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. 2026 Feb 27;40(2):813–825. doi: 10.21873/invivo.14239

Methionine Restriction Alone Induces T-cell-mediated Immunotherapy of Osteosarcoma in a Syngeneic Mouse Model

KOHEI MIZUTA 1,2,3, YUTARO KUBOTA 1,2, YUSUKE AOKI 1,2,3, SEI MORINAGA 1,2, YOHEI ASANO 1,2, YUTA MIYASHI 1,2,3, MICHAEL BOUVET 2, YASUNORI TOME 3, KOTARO NISHIDA 3, ROBERT M HOFFMAN 1,2
PMCID: PMC12949883  PMID: 41760305

Abstract

Background/Aim

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.

Materials and Methods

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.

Results

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 (p<0.05, respectively). The MR diet significantly suppressed the growth of K7M2 tumors in immunocompetent BALB/c mice (p<0.05), but not in T-cell-deficient nu/nu mice. The MR diet enhanced CD8-positive T-cell infiltration in the K7M2 tumor growing in BALB/c mice (p<0.05).

Conclusion

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.

Keywords: Osteosarcoma, methionine addiction, Hoffman effect methionine restriction, T-cell infiltration, immunotherapy, tumor microenvironment, BALB/c mouse, nude mouse

Introduction

Osteosarcoma is a common malignant bone tumor and occurs in adolescents and young adults (1,2). Advances in chemotherapy and surgery for osteosarcoma, which occurred decades ago, have resulted in a 60-80% five-year survival rate for patients with no metastases at diagnosis (2-6). However, 18-37% of patients with osteosarcoma have metastases at diagnosis. The 5-year survival rate for patients with metastatic osteosarcoma is less than 30%, which has not improved over these decades (2,7,8).

Programmed cell death receptor ligand 1 (PD-L1) on cancer cells binds to programmed cell death 1 (PD-1) on T-cells and leads to the avoidance of immune responses. PD-L1 expression is a predictive biomarker for response to PD-1/PD-L1-targeted immune-checkpoint-inhibitor (ICI) therapy (9,10). ICIs are used clinically for patients with various cancers such as melanoma with efficacy of 33-40%; colon cancer with efficacy of 31%; lung cancer with efficacy of 45%; and renal-cell carcinoma with efficacy of 25% (11-17). However, ICIs for sarcoma patients are ineffective (18-19).

Methionine addiction is a fundamental and general hallmark of cancer, termed the Hoffman effect (20-24). Methionine restriction (MR) was first shown to improve the efficacy of chemotherapy drugs on cancer cells 40 years ago (25). It has been recently demonstrated that MR increases the response to immunotherapeutic drugs in various mouse models of various type of cancer (26-30). MR can also inhibit obesity and fatty liver (31-33). The aim of the present study was to determine if MR alone is an immunotherapeutic for osteosarcoma, as a proof-of-principle.

Materials and Methods

Cell lines and culture. The K7M2 murine osteosarcoma cell line and 143B human osteosarcoma cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). K7M2 and 143B cells were grown in Dulbecco’s Modified Eagle Medium (DMEM)/Nutrient Mixture F-12 with GlutaMAX™ supplement (GIBCO, Grand Island, NY, USA), 10% fetal bovine serum (GIBCO), and 100 IU/ml penicillin/ streptomycin (GIBCO). The methionine-free medium was made with DMEM without methionine, cystine, and glutamine (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% dialyzed fetal bovine serum, 100 IU/ml of penicillin/ streptomycin (GIBCO), 4 mM L-glutamine (GIBCO), and 100 µM L-cystine 2HCl (Sigma-Aldrich, St. Louis, MO, USA).

Determination of the efficacy of the methionine concentration on K7M2 and 143B proliferation. The proliferation of K7M2 and 143B cells as a function of the methionine concentration in vitro was determined using the WST-8 cell-viability reagent (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan). The cells (1.0×103 cells/well) were seeded in 96-well plates in normal medium, incubated overnight at 37°C with 5% CO2. Cells were rinsed with phosphate-buffered saline (PBS) (BioPioneer Inc., San Diego, CA, USA) and treated with varying concentrations of L-methionine (Sigma-Aldrich) ranging from 0 to 128µM for 96 hours at 37°C with 5% CO2. Following methionine treatment, 10 μl WST-8 solution was added to each well, and the cells were incubated for one hour. The resulting absorbance was measured using a microplate reader (Sunrise; Tecan, Männedorf, Switzerland) at 450 nm. Graghpad Prism 10.0.4 (GraphPad Software Inc., San Diego, CA, USA) was used to create dose-response curves and determine half-maximal effective concentration (EC50) values. Experiments were repeated three times, in triplicate.

Western immunoblotting. The cells (K7M2; 0.5×106 cells/dish, 143B; 1.0×106 cells/dish) were seeded in 100 mm2 dishes in normal medium and incubated overnight at 37°C with 5% CO2. PBS was used to rinse the dishes once, and the medium was changed to the methionine-free medium supplemented with 128 µM methionine as a control or the EC50 value for methionine of K7M2 and 143B for methionine restriction. Cells were incubated at 37°C with 5% CO2 for 96 h. Following cell lysis, proteins were extracted using RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) and 1% Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). After electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, proteins on the gels were transferred to 0.45 μm polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL, USA). Bullet Blocking One for Western Blotting (Nakalai Tesque Inc., Kyoto, Kyoto, Japan) was used to block the membranes. Antibodies against PD-L1 (17952-1-AP, 1:500; Proteintech, Rosemont, IL, USA) and GAPDH (60004-1-Ig, 1:25,000; Proteintech) were utilized. A loading control was applied using GAPDH. Horseradish-peroxidase-conjugated anti-rabbit IgG (SA00001-2, 1:5,000; Proteintech) and anti-mouse IgG (SA00001-1, 1:5,000; Proteintech) were utilized as secondary antibodies. To qualitatively visualize immunoreactivity, the signals were detected with the UVP ChemStudio (Analytik Jena, Jena, Germany) after application of the Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). Quantitative comparisons of band expression were performed using ImageJ ver. 1.53t (National Institutes of Health, Bethesda, MD, USA). Experiments were performed using three independent samples.

