Induction of the DNA-Repair Gene POLQ only in BRCA1-mutant Breast-Cancer Cells by Methionine Restriction.

BACKGROUND/AIM: BRCA1/2 mutations in breast cancer cells impair homologous recombination and promote alternative end joining (Alt-EJ) for DNA-damage repair. DNA polymerase theta, encoded by POLQ, plays a crucial role in Alt-EJ, making it a potential therapeutic target, particularly in BRCA1/2-mutant cancers. Methionine restriction is a promising approach to target cancer cells due to their addiction to this amino acid. The present study investigated the expression of POLQ in BRCA1/2 wild-type and BRCA1-mutant breast cancer cells under methionine restriction. MATERIALS AND METHODS: POLQ mRNA expression was measured using qRT-PCR in BRCA1/2 wild-type (MDA-MB-231) and BRCA1- mutant (HCC1937 and MDA-MB-436) breast-cancer cells under normal, or serum-restricted, or serum- and methionine-restricted conditions. RESULTS: Compared to BRCA1/2 wild-type cells, BRCA1-mutant cells displayed significantly higher basal POLQ expression in normal medium. Methionine restriction further increased POLQ expression in the BRCA1-mutant cells but decreased it in the BRCA1/2 wild-type cells. CONCLUSION: The present findings suggest that methionine restriction showed differential effects on POLQ expression, potentially impacting Alt-EJ activity, in BRCA1/2 wild-type and BRCA1-mutant breast-cancer cells. Further investigation is needed to explore the potential of combining methionine restriction with DNA-repair inhibitors, such as PARP inhibitors, to overcome drug resistance in BRCA1/2 mutant cancers.

Target-Oriented Classification of Triple-negative Breast Cancer.

BACKGROUND/AIM: Breast cancer that is estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and human epidermal growth factor receptor-2 (HER2)-negative is termed triple-negative breast cancer (TNBC). Cytotoxic chemotherapy remains the first choice of treatment against TNBC due to lack of specific therapeutic targets. TNBC is not classified based on therapeutic targets, but recently, the development of targeted therapies - including immune checkpoint inhibitors and poly (adenosine diphosphate-ribose) polymerase inhibitors - has gained attention. This study aimed to examine a novel target-oriented TNBC classification to further facilitate targeted therapy by classifying TNBC based on the breast cancer 1 (BRCA1)-like as well as the protein expression of HER2, programmed death ligand 1 (PD-L1), androgen receptor (AR), cytokeratin 5/6, and epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: We enrolled 17 patients with primary TNBC who did not receive preoperative chemotherapy and underwent surgery at the Kobe University Hospital, Japan, between January 1, 2018, and July 31, 2019. Immunohistochemical staining was performed on tumor specimens, while a BRCAness test was performed using multiplex ligation-dependent probe amplification (MLPA) analysis. A BRCAness score 0.5 or higher was considered BRCA1-like. RESULTS: Tumors were classified as HER2-low (immunohistochemistry score 1+ or 2+ and FISH negative), PD-L1 positive, AR positive, or BRCA1-like. HER2-low, PD-L1 positive, AR positive, and BRCA1-like were detected in 11 (64.7%), 4 (23.5%), 6 (35.3%), and 6 (35.3%) samples. The tumor of only one patient could not be classified into any of these categories. CONCLUSION: Almost all TNBC cases can be classified according to treatable targets.

Methionine Restriction Increases Exosome Production and Secretion in Breast Cancer Cells.

