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Background/aim: Methotrexate (MTX) resistance in osteosarcoma results in a very poor patient prognosis. We previously reported that super MTX-resistant osteosarcoma (143B-MTX SR ) cells, selected from parental 143B osteosarcoma (143B-P) cells by culturing them with increasing concentrations of MTX, exhibited reduced malignancy, despite the over-expression of oncogenes. The present study explored the mechanism of reduced malignancy in the super MTX-resistant osteosarcoma cells., Materials and Methods: Previously selected 143B-MTX SR cells which are 5,500 times more MTX resistant than parental cells, were used for this study. The status of methylated histone H3K9me3 and H3K27me3 marks was examined with western immunoblotting and compared between 143B-MTX SR and parental 143B-P cells., Results: Histone H3K9me3 and H3K27me3 marks were over-expressed in 143B-MTX SR compared to 143B-P (p<0.05, p<0.01, respectively)., Conclusion: Over-expression of histone H3K9me3 and H3K27me3 marks may be related to super-MTX resistance and to the loss of malignancy of super MTX-resistant osteosarcoma cells due to the fundamental relationship of methylation and cancer., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The present study aimed to validate the accuracy of a tumor-specific antibody to target liver metastases of colorectal cancer., Methods: A humanized anti-CEA antibody conjugated to a fluorescent dye (M5A-IR800) was tested for targeting human colorectal cancer liver metastases (CRLMs) expressing luciferase in an orthotopic mouse model. Orthotopic mouse models of CRLMs were established by implanting fragments of a luciferase-expressing human colorectal cancer cell line, LS174T, in the liver of nude mice. Mice received 50 µg M5A-IR800 72 h prior to imaging. To test co-localization, bioluminescence imaging was performed using D-luciferin, which was given via intraperitoneal injection just prior to imaging., Results: Tumors were able to be visualized non-invasively through the skin with the luciferase-luciferin signal. Intra-abdominal imaging showed accurate labeling of CRLMs with M5A-IR800, which co-localized with the luciferase-luciferin signal., Conclusions: The present results validate the accuracy of a tumor-specific anti-CEA antibody in targeting liver metastases of colorectal cancer.

Background/aim: Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument., Materials and Methods: Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP., Results: For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system., Conclusion: Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment., Competing Interests: AW, NC, and SG are employees of Analytik Jena. JR is an employee of Anticancer Inc. KM, SM, MS, BMK, and RMH are non-salaried associates of AntiCancer Inc. AntiCancer Inc. uses mouse models of cancer for contract research., (©2024 The Author(s). Published by the International Institute of Anticancer Research.)

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research., Competing Interests: The Authors declare no competing interests., (©2024 The Author(s). Published by the International Institute of Anticancer Research.)

Background/aim: A major challenge in treating soft-tissue sarcoma is the development of drug resistance. Eribulin, an anti-tubulin agent, is used as a second-line chemotherapy for patients with unresectable or metastatic soft-tissue sarcoma. However, most patients with advanced soft-tissue sarcoma are resistant to eribulin and do not survive. Recombinant methioninase (rMETase) targets the fundamental and general hallmark of cancer, methionine addiction, termed the Hoffman Effect. The present study aimed to show how much rMETase could increase the efficacy of eribulin on eribulin-resistant fibrosarcoma cells in vitro., Materials and Methods: HT1080 human fibrosarcoma cells were exposed to step-wise increasing concentrations of eribulin from 0.15-0.4 nM to establish eribulin-resistant HT1080 (ER-HT1080). ER-HT1080 cells were cultured in vitro and divided into four groups: untreated control; eribulin treated (0.15 nM); rMETase treated (0.75 U/ml); and eribulin (0.15 nM) plus rMETase (0.75 U/ml) treated., Results: The IC 50 of eribulin on ER-HT1080 cells was 0.95 nM compared to the IC 50 of 0.15 nM on HT1080 cells, a 6-fold increase. The IC 50 of rMETase on ER-HT1080 and HT1080 was 0.87 U/ml and 0.75 U/ml, respectively. The combination of rMETase (0.75 U/ml) and eribulin (0.15 nM) was synergistic on ER-HT1080 cells resulting in an inhibition of 80.1% compared to eribulin alone (5.0%) or rMETase alone (47.1%) (p<0.05). rMETase thus increased the efficacy of eribulin 16-fold on eribulin-resistant fibrosarcoma cells., Conclusion: The present study showed that the combination of eribulin and rMETase can overcome high eribulin resistance of fibrosarcoma. The present results demonstrate that combining rMETase with first- or second-line therapy for soft-tissue sarcoma has the potential to overcome the intractable clinical problem of drug-resistant soft-tissue sarcoma., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Drug resistance has been a recalcitrant problem for sarcoma patients for many decades. Trabectedin is a second-line chemotherapy for soft-tissue sarcoma that often leads to resistance and death of the patients. The objective of the present study was to address the issue of trabectedin-chemoresistance in HT1080 fibrosarcoma cells by combining recombinant methioninase (rMETase) with trabectedin and examining their efficacy on trabectedin-resistant fibrosarcoma cells in vitro., Materials and Methods: Trabectedin-resistant HT1080 (TR-HT1080) cells were generated by subjecting HT1080 human fibrosarcoma cells to increasing trabectedin concentrations (3.3-8 nM). IC 50 values for trabectedin and rMETase were compared for HT1080 and TR-HT1080 cells. TR-HT 1080 cells were placed into four groups to determine synergy of rMETase and trabectedin on TR-HT1080 cells: a control group with no treatment; a group treated with trabectedin (3.3 nM); a group treated with rMETase (0.75 U/ml); and a group treated with both trabectedin (3.3 nM) and rMETase (0.75 U/ml)., Results: The IC 50 value of trabectedin- on TR-HT1080 cells was 42.9 nM, whereas the IC 50 value of trabectedin on the parental HT1080 cells was 3.3 nM, indicating a 13-fold increase. The combination of rMETase (0.75 U/ml) and trabectedin (3.3 nM) was synergistic on TR-HT1080 cells resulting in an inhibition of 64.2% compared to trabectedin alone (5.7%) or rMETase alone (50.5%) (p<0.05). rMETase increased the efficacy of trabectedin 11-fold on trabectedin-resistant fibrosarcoma cells., Conclusion: The combined administration of trabectedin and rMETase was synergistic on the viability of TR-HT1080 cells in vitro. The combination of rMETase and trabectedin has promising clinical potential for overcoming chemo-resistance of soft-tissue sarcoma., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Pancreatic cancer has a very poor prognosis with a 5-year survival rate of less than 5% among patients with distant metastasis, a figure that has not