Mice. Immunocompetent BALB/c mice aged 6-8 weeks and athymic T-cell-deficient nude (nu/nu) mice aged 6-8 weeks (AntiCancer Inc., San Diego, CA, USA) were used in the present study. All mice were bred in a barrier facility with a high efficacy particulate air (HEPA) filter and 12-h light/dark cycles. The present mouse study was approved by the AntiCancer Inc. Institutional Animal Care and Use Committee protocol outlined in the National Institutes of Health Guide for the Care and Use of Animals.

Dietary methionine restriction in BALB/c and in nude mice implanted with K7M2 cells. K7M2 cells (1.0×106 cells / 100 µl PBS) were injected subcutaneously into the nude-mouse or BALB/c mouse flank. After the tumor volume reached 50-100 mm3, the mice were divided into two groups (n=8 mice per group for BALB/c or n=10 mice per group for nude mice). A normal diet containing 0.65% methionine (Inotiv, West Lafayette, IN, USA) was switched to a methionine-restricted diet containing 0.12% methionine (Inotiv) for the MR group, which has been used in various mouse models and shown to significantly reduce the level of circulating methionine (31-35). Tumor length, width, and the mouse body weight were measured on Day 1, 8, and 15. The mice were sacrificed on day 15, and the tumors were dissected. Tumor volume was calculated with the following formula: Tumor volume (mm3)=length (mm)×width (mm)×width (mm)×1/2.

Immunohistochemical (IHC) identification of CD8α-positive T-cells in the osteosarcoma tumors in BALB/c mice. Fresh tumor samples from the dissected mice (5 mice in each group) on day 15 were fixed in 10% formalin 10% formalin (Thermo Fisher Scientific), dehydrated in an ethanol and Clear-Rite 3® (Thermo Fisher Scientific) series, and embedded in paraffin before sectioning and staining. Tissue sections (4 µm thick) were deparaffinized in Clear-Rite 3® and rehydrated in an ethanol series. We performed IHC staining using the ImmPRESS® HRP Horse Anti-rabbit IgG Polymer Detection Kit (Vector Laboratories, Newark, CA, USA) following the manufacturer’s procedural guidelines. Antigen retrieval was performed by incubating slides with tris-EDTA buffer (pH 9.0) at 96°C, then blocking with BLOXALL® Endogenous Blocking Solution (Vector Laboratories) protein-blocking reagent. Anti-CD8α antibody (#98941, 1:500; Cell Signaling Technology, Danvers, MA, USA) was used as the primary antibody and incubated overnight at 4°C. An HRP-conjugated secondary-antibody reagent was applied, and the sections were treated with ImmPACT® DAB EqV Substrate Kit (Vector Laboratories). The slides were counterstained with hematoxylin, dehydrated, and sealed with a mounting medium. CD8α-positive cells detected in tumor tissue were defined as tumor-infiltrating lymphocytes (TILs) at ×200 magnification. Five microscopic fields at ×200 magnification were selected, and the cells were counted one by one using ImageJ ver. 1.53t (National Institutes of Health, Bethesda, MD, USA). Their average was utilized as the TILs score.

Statistical analysis. Welch’s t-test was used for comparison between groups. Data are presented as mean±standard deviation. Statistical analyses were performed with GraphPad Prism 10.0.4 (GraphPad Software, Inc.). p<0.05 was considered as significant difference.

Results

Effect of methionine concentration on K7M2 and 143B osteosarcoma cell proliferation in vitro. Viability of K7M2 and 143B cells at varying concentrations of methionine in the medium was assessed over 96 h. The K7M2 and 143B cells showed a steep increase in cell number between 4 and 32 µM methionine, suggesting a strong dependence on methionine. The EC50 of methionine in K7M2 and 143B cells was 14.08 µM and 20.85 µM, respectively (Figure 1).

Figure 1.

Figure 1

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K7M2 murine osteosarcoma cells and 143B human osteosarcoma cells are highly dependent on methionine in vitro. The EC50 of methionine of K7M2 and 143B cells was determined by treating the cells with various concentrations of methionine for 96 h. Cell viability was measured using the WST-8 cell-viability reagent. Experiments were repeated three times, in triplicate. Data are shown as mean±standard deviation. The concentration axis is scaled in log2. EC50: Half-maximal effective concentration. Please see the Materials and Methods for details.

Methionine restriction decreases the expression of PD-L1 in K7M2 and 143B. The protein expression of PD-L1 was determined in K7M2 and K7M2 cells with western immunoblotting. K7M2 and 143B cells were treated at the EC50 of methionine, as MR for 96 h and compared to 128 µM methionine in DMEM. MR at the methionine EC50 decreased the expression of PD-L1 in K7M2 by 44.8%, and in 143B by 19.4% compared to untreated control cells in 128 μM methionine (p<0.05, respectively) (Figure 2).

Figure 2.

Figure 2

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Expression of PD-L1 in K7M2 and 143B cells decreased with methionine-restricted (MR) medium. (A) The expression of PD-L1 was measured in K7M2 and 143B cells treated with the EC50 of methionine as MR for 96 h. Western blotting was performed for analysis of PD-L1 in three independent samples. GAPDH was used as a loading control. (B) Quantitative comparisons of PD-L1 expression in 143B and K7M2 cells determined by western blot are shown as mean±standard deviation. EC50: Half-maximal effective concentration; MR: methionine restriction. *p<0.05. Please see the Materials and Methods for details.

