Targeting C/EBPβ/SCD1/C16:1 loop-driven macrophage M2 polarization by Bruceine D improves anti-PD-1 immunotherapy in colorectal cancer

The immunosuppressive tumor microenvironment, particularly M2-like tumor-associated macrophages (TAM), critically contributes to colorectal Cancer (CRC) progression and resistance to immune checkpoint inhibitors. Here, we identify Bruceine D (BD), a natural compound, as a potent regulator of macrophage-associated immunometabolism in CRC. BD suppressed tumor progression and enhanced anti-programmed cell death protein 1 (anti-PD-1) immunotherapy efficacy in both MC38 and CT26 syngeneic mouse models without inducing significant systemic toxicity. Mechanistically, BD inhibited M2-like macrophage polarization in THP-1-derived macrophages, bone marrow-derived macrophages (BMDMs), and human monocyte-derived macrophages (MDMs), while attenuating the tumor-promoting effects mediated by macrophage-conditioned medium. Integrated transcriptomic, metabolic, and mechanistic analyses indicated that BD interacts with CCAAT/enhancer-binding protein beta (C/EBPβ) and suppresses stearoyl-CoA desaturase 1 (SCD1) transcription, thereby reducing the production of palmitoleic acid (C16:1). In turn, C16:1 maintained C/EBPβ protein stability, forming a positive feedback loop that sustained M2-associated macrophage phenotypes. Molecular docking, molecular dynamics simulation, cellular thermal shift assay, and surface plasmon resonance collectively supported the direct interaction between BD and C/EBPβ. Multiplex immunohistochemistry analysis of human CRC tissue microarrays further demonstrated positive correlations among C/EBPβ, SCD1, and the M2 marker CD206 in clinical specimens. Collectively, our findings support the C/EBPβ/SCD1/C16:1 axis as an important immunometabolic pathway involved in regulating M2-like macrophage phenotypes and suggest that BD represents a promising therapeutic strategy for improving anti-PD-1 immunotherapy in CRC.