improved over many decades. Only 10 to 20% patients are candidates for curative surgery at presentation due to the aggressive nature and asymptomatic progression of pancreatic cancer. Although first-line chemotherapy, such as FOLFIRINOX and gemcitabine + nab paclitaxel, improved the median survival from 8.5 to 11.1 months, more effective treatments are immediately needed. The aim of the present study was to evaluate the efficacy of methionine restriction with oral rMETase (o-rMETase) and a low-methionine diet combined with first-line chemotherapy on a patient with stage IV metastatic pancreatic cancer., Case Report: A 63-year-old female was diagnosed with metastatic pancreatic cancer in October 2023. The patient started FOLFIRINOX as first-line chemotherapy in combination with methionine restriction, which comprised o-rMETase 250 units twice a day and a low-methionine diet. The patient was monitored using computed tomography and CA19-9 blood tests. After five months from the start of combination therapy, the size of the primary tumor decreased by 40% along with liver-metastasis regression. The CA19-9 blood marker decreased by 86%. The patient sustains a high performance status and continues the combination therapy without severe side effects., Conclusion: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with first-line chemotherapy, was highly effective in a patient with inoperable stage IV pancreatic cancer., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Positron emission tomography (PET) is an important imaging modality, especially in oncology. [ 18 F]fluorodeoxyglucose PET (FDG-PET) is the most used cancer PET imaging. However, since the elevated glucose use by cancers, termed the Warburg effect, is usually only moderate, FDG often does not provide a strong or well-delineated signal. Malignancies have a stronger addiction to methionine, known as the Hoffman effect, and thus [ 11 C]methionine PET (MET-PET) has demonstrated superiority over FDG-PET in gliomas and other brain tumors. Our team is pioneering the use of MET-PET for tumors of the trunk for both better detection of cancer and to determine candidates for methionine-restriction therapy. The present study provides examples of cancers of organs in the trunk in which MET-PET outperforms FDG-PET in detecting and delineating primary and metastatic cancer., Patients and Methods: In all cases, MET-PET and FDG-PET were performed simultaneously. An evaluation of the images was conducted by a nuclear medicine physician., Results: Four cases, including prostate, bladder, esophageal, and breast cancer demonstrated the superiority of MET-PET compared to FDG-PET., Conclusion: MET-PET can out-perform FDG PET for accurate detection of primary and metastatic cancer in the trunk and can determine the extent of methionine addiction of cancer, thereby indicating whether cancer patients can benefit from methionine-restriction therapy., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: In vivo imaging with luciferase-luciferin has been limited by the inability to visualize the low emitted light, with the signal quantified only by photon counting using a cumbersome highly-cooled CCD camera in a dark room. In the present study, we demonstrate direct visualization of the luciferase-luciferin signal from an orthotopic lung cancer in a nude-mouse xenograft model with a sensitive low-light camera and optics., Materials and Methods: Mouse Lewis-lung carcinoma cells expressing luciferase (LL/2-Luc2) were injected transcutaneously into the lung of a nude mouse. One week later after cell injection, luciferase imaging for emission at 560 nm was performed using the UVP Biospectrum Advanced system after i.v. injection of D-luciferin potassium salt. The intensity of the visualized light was measured and quantified with the instrument., Results: A week following the implantation of LL/2-Luc2 cells in nude mice, the luciferase-luciferin signal from LL/2-Luc2 tumors in the lung was sufficiently visible through the skin to produce true images. At fifteen minutes, the intensity peaked and then progressively dropped due to clearance of luciferin from the tumor., Conclusion: Using the UVP Biospectrum Advanced system we demonstrated non-invasive visualization of true images from luciferase-luciferin signals from an orthotopic lung-cancer mouse model. The luciferase-luciferin emitted light was directly visible through the skin which is a major improvement over previous photon counting to detect the luciferase-luciferin signal., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: We and others have previously shown that cell fusion plays an important role in cancer metastasis. Color coding of cancer and stromal cells with spectrally-distinct fluorescent proteins is a powerful tool, as pioneered by our laboratory to detect cell fusion. We have previously reported color-coded cell fusion between cancer cells and stromal cells in metastatic sites by using color-coded EL4 murine lymphoma cells and host mice expressing spectrally-distinct fluorescent proteins. Cell fusion occurred between cancer cells or, between cancer cells and normal cells, such as macrophages, fibroblasts, and mesenchymal stem cells. In the present study, the aim was to morphologically classify the fusion-hybrid cells observed in the primary tumor and multiple metastases EL4 formed from cells expressing red fluorescent protein (RFP) in transgenic mice expressing green fluorescent protein (GFP), in a syngeneic model., Materials and Methods: RFP-expressing EL4 murine lymphoma cells were cultured in vitro. EL4-RFP cells were harvested and injected intraperitoneally into immunocompetent transgenic C57/BL6-GFP mice to establish a syngeneic model. Two weeks later, mice were sacrificed and each organ was harvested, cultured, and observed using confocal microscopy., Results: EL4 intraperitoneal tumors (primary) and metastases in the lung, liver, blood, and bone marrow were formed. All tumors were harvested and cultured. In all specimens, RFP-EL4 cells, GFP-stromal cells, and fused yellow-fluorescent hybrid cells were observed. The fused hybrid cells showed various morphologies. Immune cell-like round-shaped yellow-fluorescent fused cells had a tendency to decrease with time in liver metastases and circulating blood. In contrast fibroblast-like spindle-shaped yellow-fluorescent fused cells increased in the intraperitoneal primary tumor, lung metastases, and bone marrow., Conclusion: Cell fusion between EL4-RFP cells and GFP stromal cells occurred in primary tumors and all metastatic sites. The morphology of the fused hybrid cells varied in the primary and metastatic sites. The present results suggest that fused cancer and stromal hybrid cells of varying morphology may play an important role in cancer progression., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Gastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival., Methods: Two patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue., Results: M5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800., Conclusions: Humanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection., (© 2024. The Author(s).)

