Glycolytic Activation of Peritumoral Monocytes Fosters Immune Privilege via PFKFB3-PD-L1 axis in Human Hepatocellular Carcinoma

Abstract Background & Aims: PD-L1 expression on antigen-presenting cells (APCs) is essential for T cell impairment, and PD-L1-expressing macrophages may mechanistically shape and therapeutically predict the clinical efficacy of PD-L1/PD-1 blockade. Despite the clinical success fostered by immune checkpoint inhibitors, the mechanisms underlying PD-L1 upregulation in human tumor microenvironments remain an enigma. Methods Monocytes/macrophages were purified from peripheral blood, non-tumor, or paired tumor tissues of patients with hepatocellular carcinoma (HCC), and their possible glycolytic switch was evaluated. The underlying regulatory mechanisms and clinical significance of specific metabolic switch in these cells were studied with both ex vivo analyses and in vitro experiments. Results We found that monocytes significantly enhanced the levels of glycolysis at the peritumoral region of human HCC. The activation of glycolysis induced PD-L1 expression on these cells and subsequently attenuated CTL responses in tumor tissues. Mechanistically, tumor-derived soluble factors, including hyaluronan fragments, induced the upregulation of a key glycolytic enzyme, PFKFB3, in tumor-associated monocytes. This enzyme not only modulated the cellular metabolic switch but also mediated the increased expression of PD-L1 by activating the NF-κB signaling pathway in these cells. Consistently, the levels of PFKFB3 + CD68 + cell infiltration in peritumoral tissues were negatively correlated with overall survival and could serve as an independent prognostic factor for the survival of patients with HCC. Conclusions Our results reveal a mechanism by which the cellular metabolic switch regulates the pro-tumor functions of monocytes in a specific human tumor microenvironment, and PFKFB3 might be a potential therapeutic target for not only cancer cells but also tumor-associated monocytes in human HCC. Lay summary PD-L1 expressed on APCs, rather than tumor cells, has been reported to play essential roles in checkpoint blockade therapy. A fundamental understanding of mechanisms that regulate the expression of PD-L1 on tumor infiltrating monocytes/macrophages will undoubtedly lead to the possibility of developing novel PD-L1 blockade strategies with high specificity and efficiency. The current study provides evidence that cellular glycolysis mediates the activation of monocytes by tumor microenvironmental cues, which lead to the induction of PD-L1 expression on these cells and subsequent autologous CD8 + T cell suppression in peritumoral tissues of human HCC. Notably, a key glycolytic enzyme, PFKFB3, has been identified as an important mediator in regulating PD-L1 expression by inducing the activation of NF-κB signaling pathway. These data unveils a novel mechanism by which metabolic switch links immune activation response to immune tolerance in the tumor milieu, and thus indicate efficient targets for future immune-based anti-cancer therapies.