Yasunori Tome

Osteosarcoma is the most common malignant bone tumor in pediatric and young adult patients. Osteosarcoma is also refractory to immune checkpoint inhibitors (ICIs). It has been recently demonstrated that methionine restriction (MR) increases the response to ICIs in melanoma and colon cancer. The present study aimed to determine whether MR alone can be an immunotherapeutic for osteosarcoma. K7M2 murine osteosarcoma cells and 143B human osteosarcoma cells were used for the present study. Cell viability and the half-maximal effective concentration (EC₅₀) of methionine for K7M2 and 143B were determined with the WST-8 cell-viability reagent. Western immunoblotting was used to compare programmed cell death receptor ligand 1 (PD-L1) expression in K7M2 and 143B cells treated with and without MR. K7M2 cells were subcutaneously implanted in immunocompetent BALB/c mice and T-cell-deficient nude (nu/nu) mice to determine the efficacy of an MR diet on tumor growth and enhancing CD8-positive T-cell tumor infiltration in BALB/c mice. Tumor-infiltrating lymphocytes in the tumor of BALB/c mice were determined with immunohistochemistry. The EC₅₀ values of methionine for K7M2 and 143B were 14.18 μM and 20.85 μM, respectively. Both cell lines had a strong dependence on methionine at the concentration range of 4 to 32 μM. MR using methionine-depleted medium in vitro decreased PD-L1 expression in 143B and K7M2, compared to untreated control cells (ppp<0.05). MR alone is a potential immunotherapeutic for osteosarcoma. The present results suggest MR is a T-cell stimulant and not a cause of T-cell exhaustion.

Background/Aim: Positron emission tomography/computed tomography with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG PET/CT) is frequently used to differentiate schwannomas from malignant peripheral nerve sheath tumors. Schwannomas exhibit pathological heterogeneity, with highly cellular (Antoni A) and hypocellular (Antoni B) areas, but current PET/CT methods do not adequately reflect this heterogeneity. This study aimed to compare imaging characteristics of schwannomas in the trunk versus the extremities, with emphasis on metabolic heterogeneity. Patients and Methods: This retrospective study included patients with solitary schwannomas who underwent MRI and ¹⁸F-FDG PET/CT before surgical excision (June 2013-September 2023). Exclusion criteria were plexiform, multiple, biopsy-only lesions, and tumors originating from internal organs. Tumors were classified as trunk or extremity lesions. MRI was used to determine size and volume, while PET/CT measured SUV_max, SUV_mean, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). Heterogeneity was assessed using three indices: MTV-to-volume ratio (MTV/volume), SUV-based heterogeneity index (HI_SUV), and metabolic region-adjusted SUV-based heterogeneity index (MRA-HI_SUV). Results: Fifty-six patients were included. Trunk schwannomas were larger than extremity tumors in diameter (4.33 cm vs. 2.77 cm; p3vs. 6.25 cm³; pmax (4.09 vs. 3.71) and SUV_mean (2.47 vs. 2.22) did not differ significantly. MTV (18.43 cm³vs. 6.19 cm³, pvs. 14.40, pvs. 1.12, pSUV and MRA-HI_SUV were higher in trunk schwannomas (1.79 vs. 1.65 and 2.36 vs. 1.49, pSUV may enhance the characterization of schwannoma heterogeneity.

Background/Aim: Tumor immunology and immunotherapy have been intensely researched recently, especially with the development of immune checkpoint inhibitors (ICIs). However, only a small percentage of patients respond to ICIs, and this response is limited to a minority of cancer types, such as colon cancer, lung cancer, and melanoma. The aim of the present study was to develop an imageable in-vivo-like in vitro system to directly visualize tumor immunology and immunotherapy in real-time, to further understand tumor immunology and develop improved tumor immunotherapy. Materials and Methods: Lewis lung carcinoma cells labeled with red fluorescent protein (LLC-RFP) and peripheral blood mononuclear cells labeled with green fluorescent protein (PBMCs-GFP), derived from transgenic GFP mice, were cultured in two dimensions (2-D) on plastic or in three dimensions (3-D) in Gelfoam^® histoculture, using RPMI-1640 culture medium with 10% fetal bovine serum, and 1% penicillin/streptomycin, and with concanavalin-A. Results: LLC-RFP cells seeded on Gelfoam^® formed glandular-like structures after 2 days of histoculture. PBMCs-GFP were seeded on Gelfoam^® with LLC-RFP after one day to establish a co-culture. The PBMCs-GFP co-localized with many of the structures formed by the LLC-RFP cells after one day of seeding PBMCs-GFP, suggesting a very specific interaction necessary for an immunological interaction. In contrast, only a few randomized co-localizations of PBMCs-GFP and the LLC-RFP cells were seen on plastic in 2-D culture. Conclusion: The present results demonstrate a new 3-D co-culture color-coded system to visualize in real-time the interaction of PBMCs and cancer cells using fluorescence color-coded imaging. Future experiments will test drugs that can stimulate immune reactions between the PBMCs and cancer cells.