Background/aim: Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro., Materials and Methods: The 50% inhibitory concentrations (IC 50 ) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope., Results: The IC 50 for doxorubicin was 3.3 μM for HT1080 cells, 12.4 μM for DR-HT1080 cells, and 7.25 μM for Hs27 cells. The IC 50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells., Conclusion: The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Immune checkpoint inhibitors (ICIs) play an important role in the treatment of esophageal cancer (EC). However, few patients achieve long-term survival, and some patients develop serious immune-related adverse events (irAEs). Reliable predictive biomarkers of efficacy and safety need to be established in order to improve efficacy. We retrospectively analyzed the outcomes of nivolumab monotherapy on EC at Showa University, Department of Medicine, to identify biomarkers and characteristics of patients who benefit from ICI monotherapy., Patients and Methods: Eighty-six patients with EC who received nivolumab monotherapy were included in the present study. Patient characteristics, efficacy, and safety were analyzed. A multivariable analysis evaluated the correlation among overall survival (OS), progression-free survival (PFS), best overall response (BOR), irAEs, and the following variables: sex, age, performance status (PS), neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP) level, albumin level, and body-mass index before treatment., Results: Median PFS was 3.1 months, and median OS was 9.0 months. In multivariable analysis, pretreatment PS, NLR, and sex were significantly correlated with OS and PFS. NLR <3.3 predicted longer survival (median OS 17.5 vs. 6.4 months for NLR ≥3.3; p<0.001). Median OS was 10.6 months for PS 0-1 and 1.3 months for PS 2-3 (p<0.001). NLR remained significantly predictive in the PS 0-1 group. The development of irAEs was significantly associated with increased OS and PFS., Conclusion: Patients with low NLR and good PS before treatment may maximize the benefits of ICIs. A low NLR may be an indicator of higher immunocompetence for anti-tumor immunity, suggesting that NLR may be a convenient predictive biomarker in daily practice., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Exosome exchange between cancer cells or between cancer and stromal cells is involved in cancer metastasis. We have previously developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct fluorescent genetic reporters to demonstrate the role of exosomes in metastasis. In the present study, we studied exosome transfer between different pancreatic-cancer cell lines in vivo and in vitro and its potential role in metastasis., Materials and Methods: Human pancreatic-cancer cell lines AsPC-1 and MiaPaCa-2 were used in the present study. AsPC-1 cells contain a genetic exosome reporter gene labeled with green fluorescent protein (pCT-CD63-GFP) and MiaPaCa-2 cells express red fluorescent protein (RFP). Both cell lines were co-injected into the spleen of nude mice (n=5) to further study the role of exosome exchange in metastasis. Three weeks later mice were sacrificed and tumors at the primary and metastatic sites were cultured and observed by confocal fluorescence microscopy for exosome transfer., Results: The primary tumor formed in the spleen and metastasized to the liver, as observed macroscopically. Cells were cultured from the spleen, liver, lung, bone marrow and ascites. Transfer of exosomes from AsPC-1 to MiaPaCa-2 was demonstrated in the cultured cells by confocal fluorescence microscopy. Moreover, cell fusion was also observed along with exosome transfer. Exosome transfer did not occur during in vitro co-culture between the two pancreatic-cancer cell lines, suggesting a role of the tumor microenvironment (TME) in exosome transfer., Conclusion: The transfer of exosomes between different pancreatic-cancer cell lines was observed during primary-tumor and metastatic growth in nude mice. This cell-cell communication might be a trigger of cell fusion and promotion of cancer metastasis. Exosome transfer between the two pancreatic-cancer cell lines appears to be facilitated by the TME, as it did not occur during in vitro co-culture., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Androgen-independent prostate cancer (AIPC) is resistant to androgen-depletion therapy and is a recalcitrant disease. Docetaxel is the first-line treatment for AIPC, but has limited efficacy and severe side-effects. All cancers are methionine-addicted, which is termed the Hoffman effect. Recombinant methioninase (rMETase) targets methionine addiction. The purpose of the present study was to determine if the combination of docetaxel and rMETase is effective for AIPC., Materials and Methods: The half-maximal inhibitory concentrations (IC 50 ) of docetaxel and rMETase alone were determined for the human AIPC cell line PC-3 and Hs27 normal human fibroblasts in vitro. The synergistic efficacy for PC-3 and Hs27 using the combination of docetaxel and rMETase at their IC 50 s for PC-3 was determined., Results: The IC 50 of docetaxel for PC-3 and for Hs27 was 0.72 nM and 0.94 nM, respectively. The IC 50 of rMETase for PC-3 and for Hs27 was 0.67 U/ml and 0.76 U/ml, respectively. The combination of docetaxel and rMETase was synergistic for PC-3 but not Hs27 cells., Conclusion: The combination of a relatively low concentration of docetaxel and rMETase was synergistic and effective for AIPC. The present results also suggest that the effective concentration of docetaxel can be reduced by using rMETase, which may reduce toxicity. The present results also suggest the future clinical potential of the combination of docetaxel and rMETase for AIPC., Competing Interests: There are no conflicts of interest, according to the Authors., (Copyright 2024, International Institute of Anticancer Research.)

Background/aim: Rapamycin inhibits the mTOR protein kinase. Methioninase (rMETase), by degrading methionine, targets the methionine addiction of cancer cells and has been shown to improve the efficacy of chemotherapy drugs, reducing their effective doses. Our previous study demonstrated that rapamycin and rMETase work synergistically against colorectal-cancer cells, but not on normal cells, when administered simultaneously in vitro. In the present study, we aimed to further our previous findings by exploring whether  synergy exists between rapamycin and rMETase when used sequentially against HCT-116 colorectal-carcinoma cells, compared to simultaneous administration, in vitro., Materials and Methods: The half-maximal inhibitory concentrations (IC 50 ) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line were previously determined using the CCK-8 cell viability assay (11). We then examined the efficacy of rapamycin and rMETase, both at their IC 50 , administered simultaneously or sequentially on the HCT-116 cell line, with rapamycin administered before rMETase and vice versa., Results: The IC 50 for rapamycin and rMETase, determined from previous experiments (11), was 1.38 nM and 0.39 U/ml, respectively, of HCT-116 cells. When rMETase was administered four days before rapamycin, both at the IC 50 , there was a 30.46% inhibition of HCT-116 cells. When rapamycin was administered four days before rMETase, both at the IC 50 , there was an inhibition of 41.13%. When both rapamycin and rMETase were simultaneously administered, both at the IC 50 , there was a 71.03% inhibition., Conclusion: Rapamycin and rMETase have synergistic efficacy against colorectal-cancer cells in vitro when administered simultaneously, but not sequentially., Competing Interests: The Authors have no conflicts of interest to declare in relation to this study., (Copyright 2024, International Institute of Anticancer Research.)

Background/aim: Methotrexate (MTX) resistance in osteosarcoma leads to a very poor prognosis. In the present study, in order to further understand the basis and ramifications of MTX resistance in osteosarcoma, we selected an osteosarcoma cell line that has a 5,500-fold-increased MTX IC 50 Materials and Methods: The super MTX-resistant 143B osteosarcoma cells (143B-MTX SR ) were selected from MTX-sensitive parental human 143B osteosarcoma cells (143B-P) by continuous culture with step-wise increased amounts of MTX. To compare the malignancy of 143B-MTX SR and 143B-P, colony-formation capacity was compared with clonogenic assays on plastic and in soft agar. In addition, tumor growth was compared with orthotopic xenograft mouse models of osteosarcoma. Expression of dihydrofolate reductase (DHFR), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), and myelocytomatosis oncogene (MYC) was examined with western immunoblotting and compared in 143B-MTX SR and 143B-P cells., Results: 143B-MTX SR had a 5,500-fold increase in the MTX IC 50 compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTX SR compared to 143B-P (p<0.01). 143B-MTX SR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (p<0.01) compared to 143B-P and reduced tumor growth in orthotopic xenograft mouse models (p<0.001). These results demonstrate that 143B-MTX SR had reduced malignancy. 143B-MTX SR also showed an increased expression of PI3K (p<0.01), phosphorylated (activated) AKT (p=0.031), phosphorylated mTOR (p=0.043), and c-MYC (p=0.024) compared to 143B-P., Conclusion: The present study demonstrates that the increased expression of DHFR, PI3K/AKT/mTOR and c-MYC appears to be linked to super MTX resistance and, paradoxically, to reduced malignancy. The present results suggest that DHFR may be a powerful tumor suppressor when highly amplified., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed., Materials and Methods: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin., Results: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals., Conclusion: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: It has been recently demonstrated that a methionine-restricted diet increases the response to immune checkpoint inhibitors (ICIs) via an increase in PD-L1 in a syngeneic mouse colorectal-cancer model. Our laboratory has developed recombinant methioninase (rMETase) to restrict methionine. The aim of the present study was to determine if rMETase can increase PD-L1 expression in a human colorectal cancer cell line in vitro., Materials and Methods: We evaluated the half-maximal inhibitory concentration (IC 50 ) value of rMETase on HCT-116 human colorectal cancer cells. HCT-116 cells were treated with rMETase at the IC 50 Western immunoblotting was used to compare PD-L1 expression in HCT-116 cells treated with and without rMETase., Results: The IC 50 value of rMETase on HCT-116 was 0.79 U/ml. Methionine restriction using rMETase increased PD-L1 expression compared to the untreated control (p<0.05)., Conclusion: Methionine restriction with rMETase up-regulates PD-L1 expression in human colorectal cancer cells and the combination of rMETase and ICIs may have the potential to improve immunotherapy in human colorectal cancer., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cancer cells are addicted to L-methionine (L-Met) and have a much greater requirement for L-Met than normal cells due to excess transmethylation, termed the Hoffman effect. By targeting this vulnerability through dietary restriction of L-Met, researchers have been able to achieve promising results in inhibiting tumor growth and eradicating cancer cells. Methioninase (EC 4.4.1.11; METase) catalyzes the transformation of L-Met into α-ketobutyrate, ammonia, and methanethiol. The use of METase was initially limited due to its poor stability in vivo, high immunogenicity, and enzyme-induced inactivating antibodies. These issues could be partially resolved by PEGylation, encapsulation in erythrocytes, and various site-directed mutagenesis. The big breakthrough came when it was discovered that METase is effectively administered orally. The enzyme L-asparaginase is approved by the FDA for treatment of acute lymphoblastic leukemia. METase has more potential as a therapeutic since addiction to L-Met is a general and fundamental hallmark of cancer., Competing Interests: Declaration of competing Interest The authors declare no conflict of interest. This article does not contain description of studies involving human participants or animals performed by any of the authors., (Copyright © 2024. Published by Elsevier B.V.)

Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-β1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-β1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hasegawa-Haruki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background/aim: The alkylating agent trabectedin, which binds the minor groove of DNA, is second-line therapy for soft-tissue sarcoma but has only moderate efficacy. The aim of the present study was to determine the synergistic efficacy of recombinant methioninase (rMETase) and trabectedin on fibrosarcoma cells in vitro, compared with normal fibroblasts., Materials and Methods: HT1080 human fibrosarcoma cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and Hs27 normal human fibroblasts, were used. Each cell line was cultured in vitro and divided into four groups: no-treatment control; trabectedin treated; rMETase treated; and trabectedin plus rMETase treated. The dual-color HT1080 cells were used to quantitate nuclear fragmentation in each treatment group., Results: The combination of rMETase and trabectedin was highly synergistic to decrease HT1080 cell viability. In contrast, there was no synergy on Hs27 cells. Moreover, nuclear fragmentation occurred synergistically with the combination of trabectedin and rMETase on dual-color HT1080 cells., Conclusion: The combination treatment of trabectedin plus rMETase was highly synergistic on fibrosarcoma cells in vitro suggesting that the combination can improve the outcome of trabectedin alone in future clinical studies. The lack of synergy of rMETase and trabectedin on normal fibroblasts suggests the combination is not toxic to normal cells. Synergy of the two drugs may be due to the high rate of nuclear fragmentation on treated HT1080 cells, and the late-S/G 2 cell-cycle block of cancer cells by rMETase, which is a target for trabectedin. The results of the present study suggest the future clinical potential of the combination of rMETase and trabectedin for soft-tissue sarcoma., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The present study utilized the three-dimensional histoculture drug response assay (HDRA) to determine the efficacy of recombinant methioninase (rMETase) on tumor tissue resected from patients with late-stage cancer, as a functional biomarker of sensitivity to methionine restriction therapy., Patients and Methods: Resected peritoneal-metastatic cancer, including colorectal cancer, pancreatic cancer, ovarian cancer, and pseudomyxoma were placed on Gelform in RPMI 1640 medium for seven days and treated with rMETase from 2.5 U/ml to 20 U/ml. Cell viability was determined using the MTT assay. A total of 48 patients with late-stage cancer underwent testing for rMETase responsiveness using the HDRA., Results: Colorectal cancer and pseudomyxoma had the highest sensitivity to rMETase. Pancreatic and ovarian cancer also responded to rMETase, but to a lesser degree., Conclusion: Patients with tumors with at least 40% sensitivity to rMETase in the HDRA are being considered as candidates for methionine restriction therapy, which includes the use of rMETase in combination with a low-methionine diet., Competing Interests: The Authors declare no competing interests regarding this work., (Copyright 2024, International Institute of Anticancer Research.)

Background/aim: Gliomas are the most common and recalcitrant malignant primary brain tumors. All cancer types are addicted to methionine, which is a fundamental and general hallmark of cancer known as the Hoffman effect. Particularly glioma cells exhibit methionine addiction. Because of methionine addiction, [ 11 C]-methionine positron emission tomography (MET-PET) is widely used for glioma imaging in clinical practice, which can monitor the extent of methionine addiction. Methionine restriction including recombinant methioninase (rMETase) and a low-methionine diet, has shown high efficacy in preclinical models of gliomas, especially in combination with chemotherapy. The aim of the present study was to determine the efficacy of methionine restriction with oral rMETase (o-rMETase) and a low-methionine diet, combined with radiation and temozolomide (TMZ), on a teenage female patient with high-grade glioma., Case Report: A 16-year-old girl was diagnosed with high-grade glioma. Magnetic resonance imaging (MRI) showed a left temporal-lobe tumor with compression to the left lateral ventricle and narrowing of sulci in the left temporal lobe. After the start of methionine restriction with o-rMETase and a low-methionine diet, along with TMZ combined with radiotherapy, the tumor size shrunk at least 60%, with improvement in the left lateral ventricle and sulci. The patient's condition remains stable for 19 months without severe adverse effects., Conclusion: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with radiation and TMZ as first-line chemotherapy, were highly effective in a patient with high-grade glioma., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Hair-follicle keratinocytes contain high levels of cysteine, which is derived from methionine, rapidly proliferate, and form the hair shaft. The high proliferation rate of hair-follicle keratinocytes resembles that of aggressive cancer cells. In the present study, we determined the effect of a methionine-deficient diet on hair loss (alopecia) in mice with or without homocysteine supplementation., Materials and Methods: Mice were fed a normal rodent diet (2020X, ENVIGO) (Group 1); a methionine-choline-deficient diet (TD.90262, ENVIGO) (Group 2); a methionine-choline-deficient diet with a 10 mg/kg/day supply of homocysteine administered by intra-peritoneal (i.p.) injection for 2 weeks (Group 3). In Group 2, mice were fed a methionine-choline-deficient diet for an additional 2 weeks but with 10 mg/kg/day of i.p. l-homocysteine and the mice were observed for two additional weeks. Subsequently, the mice were fed a standard diet that included methionine. Hair loss was monitored by photography., Results: After 14 days, hair loss was observed in Group 2 mice on a methionine-restricted diet but not in Group 3 mice on the methionine-restricted diet which received i.p. homocysteine. In Group 2, at 2 weeks after methionine restriction, hair loss was not rescued by homocysteine supplementation. However, after restoration of methionine in the diet, hair growth resumed. Thus, after 2 weeks of methionine restriction, only methionine restored hair loss, not homocysteine., Conclusion: Hair maintenance requires methionine in the diet. Future experiments will determine the effects of methionine restriction on hair-follicle stem cells., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Colorectal cancer (CRC) is the third-leading cause of death in the world. Although the prognosis has improved due to improvement of chemotherapy, metastatic CRC is still a recalcitrant disease, with a 5-year survival of only 13%. Irinotecan (IRN) is used as first-line chemotherapy for patients with unresectable CRC. However, there are severe side effects, such as neutropenia and diarrhea, which are dose-limiting. We have previously shown that methionine restriction (MR), effected by recombinant methioninase (rMETase), lowered the effective dose of IRN of colon-cancer cells in vitro. The aim of the present study was to evaluate the efficacy of the combination of low-dose IRN and MR on colon-cancer in nude mice., Materials and Methods: HCT-116 colon-cancer cells were cultured and subcutaneously injected into the flank of nude mice. After the tumor size reached approximately 100 mm 3 , 18 mice were randomized into three