BACKGROUND/AIM: Methionine addiction is the elevated requirement for exogenous methionine for growth and survival of cancer cells, termed the Hoffman effect. Methionine-addicted cancer cells synthesize normal or excess amounts of methionine but still need an external source of methionine. Methionine restriction (MR) by either a methionine-free medium or in vivo by a low-methionine diet or by methioninase, selectively arrests cancer cells in the late S/G2 cell cycle phase, but not normal cells. The present study focuses on the comparison of production and secretion of exosomes by cancer cells under MR and normal conditions. MATERIALS AND METHODS: MDA-MB-231 cells (triple-negative breast cancer), containing exosomes labeled with CD63-GFP (CD63-GFP exosomes), were visualized by fluorescence microscopy. MDA-MB-231 cells expressing exosome-specific CD63-GFP were cultured in methionine-containing (MET+) or in methionine-free (MET-) DMEM conditions. Exosomes were isolated from conditioned medium of cultured MDA-MD-231 cells by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and Western blotting. RESULTS: When MDA-MB-231-CD63-GFP cells were cultured under MR conditions, they arrested their growth and CD63-GFP-expressing exosomes were strongly increased in the cells. MR resulted in approximately a 2-fold increase in exosome production and secretion per cell, even though cell growth was arrested. Methionine restriction thus resulted in elevated exosome production and secretion per surviving cell. CONCLUSION: Exosome production and secretion in the cancer cells increased under MR, suggesting a relation between MR and exosome production and secretion.

Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation.

Methionine addiction, a fundamental and general hallmark of cancer, known as the Hoffman Effect, is due to altered use of methionine for increased and aberrant transmethylation reactions. However, the linkage of methionine addiction and malignancy of cancer cells is incompletely understood. An isogenic pair of methionine-addicted parental osteosarcoma cells and their rare methionine-independent revertant cells enabled us to compare them for malignancy, their epithelial-mesenchymal phenotype, and pattern of histone-H3 lysine-methylation. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were selected from methionine-addicted parental cells (143B-P) by their chronic growth in low-methionine culture medium for 4 passages, which was depleted of methionine by recombinant methioninase (rMETase). Cell-migration capacity was compared with a wound-healing assay and invasion capability was compared with a transwell assay in 143B-P and 143B-R cells in vitro. Tumor growth and metastatic potential were compared after orthotopic cell-injection into the tibia bone of nude mice in vivo. Epithelial-mesenchymal phenotypic expression and the status of H3 lysine-methylation were determined with western immunoblotting. 143B-P cells had an IC50 of 0.20 U/ml and 143B-R cells had an IC50 of 0.68 U/ml for treatment with rMETase, demonstrating that 143B-R cells had regained the ability to grow in low methionine conditions. 143B-R cells had reduced cell migration and invasion capability in vitro, formed much smaller tumors than 143B-P cells and lost metastatic potential in vivo, indicating loss of malignancy in 143B-R cells. 143B-R cells showed gain of the epithelial marker, ZO-1 and loss of mesenchymal markers, vimentin, Snail, and Slug and, an increase of histone H3K9me3 and H3K27me3 methylation and a decrease of H3K4me3, H3K36me3, and H3K79me3 methylation, along with their loss of malignancy. These results suggest that shifting the balance in histone methylases might be a way to decrease the malignant potential of cells. The present results demonstrate the rationale to target methionine addiction for improved sarcoma therapy.

A Rare Case of Bilateral Pectoralis Major Muscle Defect and Abnormal Muscle.

A 56-year-old female patient with left breast cancer presented at our hospital. Preoperative CT scan showed an isolated bilateral pectoralis major muscle defect and abnormal muscle originating from the entire sternum and inserting in the lower ribs and rectus sheath. Total mastectomy and axillary lymph node dissection were performed. We believe that this case is unique and that others like it have never been reported. If there is a defect in the pectoralis major muscle, reconstructive surgery with a tissue expander is contraindicated. Therefore, preoperative evaluation of the chest wall musculature on imaging is recommended.

Linkage of methionine addiction, histone lysine hypermethylation, and malignancy.