Methionine restriction inhibited the growth of K7M2 osteosarcoma in BALB/c mice but not in nu/nu mice. The MR diet suppressed the growth of K7M2 tumors in BALB/c mice. The tumor-volume ratio at day 15 relative to day 1 decreased by 36.9% in MR compared with control. The tumor volume at day 15 decreased by 41.3% in MR compared to control (p<0.05) (Figure 3A-C). However, MR did not inhibit the growth of K7M2 tumors in nude mice (Figure 4A-C). Spaghetti plots strongly distinguished tumor growth in MR and control BALB/c mice but not in nude mice (Figure 5A and B). In both BALB/c mice and nude mice, no adverse weight loss was observed following MR diet treatment. MR resulted in a slight, but significant, weight loss in BALB/c mice. Nude mice on the MR diet did not have a significant weight loss. (Figure 6A and B).

Figure 3.

Figure 3

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A methionine-restricted (MR) diet inhibited the growth of K7M2 tumors in immunocompetent BALB/c mice. K7M2 cells (1.0×106 cells/100μl PBS) were injected subcutaneously into the BALB/c mouse flank. The BALB/c mice (n=8) were treated with a normal diet containing 0.65% methionine or an MR diet containing 0.12% methionine. Tumor volume and body weight were measured. (A) Time course of the tumor-growth ratio in BALB/c mice compared to day 1 on normal and MR diets. (B) Tumor-volume ratio on day 15 was compared to day 1 in BALB/c mice on normal and MR diets. (C) Tumor volume on day 15 in BALB/c mice on normal and MR diets. MR: Methionine restriction. Data are shown as mean±standard deviation. *p<0.05. Please see the Materials and Methods for details.

Figure 4.

Figure 4

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methionine-restricted (MR) diet did not inhibits the growth of the K7M2 tumors in T-cell-deficient nude mice. K7M2 cells (1.0×106 cells/100μl PBS) were injected subcutaneously into the nude-mouse flank. The nude-mice (n=10) were treated with a normal diet containing 0.65% methionine or an MR diet containing 0.12% methionine. Tumor volume and body weight were measured. (A) Time course of tumor-growth ratio in nude mice compared to day 1. (B) Tumor-volume ratio on day 15 was compared to day 1. (C) Tumor volume on day 15 in nude mice. MR: methionine restriction. Data are shown as mean±standard deviation. Please see the Materials and Methods for details.

Figure 5.

Figure 5

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Time course of K7M2 tumor growth as absolute volume in spaghetti plots in BALB/c or nude mice on normal or MR diets. (A) Individual-mouse time course of tumor growth as absolute volume in BALB/c mice. (B) Individual mouse time course of tumor growth as absolute volume in nude mice. MR: Methionine restriction. Data are shown as mean±standard deviation. Please see the Materials and Methods for details.

Figure 6.

Figure 6

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No adverse weight loss was observed following methionine-restriction (MR)-diet treatment in BALB/c and nude mice with implanted osteosarcoma. (A) Time course of the body-weight ratio of K7M2 tumor-bearing BALB/c mice treated with MR or control diets. (B) Time course of the body-weight ratio in K7M2 tumor-bearing nude mice treated with MR or control diets. Data are shown as mean±standard deviation. *p<0.05. Please see the Materials and Methods for details.

MR diet enhanced infiltration of CD8-positive T-cells into K7M2 osteosarcoma tumors in BALB/c mice. The MR diet enhanced CD8-positive T-cell TIL infiltration in the K7M2 tumor growing in BALB/c mice 1.6 fold (p<0.05) (Figure 7).

Figure 7.

Figure 7

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A methionine-restricted (MR) diet enhanced infiltration of CD8-positive cells in the K7M2 osteosarcoma in BALB/c mice. After sacrificing the mice, the tumor tissues were dissected. The tumor tissues were stained brown using an anti-CD8α antibody for immunohistochemistry. (A) Representative immunohistochemistry images of CD8α-positive cells in the control-diet and methionine-restriction (MR-diet) group at ×200 magnification. (B) CD8-positive T-cells detected in tumor tissue were defined as tumor-infiltrating lymphocytes (TILs). MR: Methionine restriction; TILs: tumor-infiltrating lymphocytes. Data are shown as mean±standard deviation. *p<0.05. Please see the Materials and Methods for details.

Discussion

The present study showed that K7M2 mouse osteosarcoma cells and 143B human osteosarcoma cells were strongly dependent on the methionine concentration in vitro. MR medium decreased the expression of PD-L1 in vitro. An MR diet suppressed the growth of K7M2 tumors in BALB/c mice but not in nude mice demonstrating T-cells mediated the efficacy of MR. The MR diet enhanced infiltration of CD8-positive T-cell in the K7M2 tumors in BALB/c mice. These results support the concept that MR alone can enhance T-cell mediated antitumor immunity against osteosarcoma, and that immunotherapy of osteosarcoma with MR is feasible. Thus, MR itself has potential as an immunotherapeutic.

Cancer cells are addicted to methionine, called the Hoffman effect (20-24), and MR selectively reduces tumor growth. In a previous study treating mice with a 0.12% MR diet, circulating methionine was significantly reduced, and the MR diet inhibited tumor growth in colorectal-cancer patient-derived xenograft (PDX) models (34,35). It has also been reported that MR diet is not toxic on immune cells in mice for up to 8 weeks (35).

Several reports have described PD-L1 expression in cells treated with MR medium in vitro. Li et al. demonstrated reduced PD-L1 expression in human colorectal-cancer cell lines treated with 10 µM methionine for 12 h compared to cells treated with 200 µM methionine (27). Morehead et al. reported that treating human colorectal-cancer cell lines with 5 μM methionine for 24 h increased PD-L1 expression compared to cells treated with 200 μM methionine (26). Zhou et al. reported that treating mouse melanoma cells with 0 µM methionine for 3 days resulted in increased PD-L1 expression compared to cells treated with 100 µM (30). In the present study, mouse and human osteosarcoma cells were treated for four days at their respective methionine EC50 concentrations, and PD-L1 expression decreased compared to cells treated with 128 µM methionine. The differences in culture concentration of methionine, duration, and cancer type may have influenced these results and, therefore, further investigation is needed.

