Epigenetic demethylation sensitizes cancer cells to CAR-M triggered pyroptosis and remodels inflammatory immune responses in the tumor microenvironment to potentiate CAR-M cell therapy

Background Tumor epigenetic reprogramming serves as a critical hallmark of cancer, fostering transcriptional plasticity of gene expression and driving alterations in the suppressive inflammatory responses within the tumor microenvironment, which pose significant challenges to the advancement of novel CAR-macrophage (CAR-M)-based antitumor therapies. However, it remains limited mechanistic investigations exploring how epigenetic reprogramming strategies impact the efficacy of CAR-M treatments.Methods Human epidermal growth factor receptor 2 (HER2)-targeted CAR-Ms containing either an α1β1 integrin-mediated Fc-gamma receptor I (FcγRI) or CD3ζ signaling domain were generated and compared for tumor-directed migration and antitumor activity. HER2-positive tumor cells were pretreated with decitabine (DAC). CAR-M function, tumor-cell death, transcriptomic and inflammatory pathway changes, tumor immune remodeling, and therapeutic efficacy were assessed using in vitro assays, RNA sequencing, and murine tumor and metastasis models. Full-dose and half-dose CAR-M regimens were evaluated.Results α1β1 integrin-FcγRI CAR-Ms displayed significantly greater migration toward HER2-positive tumor cells than CD3ζ CAR-Ms. DAC potentiated anti-HER2 CAR-M activity by reprogramming tumor-cell gene expression and shifting the dominant cell-death mode from apoptosis to gasdermin E (GSDME)-dependent pyroptosis. RNA sequencing demonstrated enrichment of inflammatory immune-response programs, including nuclear factor kappa B and Janus kinase-signal transducer and activator of transcription signaling, after DAC treatment. DAC also favorably remodeled the tumor immune microenvironment, thereby supporting CAR-M activity. In murine models, DAC plus CAR-M therapy enhanced inflammatory antitumor immunity and improved control of primary tumor growth and metastatic disease compared with CAR-M monotherapy. Notably, DAC pretreatment combined with half-dose CAR-Ms achieved therapeutic efficacy comparable to that of full-dose CAR-M treatment.Conclusions DAC-mediated DNA demethylation sensitizes HER2-positive tumors to CAR-M therapy through GSDME-dependent pyroptosis, activation of inflammatory signaling, and favorable remodeling of the tumor microenvironment. Epigenetic priming may improve CAR-M efficacy while reducing the required cell dose, supporting further translational development of this combination strategy for solid tumors.