Methionine addiction, found in all types of cancer investigated, is because of the overuse of methionine by cancer cells for excess transmethylation reactions. In the present study, we compared the histone H3 lysine-methylation status and degree of malignancy between methionine-addicted cancer cells and their isogenic methionine-independent revertants, selected by their growth in low concentration of methionine. The methionine-independent revertans can grow on low levels of methionine or independently of exogenous methionine using methionine precursors, as do normal cells. In the methionine-independent revertants, the excess levels of trimethylated histone H3 lysine marks found in the methionine-addicted parental cancer cells were reduced or lost, and their tumorigenicity and experimental metastatic potential in nude mice were also highly reduced. Methionine addiction of cancer is linked with malignancy and hypermethylation of histone H3 lysines. The results of the present study thus provide a unique framework to further understand a fundamental basis of malignancy.

Size Dependency of Selective Cellular Uptake of Epigallocatechin Gallate-modified Gold Nanoparticles for Effective Radiosensitization.

The high incidence and mortality of cancer make it a global health issue. However, conventional cancer therapies have several disadvantages, especially serious side effects due to low selective toxicity to cancer cells. Gold nanoparticles (AuNPs) are an excellent drug carrier, enhance drug delivery efficiency, and hold promise for photothermal and radiation therapies. (-)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic antioxidant constituent of green tea, has a potent antitumor effect, and binds specifically to the 67 kDa laminin receptor, which is overexpressed on the surface of several cancer cell lines such as HeLa and MDA-MB-231 cells. We synthesized EGCG-modified AuNPs (EGCG-AuNPs) using ratios (nEGCG/ngold) from 1:2 to 10:1 and evaluated their size, morphology, stability, antioxidant ability, cytotoxicity, cellular uptake, and uptake mechanisms in vitro in comparison with the conventional AuNPs prepared by using citrate as the reducing agent (citrate-AuNPs). In HeLa cells, EGCG-AuNPs (10:1) (135 nm diameter, sea-urchin-like shape) exhibited the highest cellular uptake. Conversely, EGCG-AuNPs (1:2) (39 nm diameter, spherical shape) were preferentially taken up by MDA-MB-231 cells. Cellular uptake of EGCG-AuNPs toward normal cells (NIH3T3 cells) was found to be in a nonspecific manner, and the amount of uptake was suppressed. X-ray irradiation after cellular uptake of EGCG-AuNPs (1:2) in MDA-MB-231 cells significantly enhanced irradiation-induced cell death. These findings suggest enhanced cellular uptake of EGCG-AuNPs with a 39 nm diameter and their potential use in combinatorial therapeutics of EGCG-AuNPs for breast cancer.

Extent and Instability of Trimethylation of Histone H3 Lysine Increases With Degree of Malignancy and Methionine Addiction.

BACKGROUND/AIM: Methionine addiction is a fundamental and general hallmark of cancer, termed the Hoffman effect. Methionine addiction is due to excessive use of and dependence on methionine by cancer cells. In the present report, we correlated the extent of methionine addiction and degree of malignancy with the amount and stability of methylated histone H3 lysine marks. MATERIALS AND METHODS: We established low- and high-malignancy variants from a parental human pancreatic-cancer cell line and compared their sensitivity to methionine restriction and histone H3 lysine methylation status. RESULTS: A low-malignancy, low-methionine-addiction revertant of the parental pancreatic-cancer cell line had less methylated H3K9me3 and was less sensitive to methionine restriction effected by recombinant methioninase (rMETase) than the parental cell line. A high-malignancy variant of the pancreatic cancer cell line had increased methylated H3K9me3 and was more sensitive to methionine restriction by rMETase with regard to inhibition of proliferation and to instability of histone H3 lysine methylation than the parental cell line. Orthotopic malignancy in nude mice was reduced in the low-methionine-addiction revertant and greater in the high-malignancy variant than in the parental cell line. CONCLUSION: The present study indicates that the degree of malignancy is linked to the extent of methionine addiction and the level and instability of trimethylation of histone H3, suggesting these phenomena are linked as a fundamental basis of oncogenic transformation.

Over-methylation of Histone H3 Lysines Is a Common Molecular Change Among the Three Major Types of Soft-tissue Sarcoma in Patient-derived Xenograft (PDX) Mouse Models.