MR has previously been found to enhance tumor immunotherapy using a variety of methods and cancer-cell types. Previous reports have suggested that MR with a low-methionine diet improved the response of colon cancer to ICIs (26-29). Morehead et al. reported that an MR diet increased CD8-positive-cell infiltration into colorectal cancer tumors, and enhanced the efficacy to ICIs in a mouse colorectal-cancer model (26). MR reduced S-adenosylmethionine derived from methionine, resulting in reduced N6-methyladenosine (m6A) affecting immune checkpoints in a colorectal-cancer mouse model (27). Fang et al. demonstrated that MR increased antitumor immunity by enhancing cyclic GMP-AMP synthase (cGAS) activation and chromatin untethering through demethylation in colorectal-cancer and melanoma mouse models (28). Xue et al. showed intermittent MR accelerated ferroptosis by stimulating cation transport regulator homolog 1 (CHAC1) transcription thereby increasing the immune response to cancer cells and CD8-positive TIL cytotoxicity in a melanoma mouse model (29). Zhou et al. showed that recombinant Salmonella expressing an L-methioninase to degrade methionine, enhanced the efficacy of an ICI in a mouse model of melanoma (30). Only one study showed a negative effect of MR using a colon-cancer mouse model that was attributed to the inhibition of microbiota (36).

In osteosarcoma, poor response to ICIs has been reported in patients deficient in methylthioadenosine phosphorylase (MTAP). However, combination therapy with a methionine-restricted diet and ICIs has been shown to increase CD8-positive T-cell infiltration and to decrease growth of osteosarcoma tumors consisting of cells with knockdown of MTAP in mice (37). Thus, many studies have shown that MR increases the CD8-positive T-cell infiltration in tumors (26-30,37).

The present study showed that MR inhibited osteosarcoma cell proliferation in vitro. MR alone inhibited osteosarcoma growth in immunocompetent mice and enhanced CD8-positive T-cell infiltration in the osteosarcoma tumor, but did not inhibit growth in T-cell-deficient nude mice. The results demonstrate that the combination of a methionine-restricted diet and an intact immune system providing T-cells for tumor-infiltration were necessary to inhibit osteosarcoma growth.

Ultimately, these findings suggest that MR alone has potential as an immunotherapeutic and that immunotherapy of osteosarcoma is feasible. MR is effective because it targets methionine addiction, a fundamental and general hallmark of cancer (20-25, 38-71). Recombinant methioninase (rMETase) is showing clinical promise for cancer patients (67). The Hoffman effect appears stronger than the Warburg effect as shown by comparison of methionine-based and glucosebased PET imaging, respectively (68). The present results indicate MR stimulates T-cell infiltration in tumors and does not cause T-cell exhaustion as has been claimed (36, 69-71).

Conflicts of Interest

The 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.

Authors’ Contributions

KM, YT, and RMH designed experiments. KM and RMH wrote this article. KM, YA, and YM performed experiments. KM, YA, YM, and RMH analyzed and interpreted data. YK and YA provided technical support and conceptual advice. YK, YA, SM, YA, YM, MB, YT, and KN reviewed this article. All Authors contributed to the article and approved the submitted version.

Acknowledgements

This paper is dedicated to the memory of A. R. Moossa, MD; Professor Philip Miles; Sun Lee, MD; Richard Erbe, MD; Professor Milton Plesur; Professor Gordon H. Sato; Professor Li Jiaxi; Masaki Kitajima, MD; Shigeo Yagi, PhD; Jack Geller, MD; Joseph R. Bertino, MD; J.A.R. Mead PhD; Eugene P. Frenkel, MD; John Mendelsohn, MD; Professor I. J. Fidler; Professor Lev Bergelson; Professor John R. Raper; Professor J.D. Watson and Joseph Leighton, MD.

Funding

The Robert M. Hoffman Foundation for Cancer Research provided funding of this study.

