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.

低剂量甲氨蝶呤（MTX）是作为免疫抑制剂治疗类风湿性关节炎的一线药物。我们发现，低剂量的MTX表现出抗肿瘤免疫活性。在小鼠中，MTX治疗减少了肿瘤转移，并增强了放射疗法和免疫检查点阻断疗法的效果。从机制上讲，MTX选择性地诱导了癌细胞中的DNA损伤、cGAS-STING [环状鸟苷酸-腺苷酸（cGAMP）合成酶（cGAS）-干扰素基因刺激物（STING）]通路激活和cGAMP生成。此外，MTX与胞外核苷焦磷酸酶/磷酸二酯酶1（ENPP1）的底物结合口袋相结合，抑制了ENPP1介导的cGAMP水解及腺苷生成。因此，MTX减少了细胞外腺苷，并增强了宿主STING介导的抗肿瘤免疫反应。此外，在初步临床试验中，低剂量MTX与免疫疗法和放疗联合使用对不可切除或转移性实体瘤患者显示出有前景的有效性和安全性，其结果优于历史对照组。这些结果强调了之前未被认识的MTX免疫刺激功能，并为在临床上将MTX与肿瘤免疫治疗和放疗结合提供了一个合理的理由。