BACKGROUND/AIM: Sarcomas are considered a heterogeneous disease with incomplete understanding of its molecular basis. In the present study, to further understand general molecular changes in sarcoma, patient-derived xenograft (PDX) mouse models of the three most common soft-tissue sarcomas: myxofibrosarcoma, undifferentiated pleomorphic sarcoma (UPS) and liposarcoma were established and the methylation status of histone H3 lysine marks was studied. MATERIALS AND METHODS: Immunoblotting and immunohistochemical staining were used to quantify the extent of methylation of histone H3K4me3 and histone H3K9me3. RESULTS: In all 3 sarcoma types in PDX models, histone H3K4me3 and H3K9me3 were found highly over-methylated compared to normal muscle tissue. CONCLUSION: Histone H3 lysine over-methylation may be a general basis of malignancy of the major sarcoma types.

Exosome-mediated radiosensitizing effect on neighboring cancer cells via increase in intracellular levels of reactive oxygen species.

The precise mechanism of intercellular communication between cancer cells following radiation exposure is unclear. Exosomes are membrane­enclosed small vesicles comprising lipid bilayers and are mediators of intercellular communication that transport a variety of intracellular components, including microRNAs (miRNAs or miRs). The present study aimed to identify novel roles of exosomes released from irradiated cells to neighboring cancer cells. In order to confirm the presence of exosomes in the human pancreatic cancer cell line MIAPaCa­2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosome­specific surface markers CD9 and CD63. Subsequent endocytosis of exosomes was confirmed by fluorescent microscopy. Cell survival following irradiation and the addition of exosomes was evaluated by colony forming assay. Expression levels of miRNAs in exosomes were then quantified by microarray analysis, while protein expression levels of Cu/Zn­ and Mn­superoxide dismutase (SOD1 and 2, respectively) enzymes in MIAPaCa­2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of non­irradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of reactive oxygen species (ROS) and a higher frequency of DNA damage in MIAPaCa­2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six up­ and five downregulated miRNAs were identified in 5 and 8 Gy­irradiated cells using miRNA microarray analyses. Further analysis using miRNA mimics and reverse transcription­quantitative PCR identified miR­6823­5p as a potential candidate to inhibit SOD1, leading to increased intracellular ROS levels and DNA damage. To the best of our knowledge, the present study is the first to demonstrate that irradiated exosomes enhance the radiation effect via increasing intracellular ROS levels in cancer cells. This contributes to improved understanding of the bystander effect of neighboring cancer cells.

Reversion from Methionine Addiction to Methionine Independence Results in Loss of Tumorigenic Potential of Highly-malignant Lung-cancer Cells.

BACKGROUND/AIM: Methionine addiction, a fundamental and general hallmark of cancer, is due to the excess use of methionine for transmethylation, and is described as the Hoffman-effect. Methionine-addicted cancer cells can revert at low frequency to methionine independence when selected under methionine-restriction. We report here that highly-malignant methionine-addicted H460 human lung-cancer cells, when selected for methionine independence, have greatly-reduced tumorigenic potential. MATERIALS AND METHODS: Methionine-addicted H460 parental cancer cells and methionine-independent revertant H460-R1 cells were injected in nude mice subcutaneously. RESULTS: When the parental H460 methionine-addicted cells were injected in nude mice at 2.5×105, 1×105 and 5×104, the cells could form tumors. In contrast, the H460-R1 methionine-independent revertant cells could not form tumors when the above-listed cell numbers were injected in nude mice. CONCLUSION: There is a tight linkage between methionine addiction and malignancy.

Triple-Methyl Blockade With Recombinant Methioninase, Cycloleucine, and Azacitidine Arrests a Pancreatic Cancer Patient-Derived Orthotopic Xenograft Model.