Artificial Intelligence (AI) Disclosure

No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

References

  • 1.Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer. 2009;115(7):1531–1543. doi: 10.1002/cncr.24121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Durfee RA, Mohammed M, Luu HH. Review of osteosarcoma and current management. Rheumatol Ther. 2016;3(2):221–243. doi: 10.1007/s40744-016-0046-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bacci G, Longhi A, Versari M, Mercuri M, Briccoli A, Picci P. Prognostic factors for osteosarcoma of the extremity treated with neoadjuvant chemotherapy: 15-year experience in 789 patients treated at a single institution. Cancer. 2006;106(5):1154–1161. doi: 10.1002/cncr.21724. [DOI] [PubMed] [Google Scholar]
  • 4.Iwamoto Y, Tanaka K, Isu K, Kawai A, Tatezaki S, Ishii T, Kushida K, Beppu Y, Usui M, Tateishi A, Furuse K, Minamizaki T, Kawaguchi N, Yamawaki S. Multiinstitutional phase II study of neoadjuvant chemotherapy for osteosarcoma (NECO study) in Japan: NECO-93J and NECO-95J. J Orthop Sci. 2009;14(4):397–404. doi: 10.1007/s00776-009-1347-6. [DOI] [PubMed] [Google Scholar]
  • 5.Allison DC, Carney SC, Ahlmann ER, Hendifar A, Chawla S, Fedenko A, Angeles C, Menendez LR. A meta-analysis of osteosarcoma outcomes in the modern medical era. Sarcoma. 2012;2012:704872. doi: 10.1155/2012/704872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Saraf AJ, Fenger JM, Roberts RD. Osteosarcoma: accelerating progress makes for a hopeful future. Front Oncol. 2018;8:4. doi: 10.3389/fonc.2018.00004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Tsuchiya H, Kanazawa Y, Abdel-Wanis ME, Asada N, Abe S, Isu K, Sugita T, Tomita K. Effect of timing of pulmonary metastases identification on prognosis of patients with osteosarcoma: The Japanese Musculoskeletal Oncology Group Study. J Clin Oncol. 2002;20(16):3470–3477. doi: 10.1200/JCO.2002.11.028. [DOI] [PubMed] [Google Scholar]
  • 8.Vasquez L, Tarrillo F, Oscanoa M, Maza I, Geronimo J, Paredes G, Silva JM, Sialer L. Analysis of prognostic factors in high-grade osteosarcoma of the extremities in children: a 15-year single-institution experience. Front Oncol. 2016;6:22. doi: 10.3389/fonc.2016.00022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Blank C, Mackensen A. Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunol Immunother. 2007;56(5):739–745. doi: 10.1007/s00262-006-0272-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Patel SP, Kurzrock R. PD-L1 expression as a predictive biomarker in cancer immunotherapy. Mol Cancer Ther. 2015;14(4):847–856. doi: 10.1158/1535-7163.MCT-14-0983. [DOI] [PubMed] [Google Scholar]
  • 11.Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, Hassel JC, Rutkowski P, McNeil C, Kalinka-Warzocha E, Savage KJ, Hernberg MM, Lebbé C, Charles J, Mihalcioiu C, Chiarion-Sileni V, Mauch C, Cognetti F, Arance A, Schmidt H, Schadendorf D, Gogas H, Lundgren-Eriksson L, Horak C, Sharkey B, Waxman IM, Atkinson V, Ascierto PA. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015;372(4):320–330. doi: 10.1056/NEJMoa1412082. [DOI] [PubMed] [Google Scholar]
  • 12.Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, Daud A, Carlino MS, McNeil C, Lotem M, Larkin J, Lorigan P, Neyns B, Blank CU, Hamid O, Mateus C, Shapira-Frommer R, Kosh M, Zhou H, Ibrahim N, Ebbinghaus S, Ribas A, KEYNOTE-006 investigators Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med. 2015;372(26):2521–2532. doi: 10.1056/NEJMoa1503093. [DOI] [PubMed] [Google Scholar]
  • 13.Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, Tykodi SS, Sosman JA, Procopio G, Plimack ER, Castellano D, Choueiri TK, Gurney H, Donskov F, Bono P, Wagstaff J, Gauler TC, Ueda T, Tomita Y, Schutz FA, Kollmannsberger C, Larkin J, Ravaud A, Simon JS, Xu LA, Waxman IM, Sharma P, CheckMate 025 Investigators Nivolumab versus everolimus in advanced renal-cell carcinoma. N Engl J Med. 2015;373(19):1803–1813. doi: 10.1056/NEJMoa1510665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Reck M, Rodríguez-Abreu D, Robinson AG, Hui R, Csőszi T, Fülöp A, Gottfried M, Peled N, Tafreshi A, Cuffe S, O'Brien M, Rao S, Hotta K, Leiby MA, Lubiniecki GM, Shentu Y, Rangwala R, Brahmer JR, KEYNOTE-024 Investigators Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med. 2016;375(19):1823–1833. doi: 10.1056/NEJMoa1606774. [DOI] [PubMed] [Google Scholar]
  • 15.De Velasco G, Je Y, Bossé D, Awad MM, Ott PA, Moreira RB, Schutz F, Bellmunt J, Sonpavde GP, Hodi FS, Choueiri TK. Comprehensive meta-analysis of key immune-related adverse events from CTLA-4 and PD-1/PD-L1 inhibitors in cancer patients. Cancer Immunol Res. 2017;5(4):312–318. doi: 10.1158/2326-6066.CIR-16-0237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz HJ, Morse MA, Desai J, Hill A, Axelson M, Moss RA, Goldberg MV, Cao ZA, Ledeine JM, Maglinte GA, Kopetz S, André T. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol. 2017;18(9):1182–1191. doi: 10.1016/S1470-2045(17)30422-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Davis AA, Patel VG. The role of PD-L1 expression as a predictive biomarker: an analysis of all US Food and Drug Administration (FDA) approvals of immune checkpoint inhibitors. J Immunother Cancer. 2019;7(1):278. doi: 10.1186/s40425-019-0768-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Tawbi HA, Burgess M, Bolejack V, Van Tine BA, Schuetze SM, Hu J, D'Angelo S, Attia S, Riedel RF, Priebat DA, Movva S, Davis LE, Okuno SH, Reed DR, Crowley J, Butterfield LH, Salazar R, Rodriguez-Canales J, Lazar AJ, Wistuba II, Baker LH, Maki RG, Reinke D, Patel S. Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): a multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol. 2017;18(11):1493–1501. doi: 10.1016/S1470-2045(17)30624-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Le Cesne A, Marec-Berard P, Blay JY, Gaspar N, Bertucci F, Penel N, Bompas E, Cousin S, Toulmonde M, Bessede A, Fridman WH, Sautes-Fridman C, Kind M, Le Loarer F, Pulido M, Italiano A. Programmed cell death 1 (PD-1) targeting in patients with advanced osteosarcomas: results from the PEMBROSARC study. Eur J Cancer. 2019;119:151–157. doi: 10.1016/j.ejca.2019.07.018. [DOI] [PubMed] [Google Scholar]
