underlying SSTR2 expression regulation and may aid in developing therapeutic approaches for enhancing the response to octreotide, particularly in patients unresponsive to SSTR2-targeted somatostatin analog treatment.