groups; Group 1: untreated control on a normal diet; Group 2: high-dose IRN on a normal diet (2 mg/kg, i.p.); Group 3: low-dose IRN (1 mg/kg i.p.) on MR effected by a methionine-depleted diet., Results: There was no significant difference between the control mice and the mice treated with high-dose IRN, without MR. However, low-dose IRN combined with MR was significantly more effective than the control and arrested colon-cancer growth (p=0.03). Body weight loss was reversible in the mice treated by low-dose IRN combined with MR., Conclusion: The combination of low-dose IRN and MR acted synergistically in arresting HCT-116 colon-cancer grown in nude mice. The present study indicates the MR has the potential to reduce the effective dose of IRN in the clinic., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The prognosis of ovarian cancer (OC) patients is especially poor for patients with chemotherapy resistance. Anlotinib, a novel multi-targeted tyrosine kinase inhibitor, has shown encouraging clinical efficacy in several tumor types. The aim of the present study was to examine the inhibitory efficacy and mechanism of anlotinib on the proliferation and chemosensitivity of OC cells., Materials and Methods: The inhibitory effects of Anlotinib on SKOV3 and OVCAR3 OC cells were examined using CCK-8 cell-viability, colony-formation, flow-cytometry, transwell-migration and sphere-formation assays. A xenograft mouse model was used for in vivo studies. RT-qPCR and western blotting were used to detect gene expression., Results: Molecular targets of anlotinib were elevated in OC patient tumors. Anlotinib significantly inhibited ovarian cancer cell proliferation and migration in vitro. Anlotinib enhanced the sensitivity of ovarian cancer cells to cisplatinum both in vitro and in vivo. Anlotinib suppressed sphere formation and the stemness phenotype of OC cells by inhibiting NOTCH2 expression., Conclusion: Anlotinib inhibits ovarian cancer and enhances cisplatinum sensitivity, suggesting its future clinical promise., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Breast cancer is the most common and the deadliest cancer among women in the world. Treatment options for HER2-positive metastatic breast cancer patients are limited. Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate (ADC), has recently been introduced as second-line chemotherapy for HER2-positive metastatic breast cancer. The aim of the present study was to evaluate the efficacy of methionine restriction with oral recombinant methioninase (o-rMETase) and a low-methionine diet combined with T-DXd, on a patient with HER2-positive recurrent stage IV breast cancer., Case Report: A 66-year-old female was diagnosed with HER2-positive metastatic breast cancer. Computed tomography (CT) indicated peritoneal dissemination, thickening of the sigmoid colon and splenic flexure and widespread bone metastases. The patient was previously treated with fulvestrant, trastuzumab, pertuzumab, paclitaxel and capecitabine which were ineffective. T-DXd was administered as a second-line chemotherapy. Since the patient experienced strong side effects, the dose of T-Dxd was decreased. The patient began methionine restriction using o-rMETase and a low-methionine diet along with T-DXd. After the start of the combined treatment, CA15-3 and CA27.29, tumor markers for breast cancer, decreased rapidly from a very high level. The levels of both tumor markers are currently normal. Additionally, peritoneal-dissemination nodules, ascites and the thickness of the sigmoid colon and splenic flexure are no longer detected on CT. The patient maintains a high performance status, without severe side effects of the combination treatment., Conclusion: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with T-DXd as second-line chemotherapy, was highly effective in a patient with HER2-positive stage IV breast cancer., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The aim of the present study was to determine the synergy of recombinant methioninase (rMETase) and the anti-tubulin agent eribulin on fibrosarcoma cells, in comparison to normal fibroblasts, in vitro., Materials and Methods: HT1080 human fibrosarcoma cells and HS27 human fibroblasts were used for in vitro experiments. Four groups were analyzed in vitro: No-treatment control; eribulin; rMETase; eribulin plus rMETase. Dual-color HT1080 cells which express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize cytoplasmic and nuclear dynamics during treatment., Results: Eribulin combined with rMETase greatly decreased the viability of HT 1080 cells. In contrast, eribulin combined with rMETase did not show synergy on Hs27 normal fibroblasts. Eribulin combined with rMETase also caused more fragmentation of the nucleus than all other treatments., Conclusion: The combination treatment of eribulin plus rMETase demonstrated efficacy on fibrosarcoma cells in vitro. In contrast, normal fibroblasts were resistant to this combination, indicating the potential clinical applicability of the treatment., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Rapamycin and recombinant methioninase (rMETase) have both shown efficacy to target cancer cells. Rapamycin prevents cancer-cell growth by inhibition of the mTOR protein kinase. rMETase, by degrading methionine, targets the methionine addiction of cancer and has been shown to improve the efficacy of chemotherapy drugs. In the present study, we aimed to determine if a synergy exists between rapamycin and rMETase when used in combination against a colorectal-carcinoma cell line, compared to normal fibroblasts, in vitro., Materials and Methods: The half-maximal inhibitory concentrations (IC 50 ) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line and Hs-27 human fibroblasts were determined using the CCK-8 Cell Viability Assay. After calculating the IC 50 of each drug, we determined the efficacy of rapamycin and rMETase combined on both HCT-116 and Hs-27., Results: Hs-27 normal fibroblasts were more sensitive to rapamycin than HCT-116 colon-cancer cells (IC 50 =0.37 nM and IC 50 =1.38 nM, respectively). HCT-116 cells were more sensitive to rMETase than Hs-27 cells (IC 50 0.39 U/ml and IC 50 0.96 U/ml, respectively). The treatment of Hs-27 cells with the combination of rapamycin (IC 50 =0.37 nM) and rMETase (IC 50 =0.96 U/ml) showed no significant difference in their effect on Hs-27 cell viability compared to the two drugs being used separately. However, the treatment of HCT-116 cells with the combination of rapamycin (IC 50 =1.38 nM) and rMETase (IC 50 =0.39 U/ml) was able to decrease cancer-cell viability significantly more than either single-drug treatment., Conclusion: Rapamycin and rMETase, when used in combination against colorectal-cancer cells, but not normal fibroblasts, in vitro, have a cancer-specific synergistic effect, suggesting that the combination of these drugs can be used as an effective, targeted cancer therapy., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Claudins (CLDN1-CLDN24) are a family of tight junction proteins whose dysregulation has been implicated in tumorigeneses of many cancer types. In colorectal cancer (CRC), CLDN1, CLDN2, CLDN4, and CLDN18 have been shown to either be upregulated or aberrantly expressed. In the normal colon, CLDN1 and CLDN3-7 are expressed. Although a few claudins, such as CLDN6 and CLDN7, are expressed in CRC their levels are reduced compared to the normal colon. The present review outlines the expression profiles of claudin proteins in CRC and those that are potential biomarkers for prognostication.

Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O 6 -methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Takaoka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background/aim: Pancreatic cancer is a recalcitrant disease with 5-year survival of only 12%. Improved mouse models of pancreatic cancer are critical for discovery of effective therapeutics., Materials and Methods: Orthotopic mouse nude-mouse models of pancreatic cancer were established with the human pancreatic-cancer cell line Panc-1 expressing green fluorescent protein (GFP) by transplanting tumor fragments into the pancreas, using the procedure of surgical orthotopic implantation (SOI). Four weeks after establishment of the orthotopic models, the mice were imaged with the Analytik Jena UVP Biospectrum Advanced with a very-narrow-band-width excitation at 487 nm and peak emission at 513 nm., Results: Non-invasive fluorescence imaging of the mice implanted with Panc-1-GFP showed a very bright tumor in the area of the pancreas and peritoneal cavity. The skin background autofluorescence was absent. When a laparotomy was performed on the mouse for open imaging, the tumor on the pancreas was clearly imaged. There was very clear concordance of the non-invasive image and the image obtained during laparotomy., Conclusion: A precise orthotopic mouse model of pancreatic cancer was developed in which there was high concordance between non-invasive and invasive fluorescence imaging due to the ultra-bright signal and ultra-low background using very-narrow-band-width laser fluorescence excitation. This model can be used for high-throughput in vivo screening for improved therapeutics for pancreatic cancer., Competing Interests: AW, NC, ST and SG are employees of Analytik Jena. YA, YT, NM, KO, SM and RMH are non-salaried associates of AntiCancer Inc. AntiCancer Inc. uses mouse models of cancer for contract research., (Copyright 2024, International Institute of Anticancer Research.)

Background/aim: Breast-cancer metastasis to the brain is an intractable disease. To discover improved therapy for this disease, we developed a precise non-invasively-imageable orthotopic nude-mouse model, using very-narrow-band-width laser fluorescence excitation., Materials and Methods: Female nu/nu nude mice, aged 4-8 weeks, were inoculated through the midline of the skull with triple-negative human MDA-MB-231 breast cancer cells (5×10 5 ) expressing red fluorescent protein (RFP). The mice were imaged with the Analytik Jena UVP Biospectrum Advanced at 520 nm excitation with peak emission at 605 nm., Results: Three weeks after injection of MDA-MB-231-RFP cells in the brain, non-invasive fluorescence images of the breast tumor growing on the brain were obtained. The images of the tumor were very bright, with well-defined margins with no detectable skin autofluorescence background. Images obtained at various angles showed that the extent of the tumor margins could be precisely measured. A skin flap over the skull confirmed that the tumor was growing on the surface of the brain which is a frequent occurrence in breast cancer., Conclusion: A precise orthotopic model of RFP-expressing breast-cancer metastasis to the brain was developed that could be non-invasively imaged with very-narrow-band-width laser excitation, resulting in an ultra-bright, ultra-low-background signal. The model will be useful in discovering improved therapeutics for this recalcitrant disease., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Irinotecan (IRN), a topoisomerase I inhibitor and pro-drug of SN-38, is first-line treatment of colon cancer as part of FOLFIRI and FOLFOXIRI combination chemotherapy. However, IRN causes dose-limiting adverse events such as neutropenia and diarrhea. Dose reductions are sometimes required, which reduce efficacy. Recombinant methioninase (rMETase) targets the fundamental basis of cancer, methionine addiction, known as the Hoffman effect, and enhances the efficacy of numerous chemotherapy drugs. The present study determined the efficacy of rMETase when administered in combination with IRN., Materials and Methods: Cell viability was assessed by cultivating the HCT-116 human colorectal cancer cell line in 96-well plates at 1×10 3 cells per well in Dulbecco's modified Eagle's medium (DMEM). Subsequently, HCT-116 cells were treated with increasing concentrations of SN-38, the active form of IRN, ranging from 0.5 nM to 32 nM, and/or rMETase ranging from 0.125 to 8 U/ml. After treatment for 72 h, the half-maximal inhibitory concentration (IC 50 ) of SN-38 alone and rMETase alone for HCT-116 cells were determined. Using the IC 50 concentration of rMETase, we determined the IC 50 of SN-38 in combination with rMETase. Cell viability was determined with the cell-counting Kit-8 with the WST-8 reagent.., Results: The IC 50 of rMETase alone for the HCT-116 cells was 0.55 U/ml, and the IC 50 of IRN (SN-38) alone was 3.50 nM. rMETase at 0.55 U/ml lowered the IC 50 of SN-38 to 0.232 nM (p<0.0001), a 15-fold reduction., Conclusion: rMETase and IRN are strongly synergistic, giving rise to the possibility of lowering the effective dose of IRN for the treatment of patients with colon cancer, thereby reducing its severe toxicity. This new strategy will allow more patients with cancer to be effectively treated with IRN., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Methionine addiction is a fundamental and universal hallmark of cancer, termed the Hoffman effect. Methionine addiction of cancer is greater than glucose addiction, termed the Warburg effect, as shown by the comparison of PET imaging with [ 11 C]methionine and [ 18 F]fluorodeoxyglucose. The aim of the present study was to determine whether [ 11 C]methionine PET (MET-PET) images could be a biomarker of methionine addiction of cancer and potential response to methionine-restriction-based combination chemotherapy., Patients and Methods: In the present study a patient with invasive lobular carcinoma of the breast metastatic to axillary lymph nodes was imaged by both MET-PET and [ 18 F]fluorodeoxyglucose PET (FDG-PET) before and after combination treatment with methionine restriction, comprising a low-methionine diet and methioninase, along with first-line chemotherapy., Results: MET-PET gave a much stronger and precise image of the patient's metastatic axillary lymph nodes than FDG-PET. The patient had a complete response to methionine restriction-based chemotherapy as shown by MET-PET., Conclusion: MET-PET imaging is a biomarker of methionine-addicted cancer and potential response to methionine-restriction-based chemotherapy., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The response rate to immune checkpoint inhibitors (ICIs) is approximately 10%-30% and only in a few cancer types. In the present study, we determined whether non-classical monocytes (NCMs) could enhance ICI efficacy in colon cancer using a syngeneic mouse model., Materials and Methods: The MC38 C57BL/6 mouse colon cancer model was used. Cells collected from the bone marrow of C57BL/6 mice were cultured, and NCMs were fractionated by cell sorting and administered via the tail veins to the mice implanted with MC38 cells. The anti-mouse PD-L1 antibody was administered three times, and tumor volume and overall survival were observed., Results: More tumors were eradicated and more complete response occurred, after cotreatment with ICIs and NCMs than after treatment with ICIs alone. Moreover, no efficacy was observed when NCMs were administered alone., Conclusion: NCMs enhance ICI efficacy. The underlying mechanisms and clinical applications will be studied in the future., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Introduction: Gastric cancer poses a major therapeutic challenge. Improved visualization of tumor margins at the time of gastrectomy with fluorescent tumor-specific antibodies could improve outcomes. The present report demonstrates the potential of targeting gastric cancer with a humanized anti-carcinoembryonic antigen (CEA) antibody in orthotopic mouse models., Methods: MKN45 cells were injected subcutaneously into nude mice to establish xenograft models. Tumor fragments collected from subcutaneous models were then implanted into the greater curvature of the stomach to establish orthotopic models. For tumor labeling, a humanized anti-CEA antibody (M5A) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Time (24-72 h) and dose (50-100 μg) response curves were performed in subcutaneous models. Orthotopic models received 50 μg of M5A-IR800 or 50 μg IgG-IR800 as a control and were imaged after 72 h. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System., Results: In subcutaneous models, tumor to background ratios (TBRs) reached 8.85 at 72 h. Median TBRs of orthotopic model primary tumors were 6.25 (interquartile range [IQR] 6.03-7.12) for M5A-IR800 compared to 0.42 (IQR 0.38-0.54) for control. Abdominal wall metastasis median TBRs were 13.52 (IQR 12.79-13.76) for M5A-IR800 and 3.19 (IQR 2.65-3.73) for the control. Immunohistochemistry confirmed CEA expression within tumors., Conclusions: Humanized anti-CEA antibodies conjugated to near-infrared dyes provide specific labeling of gastric cancers in mouse models. Orthotopic models demonstrated bright and specific labeling with TBRs greater than ten times that of control. This tumor-specific fluorescent antibody is a promising potential clinical tool for improving visualization of gastric cancer margins at time of surgical resection., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The methionine addiction of cancer cells is known as the Hoffman effect. While non-cancer cells in culture can utilize homocysteine in place of methionine for cellular growth, most cancer cells require exogenous methionine for proliferation. It has been suggested that a biochemical basis of this effect is the increased utilization of methionine for S-adenosylmethionine, the major methyl donor for a variety of cellular methyltransferases. Recent studies have pointed to the role of S-adenosylmethionine-dependent protein arginine methyltransferases (PRMTs) in cell proliferation and cancer. To further understand the biochemical basis of the methionine addiction of cancer cells, we compared protein arginine methylation in two previously described isogenic cell lines, a methionine-addicted 143B human osteosarcoma cell line and its less methionine-dependent revertant. Previous work showed that the revertant cells were significantly less malignant than the parental cells. In the present study, we utilized antibodies to detect the asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) products of PRMTs in polypeptides from cellular extracts and purified histone preparations of these cell lines fractionated by SDS-PAGE. Importantly, we observed little to no differences in the banding patterns of ADMA- and SDMA-containing species between the osteosarcoma parental and revertant cell lines. Furthermore, enzymatic activity assays using S-adenosyl-ʟ-[methyl-3H] methionine, recombinantly purified PRMT enzymes, cell lysates, and specific PRMT inhibitors revealed no major differences in radiolabeled polypeptides on SDS-PAGE gels. Taken together, these results suggest that changes in protein arginine methylation may not be major contributors to the Hoffman effect and that other consequences of methionine addiction may be more important in the metastasis and malignancy of osteosarcoma and potentially other cancers., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Y.A., Y.K., and R.M.H. are or were unsalaried associates of AntiCancer, Inc. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright: © 2023 Holtz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background/aim: The fundamental and general hallmark of cancer cells, methionine addiction, termed the Hoffman effect, is due to overuse of methionine for highly-increased transmethylation reactions. In the present study, we tested if the combination efficacy of recombinant methioninase (rMETase) and a methionine analogue, ethionine, could eradicate osteosarcoma cells and down-regulate the expression of c-MYC., Materials and Methods: 143B osteosarcoma cells and Hs27 normal human fibroblasts were tested. The efficacy of rMETase alone and ethionine, alone and in their combination, on cell viability was determined with the WST-8 assay on 143B cells and Hs27 cells. c-MYC expression was examined with western immunoblotting and compared in 143B cells treated with/without rMETase, ethionine, or the combination of both rMETase and ethionine., Results: 143B cells were more sensitive to both rMETase and ethionine than Hs 27 cells, with the following IC 50 s: rMETase (143B: 0.22 U/ml; Hs27: 0.82 U/ml); ethionine (143B: 0.24 mg/ml; Hs27: 0.42 mg/ml). The combination of rMETase and ethionine synergistically eradicated 143B cells, lowering the IC50 for ethionine 14-fold compared to ethionine alone (p<0.001). In contrast, Hs27 fibroblasts were relatively resistant to the combination. The expression of c-MYC was significantly down-regulated only by the combination of rMETase and ethionine in 143B cells (p<0.001)., Conclusion: In the present study, we showed, for the first time, the synergistic combination efficacy of rMETase and ethionine on osteosarcoma cells in contrast to normal fibroblasts, which were relatively resistant. The combination of rMETase and ethionine down-regulated c-MYC expression in the cancer cells. The present results indicate the combination of rMETase and ethionine may reduce the malignancy of osteosarcoma cells and can be a potential future clinical strategy., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Regorafenib is a multi-kinase inhibitor, targeting vascular endothelial growth factor receptor 2, fibroblast growth factor receptor 1 and other oncogenic kinases. Regorafenib has efficacy in metastatic colon cancer, but has severe dose-limiting toxicities which cause patients to stop taking the drug. The aim of the present study was to determine if recombinant methioninase (rMETase) could lower the effective concentration of regorafenib in vitro against a colorectal-cancer cell line., Materials and Methods: Firstly, we examined the half-maximal inhibitory concentration (IC 50 ) of regorafenib alone and rMETase alone for the HCT-116 human colorectal-cancer cell line. After that, using the IC 50 concentration of each drug, we investigated the efficacy of the combination of regorafenib and rMETase., Results: While both methioninase alone (IC 50 =0.61 U/ml) and regorafenib alone (IC 50 =2.26 U/ml) inhibited the viability of HCT-116 cells, the combination of the two agents was more than twice as effective as either alone. Addition of rMETase at 0.61 U/ml lowered the IC 50 of regorafenib from 2.26 μM to 1.46 μM., Conclusion: rMETase and regorafenib are synergistic, giving rise to the possibility of lowering the effective dose of regorafenib in patients, thereby reducing its severe toxicity, allowing more cancer patients to be treated with regorafenib., Competing Interests: The Authors declare no competing interests regarding this work., (Copyright 2023, International Institute of Anticancer Research.)