OBJECTIVES: Methionine addiction is a fundamental and general hallmark of cancer caused by enhanced methyl flux. In the present study, we effected a novel methionine-methylation blockade to target a patient-derived orthotopic xenograft model of pancreatic cancer. METHODS: The pancreatic cancer patient-derived orthotopic xenograft mouse models were randomized into 6 groups of 8 mice each and treated for 2 weeks: untreated control; azacitidine; oral recombinant methioninase (o-rMETase); o-rMETase plus cycloleucine; o-rMETase plus cycloleucine plus azacitidine (triple-methyl blockade therapy); and gemcitabine (positive control). RESULTS: Triple-methyl blockade therapy arrested tumor growth (mean relative tumor volume, 1.03 [standard deviation, 0.36]) and was significantly more effective compared with azacitidine (P = 0.0001); o-rMETase (P = 0.007); or o-rMETase plus cycloleucine (P = 0.04). Gemcitabine alone also inhibited but did not arrest tumor growth (mean relative tumor volume, 1.50 [standard deviation, 0.30]). The percentage of cancer cells that were negative for 5-methylcytosine staining in immunohistochemistry, indicating reduction of DNA methylation, increased with triple-methyl blockade therapy (37.5%), compared with gemcitabine (1.8%); o-rMETase (2.8%); azacitidine (9.0%); or o-rMETase plus cycloleucine (10.6%). CONCLUSIONS: This new concept of triple-methyl blockade therapy has clinical potential for pancreatic cancer, which is currently a recalcitrant disease.

A Universal Gelfoam 3-D Histoculture Method to Establish Patient-derived Cancer Cells (3D-PDCC) Without Fibroblasts from Patient-derived Xenografts.

BACKGROUND/AIM: The direct placement of patient tumors in 2-D culture on plastic or glass surfaces has inhibited the establishment of patient-derived cancer cells (PDCCs). The aim of the present study was to develop universal and efficient methods to prepare PDCCs. MATERIALS AND METHODS: Fragments of patient-derived xenograft (PDX) tumors established form colon cancer liver metastasis (1 mm3) were placed on Gelfoam and cultured in DMEM. RESULTS: PDX tumor fragments were cultured on Gelfoam. Cancer cells migrated from the explant and formed distinct 3-D structures in the Gelfoam. Each of the three PDCCs showed a distinct morphology. The cultures were essentially all cancer cells without fibroblasts, the opposite of what usually occurs in 2-D culture on plastic or glass. Gelfoam cultures could be readily passaged from one Gelfoam cube to anothers suggesting indefinite culture potential. CONCLUSION: A potentially universal method to establish PDCC using PDX tumors and 3-D Gelfoam histoculture was developed.

Sutureless Surgical Orthotopic Implantation Technique of Primary and Metastatic Cancer in the Liver of Mouse Models.

BACKGROUND/AIM: Surgical orthotopic implantation (SOI) is used to establish patient-derived orthotopic xenograft (PDOX) and other orthotopic mouse models. Orthotopic liver models can be challenging, as the liver parenchyma is prone to bleeding. The present report describes a sutureless method to implant tumors in the liver that reduces bleeding and procedural time. MATERIALS AND METHODS: Human HCC cell-line (Huh-7-GFP) and CM2, a patient-derived colon-cancer liver metastasis, were used for sutureless SOI of tumor fragments in the liver of nude mice. A small cavity was formed on the liver surface. A solitary tumor fragment was implanted in the cavity without suturing to create hemostasis. RESULTS: Six weeks after sutureless SOI, the tumor volume of Huh-7-GFP (n=5) was 584.41±147.64 mm3 and the tumor volume of CM2 (n=5) was 1336.54±1038.20 mm3 The engraftment rate was 100%. CONCLUSION: This novel method for establishing orthotopic liver-implantation mouse models is suitable for studies of liver cancer and liver metastases due to its simple procedure and potential high engraftment rate.

A Non-invasive Imageable GFP-expressing Mouse Model of Orthotopic Human Bladder Cancer.

