Methotrexate exerts antitumor immune activity and improves the clinical efficacy of immunotherapy in patients with solid tumors.

Low-dose methotrexate (MTX) is the first-line drug for treating rheumatoid arthritis as an immunosuppressor. We have identified that low-dose MTX exhibits antitumor immune activity. MTX treatment reduced tumor metastasis and enhanced the efficacy of radiation therapy and immune checkpoint blockade therapy in mice. Mechanistically, MTX selectively induced DNA damage, cGAS-STING [cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING)] pathway activation, and cGAMP generation in cancer cells. Furthermore, MTX bound to the substrate-binding pocket of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), inhibiting ENPP1-mediated cGAMP hydrolysis and adenosine generation. Consequently, MTX reduced extracellular adenosine and enhanced host STING-mediated antitumor immunity. In addition, a preliminary clinical trial demonstrated promising efficacy and safety of low-dose MTX in combination with immunotherapy and radiotherapy for patients with unresectable or metastatic solid tumors, showing improved outcomes compared with historical controls. These results highlight the previously unrecognized immunostimulatory functions of MTX and provide a rationale for combining MTX with tumor immunotherapy and radiotherapy in clinical settings.