  • 20.Hoffman RM, Erbe RW. High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine. Proc Natl Acad Sci U S A. 1976;73(5):1523–1527. doi: 10.1073/pnas.73.5.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Coalson DW, Mecham JO, Stern PH, Hoffman RM. Reduced availability of endogenously synthesized methionine for S-adenosylmethionine formation in methionine-dependent cancer cells. Proc Natl Acad Sci U S A. 1982;79(14):4248–4251. doi: 10.1073/pnas.79.14.4248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Stern PH, Mecham JO, Wallace CD, Hoffman RM. Reduced free-methionine in methionine-dependent SV40-transformed human fibroblasts synthesizing apparently normal amounts of methionine. J Cell Physiol. 1983;117(1):9–14. doi: 10.1002/jcp.1041170103. [DOI] [PubMed] [Google Scholar]
  • 23.Wang Z, Yip LY, Lee JHJ, Wu Z, Chew HY, Chong PKW, Teo CC, Ang HY, Peh KLE, Yuan J, Ma S, Choo LSK, Basri N, Jiang X, Yu Q, Hillmer AM, Lim WT, Lim TKH, Takano A, Tan EH, Tan DSW, Ho YS, Lim B, Tam WL. Methionine is a metabolic dependency of tumor-initiating cells. Nat Med. 2019;25(5):825–837. doi: 10.1038/s41591-019-0423-5. [DOI] [PubMed] [Google Scholar]
  • 24.Kaiser P. Methionine dependence of cancer. Biomolecules. 2020;10(4):568. doi: 10.3390/biom10040568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kubota Y, Han Q, Aoki Y, Masaki N, Obara K, Hamada K, Hozumi C, Wong ACW, Bouvet M, Tsunoda T, Hoffman RM. Synergy of combining methionine restriction and chemotherapy: the disruptive next generation of cancer treatment. Cancer Diagn Progn. 2023;3(3):272–281. doi: 10.21873/cdp.10212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Morehead LC, Garg S, Wallis KF, Simoes CC, Siegel ER, Tackett AJ, Miousse IR. Increased response to immune checkpoint inhibitors with dietary methionine restriction in a colorectal cancer model. Cancers (Basel) 2023;15(18):4467. doi: 10.3390/cancers15184467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Li T, Tan YT, Chen YX, Zheng XJ, Wang W, Liao K, Mo HY, Lin J, Yang W, Piao HL, Xu RH, Ju HQ. Methionine deficiency facilitates antitumour immunity by altering m(6)A methylation of immune checkpoint transcripts. Gut. 2023;72(3):501–511. doi: 10.1136/gutjnl-2022-326928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Fang L, Hao Y, Yu H, Gu X, Peng Q, Zhuo H, Li Y, Liu Z, Wang J, Chen Y, Zhang J, Tian H, Gao Y, Gao R, Teng H, Shan Z, Zhu J, Li Z, Liu Y, Zhang Y, Yu F, Lin Z, Hao Y, Ge X, Yuan J, Hu HG, Ma Y, Qin HL, Wang P. Methionine restriction promotes cGAS activation and chromatin untethering through demethylation to enhance antitumor immunity. Cancer Cell. 2023;41(6):1118–1133.e12. doi: 10.1016/j.ccell.2023.05.005. [DOI] [PubMed] [Google Scholar]
  • 29.Xue Y, Lu F, Chang Z, Li J, Gao Y, Zhou J, Luo Y, Lai Y, Cao S, Li X, Zhou Y, Li Y, Tan Z, Cheng X, Li X, Chen J, Wang W. Intermittent dietary methionine deprivation facilitates tumoral ferroptosis and synergizes with checkpoint blockade. Nat Commun. 2023;14(1):4758. doi: 10.1038/s41467-023-40518-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Zhou S, Zhang S, Zheng K, Li Z, Hu E, Mu Y, Mai J, Zhao A, Zhao Z, Li F. Salmonella-mediated methionine deprivation drives immune activation and enhances immune checkpoint blockade therapy in melanoma. J Immunother Cancer. 2024;12(2):e008238. doi: 10.1136/jitc-2023-008238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Ables GP, Perrone CE, Orentreich D, Orentreich N. Methionine-restricted C57BL/6J mice are resistant to diet-induced obesity and insulin resistance but have low bone density. PLoS One. 2012;7(12):e51357. doi: 10.1371/journal.pone.0051357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Malloy VL, Perrone CE, Mattocks DA, Ables GP, Caliendo NS, Orentreich DS, Orentreich N. Methionine restriction prevents the progression of hepatic steatosis in leptin-deficient obese mice. Metabolism. 2013;62(11):1651–1661. doi: 10.1016/j.metabol.2013.06.012. [DOI] [PubMed] [Google Scholar]
  • 33.McGilvrey MI, Fortier B, Cooke D, Mahdi MA, Tero B, Potts CM, Kaija A, Ryzhova L, Cora C, Richardson A, Guzior D, Pinz I, Vary C, Koza RA, Ables G, Liaw L. Shortterm dietary methionine restriction with high fat diet counteracts metabolic dysfunction in male mice. Physiol Rep. 2025;13(12):e70405. doi: 10.14814/phy2.70405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Gao X, Sanderson SM, Dai Z, Reid MA, Cooper DE, Lu M, Richie JP Jr, Ciccarella A, Calcagnotto A, Mikhael PG, Mentch SJ, Liu J, Ables G, Kirsch DG, Hsu DS, Nichenametla SN, Locasale JW. Dietary methionine influences therapy in mouse cancer models and alters human metabolism. Nature. 2019;572(7769):397–401. doi: 10.1038/s41586-019-1437-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Roy DG, Chen J, Mamane V, Ma EH, Muhire BM, Sheldon RD, Shorstova T, Koning R, Johnson RM, Esaulova E, Williams KS, Hayes S, Steadman M, Samborska B, Swain A, Daigneault A, Chubukov V, Roddy TP, Foulkes W, Pospisilik JA, Bourgeois-Daigneault MC, Artyomov MN, Witcher M, Krawczyk CM, Larochelle C, Jones RG. Methionine metabolism shapes T helper cell responses through regulation of epigenetic reprogramming. Cell Metab. 2020;31(2):250–266.e9. doi: 10.1016/j.cmet.2020.01.006. [DOI] [PubMed] [Google Scholar]
