Bufalin stimulates antitumor immune response by driving tumor-infiltrating macrophage toward M1 phenotype in hepatocellular carcinoma

Abstract

BackgroundImmunotherapy for hepatocellular carcinoma (HCC) exhibits limited clinical efficacy due to immunosuppressive tumor microenvironment (TME). Tumor-infiltrating macrophages (TIMs) account for the major component in the TME, and the dominance of M2 phenotype over M1 phenotype in the TIMs plays the pivotal role in sustaining the immunosuppressive character. We thus investigate the effect of bufalin on promoting TIMs polarization toward M1 phenotype to improve HCC immunotherapy.MethodsThe impact of bufalin on evoking antitumor immune response was evaluated in the immunocompetent mouse HCC model. The expression profiling of macrophage-associated genes, surface markers and cytokines on bufalin treatment in vitro and in vivo were detected using flow cytometry, immunofluorescence, western blot analysis, ELISA and RT-qPCR. Cell signaling involved in M1 macrophage polarization was identified via the analysis of gene sequencing, and bufalin-governed target was explored by immunoprecipitation, western blot analysis and gain-and-loss of antitumor immune response. The combination of bufalin and antiprogrammed cell death protein 1 (anti-PD-1) antibody was also assessed in orthotopic HCC mouse model.ResultsIn this study, we showed that bufalin can function as an antitumor immune modulator that governs the polarization of TIMs from tumor-promoting M2 toward tumor-inhibitory M1, which induces HCC suppression through the activation of effector T cell immune response. Mechanistically, bufalin inhibits overexpression of p50 nuclear factor kappa B (NF-κB) factor, leading to the predominance of p65-p50 heterodimers over p50 homodimers in the nuclei. The accumulation of p65-p50 heterodimers activates NF-κB signaling, which is responsible for the production of immunostimulatory cytokines, thus resulting in the activation of antitumor T cell immune response. Moreover, bufalin enhances the antitumor activity of anti-PD-1 antibody, and the combination exerts synergistic effect on HCC suppression.ConclusionsThese data expound a novel antitumor mechanism of bufalin, and facilitate exploitation of a new potential macrophage-based HCC immunotherapeutic modality.

TAM-targeted reeducation for enhanced cancer immunotherapy: Mechanism and recent progress

“…In general, TLR/NF- κ B activation promotes the M1-like polarization, but such biological process highly depends on the composition of NF- κ B subunits. For example, NF- κ B p65/p50 heterodimers can enhance pro-inflammatory cytokines production to polarize TAMs into M1-phenotype, while p50/p50 homodimers result in the M2-phenotype TAMs ( 59 , 60 ).…”

The hypoxia-driven crosstalk between tumor and tumor-associated macrophages: mechanisms and clinical treatment strategies

“…Lipopolysaccharide, for example, promotes the overexpression of p50-p50 homodimers, allowing M1 to M2 macrophage reprogramming [168][169][170]. In contrast, Bufalin promotes the overexpression of p65-p50 heterodimers, leading to the transition of macrophage from M2 to M1 [171]. Therefore, in order to better understand the multifaceted role that NF-κB plays in regulating TAMs function, it may be useful to investigate the exact functions of various NF-κB dimers.…”

Tumor Microenvironment in Hepatocellular Carcinoma: Key Players for Immunotherapy

“…Studies have evidenced that the accumulation of p50 NF-κB factors in macrophages is responsible for the TAM-governed immunosuppression in HCC. 45 , 46 Therefore, decreasing p50 NF-κB factor in TAM to reinvigorate the antitumor M1 activity may be one approach for HCC treatment.…”