Background/Aim: Drug-resistance in osteosarcoma results in a very poor clinical prognosis and has been a recalcitrant problem over many decades. We have previously reported the development of super methotrexate (MTX)-resistant osteosarcoma cells (143B-MTX^SR), selected from parental 143B osteosarcoma cells (143B-P) 143B-MTX^SR cells were previously selected by culturing the cells with increasing concentrations of MTX, resulting in osteosarcoma cells which are 5,500 times more MTX-resistant than the parental cells, due to extreme over-expression of dihydrofolate reductase (DHFR). In the present study, the potential therapeutic efficacy of methionine restriction, using recombinant methioninase (rMETase), was explored to overcome super MTX-resistant osteosarcoma cells. Materials and Methods: Previously-selected 143B-MTX^SR cells were used for the present study. Sensitivity to methionine restriction by rMETase was determined using the WST-8 assay and compared between 143B-MTX^SR and parental 143B-P cells. Results: 143B-MTX^SR cells (rMETase IC₅₀: 0.38 U/ml) were very sensitive to methionine restriction by rMETase, very similar to 143B-P (rMETase IC₅₀: 0.36 U/ml). Conclusion: rMETase overcame a 5,500-fold MTX-resistance of osteosarcoma cells. The present results suggest methionine restriction by rMETase can be a potential clinical strategy to overcome recalcitrant drug-resistance in osteosarcoma.

Chemotherapy resistance in osteosarcoma results in a very poor patient prognosis, with the 5-year survival rate of approximately 20%, which not improved for over three decades; thus, the development of novel therapeutic strategies is required. Methionine addiction is a fundamental and general hallmark of cancer, termed the Hoffman effect. Cancer cells need larger amounts of exogenous methionine in order to grow compared to normal cells, despite their ability to synthesize normal or greater amounts of methionine from homocysteine, due to increased transmethylation reactions in cancer cells. Methionine restriction therapy, including recombinant methioninase (rMETase), arrests cancer cells in the late-S/G2 phase of the cell cycle by targeting methionine addiction. First-line chemotherapy for osteosarcoma, including methotrexate (MTX), doxorubicin (DOX), and cisplatinum (CDDP), targets cells in the S/G2-phase, where cancer cells are also inhibited by methionine restriction, resulting in the synergy of methionine restriction to overcome drug resistance. In the present review, we describe the synergistic efficacy of conventional chemotherapy and methionine restriction therapy, including rMETase, in overcoming the drug resistance of osteosarcoma. The clinical potential of this new paradigm to overcome the drug resistance of osteosarcoma is discussed.

Background/Aim: The present study aimed to assess the relationship between the maximum standardized uptake value (SUVmax) on ¹⁸F-fluorodeoxyglucose positron emission tomography computed tomography (¹⁸F-FDG-PET/CT) and the geriatric nutritional risk index (GNRI) in patients with soft-tissue sarcomas (STSs). Patients and Methods: The present single-center retrospective observational study included patients who underwent ¹⁸F-FDG-PET/CT and for whom serum albumin levels, height, and body weight were measured prior to therapeutic intervention. Results: A total of 81 patients were included in the study. The mean SUVmax was 11.1±9.9. The 5-year overall survival (OS) and disease-free survival (DFS) rates were 79.7% and 52.4%, respectively, for the higher SUVmax group (≥11.1) and 91.0% and 73.0%, respectively, for the lower SUVmax group (<11.1). For the GNRI, the 5-year OS and DFS rates were 89.6% and 64.3%, respectively, for the negative-risk group, and 73.3% and 77.1% for the positive-risk group, respectively, with no significant differences. The mean SUVmax was 9.7±8.1 and 19.1±14.9 for the negative- and positive-risk groups, respectively. The positive-risk group had a significantly higher SUVmax than the negative-risk group (p=0.03). Furthermore, there was a negative correlation between the SUVmax and GNRI (r=−0.48, p<0.05). Conclusion: A higher SUVmax and lower GNRI in patients with STS may contribute to a poor prognosis. The deregulatory elevation of tumor glucose metabolic activity may affect serum albumin levels and weight loss in patients with STS, resulting in a decrease in the GNRI.

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.

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 (IC50) 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 IC50s for PC-3 was determined. Results: The IC50 of docetaxel for PC-3 and for Hs27 was 0.72 nM and 0.94 nM, respectively. The IC50 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.

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 (IC₅₀) value of rMETase on HCT-116 human colorectal cancer cells. HCT-116 cells were treated with rMETase at the IC₅₀. Western immunoblotting was used to compare PD-L1 expression in HCT-116 cells treated with and without rMETase. Results: The IC₅₀ 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.

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 IC₅₀. 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 IC₅₀ compared to the parental 143B-P cells. Expression of DHFR was increased 10-fold in 143B-MTX^SR compared to 143B-P (pSR cells had reduced colony-formation capacity on plastic (p=0.032) and in soft agar (pSR 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.