  • 36.Ji M, Xu X, Xu Q, Hsiao YC, Martin C, Ukraintseva S, Popov V, Arbeev KG, Randall TA, Wu X, Garcia-Peterson LM, Liu J, Xu X, Andrea Azcarate-Peril M, Wan Y, Yashin AI, Anantharaman K, Lu K, Li JL, Shats I, Li X. Methionine restriction-induced sulfur deficiency impairs antitumour immunity partially through gut microbiota. Nat Metab. 2023;5(9):1526–1543. doi: 10.1038/s42255-023-00854-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Mu H, Zhang Q, Zuo D, Wang J, Tao Y, Li Z, He X, Meng H, Wang H, Shen J, Sun M, Jiang Y, Zhao W, Han J, Yang M, Wang Z, Lv Y, Yang Y, Xu J, Zhang T, Yang L, Lin J, Tang F, Tang R, Hu H, Cai Z, Sun W, Hua Y. Methionine intervention induces PD-L1 expression to enhance the immune checkpoint therapy response in MTAP-deleted osteosarcoma. Cell Rep Med. 2025;6(3):101977. doi: 10.1016/j.xcrm.2025.101977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kreis W, Hession C. Biological effects of enzymatic deprivation of L-methionine in cell culture and an experimental tumor. Cancer Res. 1973;33(8):1866–1869. [PubMed] [Google Scholar]
  • 39.Stern PH, Wallace CD, Hoffman RM. Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines. J Cell Physiol. 1984;119(1):29–34. doi: 10.1002/jcp.1041190106. [DOI] [PubMed] [Google Scholar]
  • 40.Sullivan MR, Darnell AM, Reilly MF, Kunchok T, Joesch-Cohen L, Rosenberg D, Ali A, Rees MG, Roth JA, Lewis CA, Vander Heiden MG. Methionine synthase is essential for cancer cell proliferation in physiological folate environments. Nat Metab. 2021;3(11):1500–1511. doi: 10.1038/s42255-021-00486-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ghergurovich JM, Xu X, Wang JZ, Yang L, Ryseck RP, Wang L, Rabinowitz JD. Methionine synthase supports tumour tetrahydrofolate pools. Nat Metab. 2021;3(11):1512–1520. doi: 10.1038/s42255-021-00465-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Andronicos A, Yoneda KC, Lin DW, Law FV, Bae H, Basirattalab A, Graham NA, Jang C, Kaiser P. Carboxy-methylation of the catalytic subunit of protein phosphatase 2A (PP2Ac) integrates methionine availability with methionine addicted cancer cell proliferation. Biomolecules. 2025;15(9):1210. doi: 10.3390/biom15091210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Judde JG, Ellis M, Frost P. Biochemical analysis of the role of transmethylation in the methionine dependence of tumor cells. Cancer Res. 1989;49(17):4859–4865. [PubMed] [Google Scholar]
  • 44.Stern PH, Hoffman RM. Enhanced in vitro selective toxicity of chemotherapeutic agents for human cancer cells based on a metabolic defect. J Natl Cancer Inst. 1986;76(4):629–639. doi: 10.1093/jnci/76.4.629. [DOI] [PubMed] [Google Scholar]
  • 45.Tisdale MJ. Utilization of preformed and endogenously synthesized methionine by cells in tissue culture. Br J Cancer. 1984;49(3):315–320. doi: 10.1038/bjc.1984.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Tan Y, Xu M, Hoffman RM. Broad selective efficacy of recombinant methioninase and polyethylene glycol-modified recombinant methioninase on cancer cells in vitro. Anticancer Res. 2010;30(4):1041–1046. [PubMed] [Google Scholar]
  • 47.Kaiser P. Methionine dependence of cancer. Biomolecules. 2020;10(4):568. doi: 10.3390/biom10040568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Lin DW, Carranza FG, Borrego S, Lauinger L, Dantas de Paula L, Pulipelli HR, Andronicos A, Hertel KJ, Kaiser P. Nutrient control of splice site selection contributes to methionine addiction of cancer. Mol Metab. 2025;93:102103. doi: 10.1016/j.molmet.2025.102103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Sedillo JC, Cryns VL. Targeting the methionine addiction of cancer. Am J Cancer Res. 2022;12(5):2249–2276. [PMC free article] [PubMed] [Google Scholar]
  • 50.Abo Qoura L, Balakin KV, Hoffman RM, Pokrovsky VS. The potential of methioninase for cancer treatment. Biochim Biophys Acta Rev Cancer. 2024;1879(4):189122. doi: 10.1016/j.bbcan.2024.189122. [DOI] [PubMed] [Google Scholar]
  • 51.Hoffman RM, Jacobsen SJ. Reversible growth arrest in simian virus 40-transformed human fibroblasts. Proc Natl Acad Sci USA. 1980;77(12):7306–7310. doi: 10.1073/pnas.77.12.7306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Sugimura T, Birnbaum SM, Winitz M, Greenstein JP. Quantitative nutritional studies with water-soluble, chemically defined diets. VIII. The forced feeding of diets each lacking in one essential amino acid. Arch Biochem Biophys. 1959;81(2):448–455. doi: 10.1016/0003-9861(59)90225-5. [DOI] [PubMed] [Google Scholar]