BACKGROUND/AIM: A more realistic mouse model of bladder cancer is necessary to develop effective drugs for the disease. Tumor models enhanced by bright fluorescent-reporter genes to follow the disease in real-time would enhance the ability to accurately predict the efficacy of various therapeutics on this particularly-malignant human cancer. MATERIALS AND METHODS: A highly-fluorescent green fluorescent protein (GFP)-expressing bladder cancer model was orthotopically established in nude mice using the UM-UC-3 human bladder-cancer cell line (UM-UC-3-GFP). Fragments from a subcutaneous tumor of UM-UC-3-GFP were surgically implanted into the nude mouse bladder. Non-invasive and intra-vital fluorescence imaging was obtained with a simple imaging box. RESULTS: The GFP-expressing orthotopic bladder tumor was imaged in real-time non-invasively as well as intra-vitally, with the two methods correlating at r=0.99. CONCLUSION: This is the first non-invasive-fluorescence-imaging orthotopic model of bladder cancer and can be used for rapidly screening novel effective agents for this recalcitrant disease.

Co-implantation of Tumor and Extensive Surrounding Tissue Improved the Establishment Rate of Surgical Specimens of Human-Patient Cancer in Nude Mice: Toward the Goal of Universal Individualized Cancer Therapy.

BACKGROUND/AIM: The discovery of the nude mouse model enabled the experimental growth of human-patient tumors. However, the low establishment rate of tumors in nude and other immunodeficient strains of mice has limited wide-spread clinical use. MATERIALS AND METHODS: In order to increase the establishment rate of surgical specimens of patient tumors, we transplanted tumors to nude mice subcutaneously along with large amounts of surrounding tissue of the tumor. RESULTS: The new transplantation method increased the establishment rate in nude mice to 66% compared to the old method of implanting the surgical tumor specimen with surrounding tissue removed (14%). High stage and presence of metastasis in the patient donor are positively correlated to tumor engraftment in nude mice. CONCLUSION: The new method can potentially allow most cancer patients who undergo surgery or biopsy to have their own mouse model for drug-sensitivity testing.

Ischemia reperfusion-induced metastasis is resistant to PPARγ agonist pioglitazone in a murine model of colon cancer.

Ischemia reperfusion injury (IRI) during liver-metastasis resection for treatment of colon cancer may increase the risk of further metastasis. Peroxisome proliferator-activated receptor-γ (PPARγ) activation has been observed to exert a protective effect against IRI and IRI-induced metastasis of hepatocellular carcinoma. The present study aimed to investigate the effect of the PPARγ agonist pioglitazone on tumor metastasis and liver injury following IRI in a mouse model of colon cancer. Pioglitazone (30 mg/kg weight) was administered orally 1.5 h before and 2 h after the initiation of ischemia and was orally administrated daily to mice from day 0-21. SL4-cancer cells expressing red fluorescent protein (SL4-RFP) (1 × 106) were injected into the spleen. Fifteen minutes after injection, the hepatoduodenal ligament was clamped with a vessel clip, and released 5 min later. Liver, blood and tumor samples were taken from mice in order to determine if inflammation was induced by IRI. The effect of pioglitazone on liver metastasis was assessed. Furthermore, the effect of pioglitazone to control the inflammatory response during IRI progression was examined. Liver metastasis along with MMP-9 activation and the production of inflammatory cytokines were resistant to pioglitazone. Our results indicate that liver metastasis and associated inflammation in mice were resistant to pioglitazone.

Histone methylation status of H3K4me3 and H3K9me3 under methionine restriction is unstable in methionine-addicted cancer cells, but stable in normal cells.

