Thymoquinone reprograms tumor-associated macrophages toward an M1-like phenotype via the PLK1-FOXM1 axis to suppress ESCC progression

Background The clinical benefits of current treatments for esophageal squamous cell carcinoma (ESCC) are often limited, partly because of the immunosuppressive characteristics of the tumor microenvironment (TME). Natural products have attracted increasing attention in therapeutic research because of their potential immunomodulatory properties and favorable safety profiles. Thymoquinone (TQ), a bioactive quinone derived from the seeds of Nigella sativa , has shown antitumor activity in several solid tumors. However, its immunomodulatory effects in ESCC remain largely unexplored. Methods The in vivo antitumor effects of TQ and its impact on the TME were evaluated in ESCC-bearing mouse models. The contribution of macrophages to TQ-mediated tumor suppression was examined through clodronate liposome-mediated depletion. Tumor-associated macrophage (TAM) phenotypes were characterized by flow cytometry, proteomic profiling, and Seahorse analysis. Molecular docking and CETSA, together with functional assays, were used to investigate the molecular basis of TQ-mediated TAM reprogramming. The association between PLK1 expression and CD163 + TAM abundance was further assessed in ESCC specimens. Results TQ suppressed ESCC growth and modulated TAM phenotypes in vivo . In vitro , TQ reprogrammed macrophages toward an M1-like phenotype. Macrophage depletion attenuated the antitumor effect of TQ, indicating that macrophages contribute to TQ-mediated tumor suppression. This phenotypic shift was accompanied by a change in metabolic preference from OXPHOS to glycolysis. Mechanistically, TQ downregulated PLK1 and its downstream effector FOXM1, thereby shifting macrophage polarization from a suppressive state toward an immune-activating M1-like phenotype. Notably, PLK1 expression was positively associated with CD163 + TAM abundance in ESCC specimens, suggesting the potential clinical relevance of PLK1 in the ESCC TME. Conclusion Overall, these findings suggest that TQ suppresses ESCC progression, at least in part, by modulating the PLK1–FOXM1 axis to reprogram TAMs toward an M1-like phenotype. These findings provide a rationale for further investigation of TQ as a potential immunomodulatory agent for ESCC.