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

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 (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).

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.