Methionine addiction is a fundamental and general hallmark of cancer. Methionine addiction prevents cancer cells, but not normal cells from proliferation under methionine restriction (MR). Previous studies reported that MR altered the histone methylation levels in methionine-addicted cancer cells. However, no study has yet compared the status of histone methylation status, under MR, between cancer cells and normal cells. In the present study, we compared the histone methylation status between cancer cells and normal fibroblasts of H3K4me3 and H3K9me3, using recombinant methioninase (rMETase) to effect MR. Human lung and colon cancer cell lines and human normal foreskin fibroblasts were cultured in control medium or medium with rMETase. The viability of foreskin fibroblasts was approximately 10 times more resistant to rMETase than the cancer cells in vitro. Proliferation only of the cancer cells ceased under MR. The histone methylation status of H3K4me3 and H3K9me3 under MR was evaluated by immunoblotting. The levels of the H3K4me3 and H3K9me3 were strongly decreased by MR in the cancer cells. In contrast, the levels of H3K4me3 and H3K9me3 were not altered by MR in normal fibroblasts. The present results suggest that histone methylation status of H3K4me3 and H3K9me3 under MR was unstable in cancer cells but stable in normal cells and the instability of histone methylation status under MR may determine the high methionine dependency of cancer cells to survive and proliferate.

Oral recombinant methioninase increases TRAIL receptor-2 expression to regress pancreatic cancer in combination with agonist tigatuzumab in an orthotopic mouse model.

Methionine addiction is a fundamental and general hallmark of cancer. Gene expression analysis showed that methionine restriction (MR) of methionine-addicted cancer cells increases TNF-related apoptosis-induced ligand receptor-2 (TRAIL-R2) expression. Here, we determined the effects of MR on TRAIL-R2 targeted therapy in pancreatic cancer by the TRAIL-R2 agonist tigatuzumab. Human pancreatic cancer cell lines were cultured in control or methionine-free medium. The effects of MR on TRAIL-R2 expression and sensitivity to tigatuzumab were evaluated in vitro. An orthotopic pancreatic cancer mouse model was established to evaluate the efficacy of MR using oral recombinant methioninase (o-rMETase), and the efficacy of tigatuzumab and their combination. MR enabled tigatuzumab-induced apoptosis, by increasing TRAIL-R2 expression in pancreatic cancer cells in vitro. The protein expression level of the melanoma-associated antigen MAGED2, which reduces TRAIL-R2 expression, was decreased by MR. In the orthotopic pancreatic cancer mouse model, o-rMETase increased TRAIL-R2 expression level in the tumors and enabled the antitumor efficacy of tigatuzumab. MR, effected by o-rMETase, enabled the efficacy of the TRAIL-R2 agonist tigatuzumab by increasing TRAIL-R2 expression in pancreatic cancer. Our results suggest that o-rMETase has clinical potential for treating pancreatic cancer.

Novel artifact-robust and highly visible zinc solid fiducial marker for kilovoltage x-ray image-guided radiation therapy.

PURPOSE: To develop a novel biocompatible solid fiducial marker that prevents radiopaque imaging artifacts and also maintains high imaging contrast for kilovoltage x-ray image-guided radiation therapy. METHODS: The fiducial marker was made of pure zinc. An in-house water-equivalent phantom was designed to evaluate artifacts and visibility under various simulated treatment scenarios. Image artifacts were quantitatively assessed in terms of the metal artifact index (MAI) on kilovoltage computed tomography (CT) and cone-beam CT (CBCT) scans. Marker visibility was evaluated on two types of kilovoltage planar x-ray images in terms of the contrast-to-background ratio (CBR). Comparisons with a conventional gold fiducial marker were conducted. RESULTS: The use of zinc rather than a gold marker mitigates imaging artifacts. The MAI near the zinc marker decreased by 76, 79, and 77 % in CT, and by 77 (81), 74 (80), and 79 (85) % in CBCT full-fan (half-fan) scans, when using one-, two-, and three-marker phantom settings, respectively. The high-contrast part of the zinc marker exhibited CBRs above 2.00 for 28/32 exposures under four (lung, tissue, low-density bone, and high-density bone) different simulation scenarios, making its visibility comparable to that of the gold marker (30/32 exposures with CBRs > 2.00). CONCLUSIONS: We developed a biocompatible, artifact-robust, and highly visible solid zinc fiducial marker. Although further evaluation is needed in clinical settings, our findings suggest its feasibility and benefits for kilovoltage x-ray image-guided radiation therapy.