  • 53.Halpern BC, Clark BR, Hardy DN, Halpern RM, Smith RA. The effect of replacement of methionine by homocystine on survival of malignant and normal adult mammalian cells in culture. Proc Natl Acad Sci USA. 1974;71(4):1133–1136. doi: 10.1073/pnas.71.4.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Yamamoto J, Inubushi S, Han Q, Tashiro Y, Sugisawa N, Hamada K, Aoki Y, Miyake K, Matsuyama R, Bouvet M, Clarke SG, Endo I, Hoffman RM. Linkage of methionine addiction, histone lysine hypermethylation, and malignancy. iScience. 2022;25(4):104162. doi: 10.1016/j.isci.2022.104162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Tee T, Ruiter TJJ, Wu S, Zhang W, Schenau DVI, Rodionova M, Wajon D, Vervoort BMT, Grunewald KJT, Bosma M, Hagelaar R, Baker-Hernandez J, Dahaoui A, Schneider P, Verhoeven-Duif NM, Van der Meer LT, Van Leeuwen FN. S-adenosylmethionine addiction confers sensitivity to methionine restriction in KMT2A-rearranged acute lymphoblastic leukemia. Haematologica. 2025;110(11):2620–2634. doi: 10.3324/haematol.2023.284869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Mecham JO, Rowitch D, Wallace CD, Stern PH, Hoffman RM. The metabolic defect of methionine dependence occurs frequently in human tumor cell lines. Biochem Biophys Res Commun. 1983;117(2):429–434. doi: 10.1016/0006-291X(83)91218-4. [DOI] [PubMed] [Google Scholar]
  • 57.Tisdale MJ. Effect of methionine deprivation on methylation and synthesis of macromolecules. Br J Cancer. 1980;42(1):121–128. doi: 10.1038/bjc.1980.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Gao X, Sanderson SM, Dai Z, Reid MA, Cooper DE, Lu M, Richie JP Jr., Ciccarella A, Calcagnotto A, Mikhael PG, Mentch SJ, Liu J, Ables G, Kirsch DG, Hsu DS, Nichenametla SN, Locasale JW. Dietary methionine influences therapy in mouse cancer models and alters human metabolism. Nature. 2019;572(7769):397–401. doi: 10.1038/s41586-019-1437-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Yamamoto J, Han Q, Inubushi S, Sugisawa N, Hamada K, Nishino H, Miyake K, Kumamoto T, Matsuyama R, Bouvet M, Endo I, Hoffman RM. Histone methylation status of H3K4me3 and H3K9me3 under methionine restriction is unstable in methionine-addicted cancer cells, but stable in normal cells. Biochem Biophys Res Commun. 2020;533(4):1034–1038. doi: 10.1016/j.bbrc.2020.09.108. [DOI] [PubMed] [Google Scholar]
  • 60.Poirson-Bichat F, Lopez R, Bras Gonçalves RA, Miccoli L, Bourgeois Y, Demerseman P, Poisson M, Dutrillaux B, Poupon MF. Methionine deprivation and methionine analogs inhibit cell proliferation and growth of human xenografted gliomas. Life Sci. 1977;60(12):919–931. doi: 10.1016/s0024-3205(96)00672-8. [DOI] [PubMed] [Google Scholar]
  • 61.Breillout F, Antoine E, Poupon MF. Methionine dependency of malignant tumors: a possible approach for therapy. J Natl Cancer Inst. 1990;82(20):1628–1632. doi: 10.1093/jnci/82.20.1628. [DOI] [PubMed] [Google Scholar]
  • 62.Poirson-Bichat F, Gonçalves RA, Miccoli L, Dutrillaux B, Poupon MF. Methionine depletion enhances the antitumoral efficacy of cytotoxic agents in drug-resistant human tumor xenografts. Clin Cancer Res. 2000;6(2):643–653. [PubMed] [Google Scholar]
  • 63.Borrego SL, Lin DW, Kaiser P. Isolation and characterization of methionine-independent clones from methionine-dependent cancer cells. Methods Mol Biol. 2019;1866:37–48. doi: 10.1007/978-1-4939-8796-2_4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Malin D, Lee Y, Chepikova O, Strekalova E, Carlson A, Cryns VL. Methionine restriction exposes a targetable redox vulnerability of triple-negative breast cancer cells by inducing thioredoxin reductase. Breast Cancer Res Treat. 2021;190(3):373–387. doi: 10.1007/s10549-021-06398-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Aoki Y, Han Q, Tome Y, Yamamoto J, Kubota Y, Masaki N, Obara K, Hamada K, Wang JD, Inubushi S, Bouvet M, Clarke SG, Nishida K, Hoffman RM. Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation. Front Oncol. 2022;12:1009548. doi: 10.3389/fonc.2022.1009548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Stern PH, Hoffman RM. Elevated overall rates of trans-methylation in cell lines from diverse human tumors. In Vitro. 1984;20(8):663–670. doi: 10.1007/bf02619617. [DOI] [PubMed] [Google Scholar]
  • 67.Asano Y, Han Q, Li S, Mizuta K, Kang BM, Kim JS, Miyashi Y, Yamamoto N, Hayashi K, Kimura H, Miwa S, Igarashi K, Higuchi T, Morinaga S, Tsuchiya H, Demura S, Hoffman RM. Very rapid eradication of a large squamous-cell carcinoma of the head and neck treated with first-line combination chemotherapy, a low-methionine diet, and oral recombinant methioninase. Anticancer Res. 2025;45(11):5225–5231. doi: 10.21873/anticanres.17862. [DOI] [PubMed] [Google Scholar]
  • 68.Sato M, Sato T, Hozumi C, Han Q, Mizuta K, Morinaga S, Kang BM, Kobayashi N, Ichikawa Y, Nakajima A, Hoffman RM. [11C] Methionine PET vs. [18F]Fluorodeoxyglucose PET wholebody imaging to determine the extent of methionineaddiction compared to glucose-addiction of primary and metastatic cancer of the trunk in patients. Anticancer Res. 2024;44(9):3891–3898. doi: 10.21873/anticanres.17216. [DOI] [PubMed] [Google Scholar]
  • 69.Sharma P, Guo A, Poudel S, Boada-Romero E, Verbist KC, Palacios G, Immadisetty K, Chen MJ, Haydar D, Mishra A, Peng J, Babu MM, Krenciute G, Glazer ES, Green DR. Early methionine availability attenuates T cell exhaustion. Nat Immunol. 2025;26(8):1384–1396. doi: 10.1038/s41590-025-02223-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Pandit M, Kil YS, Ahn JH, Pokhrel RH, Gu Y, Mishra S, Han Y, Ouh YT, Kang B, Jeong MS, Kim JO, Nam JW, Ko HJ, Chang JH. Methionine consumption by cancer cells drives a progressive upregulation of PD-1 expression in CD4 T cells. Nat Commun. 2023;14(1):2593. doi: 10.1038/s41467-023-38316-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Hung MH, Lee JS, Ma C, Diggs LP, Heinrich S, Chang CW, Ma L, Forgues M, Budhu A, Chaisaingmongkol J, Ruchirawat M, Ruppin E, Greten TF, Wang XW. Tumor methionine metabolism drives T-cell exhaustion in hepatocellular carcinoma. Nat Commun. 2021;12(1):1455. doi: 10.1038/s41467-021-21804-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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