Background/aim: Methionine restriction by diet and recombinant methioninase (rMETase) are effective for cancer therapy by themselves or combined with chemotherapy drugs. We previously showed that oral administration of rMETase-producing Escherichia coli JM109 (E. coli JM109-rMETase) can be installed in the mouse microbiome and inhibit colon-cancer growth in a syngeneic mouse model. In the present report, we investigated the efficacy of oral administration of E. coli JM109-rMETase in an orthotopic triple-negative breast cancer (TNBC) cell-line mouse model., Materials and Methods: First, we established orthotopic 4T1 mouse triple-negative breast cancer on an abdominal mammary gland in female athymic nu/nu nude mice aged 4-6 weeks. After tumor growth, 15 mice were divided into three groups of 5. Group 1 was administered phosphate-buffered saline (PBS) orally by gavage twice daily as a control; Group 2 was administered non-recombinant E. coli JM109 competent cells orally by gavage twice daily as a control; Group 3 was administered E. coli JM109-rMETase cells by gavage twice daily for two weeks. Tumor size was measured with calipers twice per week. On day 15, blood methionine level was examined using an HPLC method., Results: Oral administration of E. coli JM109-rMETase inhibited 4T1 TNBC growth significantly compared to the PBS and E. coli JM109 control groups. On day 15, the blood methionine level was significantly lower in the mice administered E. coli JM109-rMETase than in the PBS control., Conclusion: E. coli JM109-rMETase lowered blood methionine levels and inhibited TNBC growth in an orthotopic cell-line mouse model, suggesting future clinical potential against a highly recalcitrant cancer., Competing Interests: The Authors declare no competing interests regarding this work., (Copyright 2023, International Institute of Anticancer Research.)

Fasting has been grown in popularity with multiple potential benefits. However, very few studies dynamically monitor physiological and pathological changes during long-term fasting using noninvasive methods. In the present study, we recruited 37 individuals with metabolic syndrome to engage in a 5-day water-only fasting regimen, and simultaneously captured the molecular alterations through urinary proteomics and metabolomics. Our findings reveal that water-only fasting significantly mitigated metabolic syndrome-related risk markers, such as body weight, body mass index, abdominal circumference, blood pressure, and fasting blood glucose levels in metabolic syndrome patients. Indicators of liver and renal function remained within the normal range, with the exception of uric acid. Notably, inflammatory response was inhibited during the water-only fasting period, as evidenced by a decrease in the human monocyte differentiation antigen CD14. Intriguingly, glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation underwent a sex-dependent reprogramming throughout the fasting period, whereby males exhibited a greater upregulation of carbohydrate metabolism-related enzymes than females. This disparity may be attributed to evolutionary pressures. Collectively, our study sheds light on the beneficial physiological effects and novel dynamic molecular features associated with fasting in individuals with metabolic syndrome using noninvasive methods., Competing Interests: The authors declare that they have no conflicts of interest., (© 2023 The Authors. MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.)

Introduction: Colorectal cancer (CRC) patients often develop liver metastasis. However, curative resection of liver metastasis is not always possible due to poor visualization of tumor margins. The present study reports the characterization of a humanized anti-carcinoembryonic antigen monoclonal antibody conjugated to a PEGylated near-infrared dye, that targets and brightly labels human CRC tumors in metastatic orthotopic mouse models., Methods: The hT84.66-M5A (M5A) monoclonal antibody was conjugated with a polyethylene glycol (PEG) chain that incorporated a near infrared (NIR) IR800 dye to establish M5A-IR800 Sidewinder (M5A-IR800-SW). Nude mice with CRC orthotopic primary tumors and liver metastasis both developed from a human CRC cell line, were injected with M5A-IR800-SW and imaged with the Pearl Trilogy Imaging System., Results: M5A-IR800-SW targeted and brightly labeled CRC tumors, both in primary-tumor and liver-metastasis models. M5A-IR800-SW at 75 μg exhibited highly-specific tumor labeling in a primary-tumor orthotopic model with a median tumor-to-background ratio of 9.77 and in a liver-metastasis orthotopic model with a median tumor-to-background ratio of 7.23 at 96 h. The precise labeling of the liver metastasis was due to lack of hepatic accumulation of M5A-IR800-SW in the liver., Conclusions: M5A-IR800-SW provided bright and targeted NIR images of human CRC in orthotopic primary-tumor and liver-metastasis mouse models. The results of the present study suggest the clinical potential of M5A-IR800-SW for fluorescence-guided surgery including metastasectomies for CRC. The lack of hepatic NIR signal is of critical importance to allow for precise labeling of liver tumors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Finding an effective drug for individual cancer patients among the many chemotherapies available and ruling out ineffective drugs are important challenges, especially for patients with advanced cancer. To accomplish this goal, we have pioneered and developed the patient-derived orthotopic xenograft (PDOX) nude mouse model for all cancer types, enabling the discovery and evaluation of novel therapeutics, as well as individualized therapy of patients with cancer. PDOX models can more precisely reproduce the original tumor microenvironment (TME) compared to subcutaneous-implanted xenografts including patient-derived xenograft (PDX) models. The present review presents the concordance of drug resistance in individual cancer patients and their PDOX models. There are 28 PDOX publications with 12 PDOX models from patients who were treated with chemotherapy. Sixteen chemotherapeutics were administrated to these patients and all of them were clinically ineffective. In PDOX models established from these patients' tumors, fourteen chemotherapeutics were resistant with a concordance rate of 88%. PDOX models should be established as early as possible from patients to predict and improve outcome. PDOX models mimic the clinical tumor aggressiveness, therefore enabling a high concordance with clinical outcomes. The present review shows a high concordance for drug resistance between cancer patients and their corresponding PDOX models. Future studies will include prospective clinical trials comparing both drug efficacy and resistance in patients and their PDOX models., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The choice of chemotherapy agents for RAS-mutant colorectal cancer is limited, and prognosis is poor compared to RAS-wild-type colorectal cancer. The purpose of the present study was to evaluate the effectiveness of methionine restriction combined with chemotherapy in a patient with NRAS-mutant rectal cancer., Patients and Methods: A 59-year-old female was diagnosed with lung-metastatic recurrence of NRAS-mutant rectal cancer two and a half years after resection of the primary tumor. She started chemotherapy, which consisted of fluorouracil, irinotecan (FOLFIRI), and bevacizumab, in October 2020. Eight months later, stereotactic body radiation therapy (SBRT) was performed to treat the lung metastases. She stopped chemotherapy at this point and had blood tests and computed tomography (CT) scans regularly. Her CEA level increased to 139.91 ng/ml and her lung metastasis became larger by September 2022. Therefore, she was reintroduced to FOLFIRI and bevacizumab in October 2022, and also started a low-methionine diet and oral recombinant methioninase (o-rMETase) as a supplement., Results: After starting the combination therapy with o-rMETase, a low-methionine diet, FOLFIRI, and bevacizumab, blood CEA levels very rapidly decreased and were almost within the normal limits five months later. CT findings showed the lung metastasis did not grow., Conclusion: Methionine restriction comprising o-rMETase and a low-methionine diet combined with first-line chemotherapy was effective in a patient with NRAS-mutant rectal cancer in which metastasis had re-occurred after first-line chemotherapy alone., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

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/G 2 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., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)