Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma.

Macrophages (Mϕ) represent a major component of tumor tissues and play an important role in both tumor progression and therapeutic response. Although tumor Mϕ are generally considered to be derived from circulating monocytes, emerging evidence indicates that tissue Mϕ pools can be maintained by self-renewal. We aimed to elucidate the contribution, phenotype, and regulatory mechanisms of proliferating Mϕ in human hepatocellular carcinoma (HCC). Flow cytometry analyses were performed to examine the presence and phenotype of proliferating Mϕ in fresh HCC tissues. Dual immunofluorescence staining was applied to analyze the prognostic value of proliferating Mϕ. The underlying regulatory mechanisms were examined using human monocyte-derived Mϕ. Tumor-infiltrating Mϕ exhibited a significantly higher proliferative capacity than Mϕ in non-tumor tissues. A higher level of Mϕ proliferation was positively correlated with Mϕ density in the tumor and a poor prognosis in patients with HCC. Proliferating Mϕ were less differentiated (with increased CD206 expression) and were induced by the tumor cell-derived soluble small molecule, adenosine, but not proteins, lipids, or large peptides. Mechanistic studies demonstrated that autocrine granulocyte-macrophage colony-stimulating factor (GM-CSF) released by tumor-stimulated Mϕ could enhance A2A receptor expression on Mϕ and function synergistically with adenosine to elicit Mϕ proliferation in HCC. Local Mϕ proliferation is an important mechanism for Mϕ accumulation in HCC tissues. Tumor-derived adenosine functions synergistically with autocrine GM-CSF released from activated Mϕ, which promotes Mϕ proliferation. Thus, selective modulation of Mϕ accumulation at the source may provide a novel strategy for cancer therapy. Tumor-associated macrophages (TAMs) have been reported to play an essential role in both tumor progression and therapeutic response. A fundamental understanding of the mechanisms that regulate macrophage accumulation in tumors will undoubtedly lead to the development of strategies to target macrophages with high specificity and efficiency. The current study unveils a novel mechanism by which local proliferation is linked to macrophage accumulation in the tumor milieu, identifying potential targets for future immune-based anticancer therapies.

Wang J ，Wang Y ，Chu Y ，Li Z ，Yu X ，Huang Z ，Xu J ，Zheng L ... -  《-》

An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation.

G protein-coupled receptors (GPCRs) are among the most important targets in drug discovery. In this study, we used TaqMan Low Density Arrays to profile the full GPCR repertoire of primary human macrophages differentiated from monocytes using either colony stimulating factor-1 (CSF-1/M-CSF) (CSF-1 Mϕ) or granulocyte macrophage colony stimulating factor (GM-CSF) (GM-CSF Mϕ). The overall trend was a downregulation of GPCRs during monocyte to macrophage differentiation, but a core set of 10 genes (e.g. LGR4, MRGPRF and GPR143) encoding seven transmembrane proteins were upregulated, irrespective of the differentiating agent used. Several of these upregulated GPCRs have not previously been studied in the context of macrophage biology and/or inflammation. As expected, CSF-1 Mϕ and GM-CSF Mϕ exhibited differential inflammatory cytokine profiles in response to the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS). Moreover, 15 GPCRs were differentially expressed between these cell populations in the basal state. For example, EDG1 was expressed at elevated levels in CSF-1 Mϕ versus GM-CSF Mϕ, whereas the reverse was true for EDG6. 101 GPCRs showed differential regulation over an LPS time course, with 65 of these profiles being impacted by the basal differentiation state (e.g. GPRC5A, GPRC5B). Only 14 LPS-regulated GPCRs showed asynchronous behavior (divergent LPS regulation) with respect to differentiation status. Thus, the differentiation state primarily affects the magnitude of LPS-regulated expression, rather than causing major reprogramming of GPCR gene expression profiles. Several GPCRs showing differential profiles between CSF-1 Mϕ and GM-CSF Mϕ (e.g. P2RY8, GPR92, EMR3) have not been widely investigated in macrophage biology and inflammation. Strikingly, several closely related GPCRs displayed completely opposing patterns of regulation during differentiation and/or activation (e.g. EDG1 versus EDG6, LGR4 versus LGR7, GPRC5A versus GPRC5B). We propose that selective regulation of GPCR5A and GPCR5B in CSF-1 Mϕ contributes to skewing toward the M2 macrophage phenotype. Our analysis of the GPCR repertoire expressed during primary human monocyte to macrophage differentiation and TLR4-mediated activation provides a valuable new platform for conducting future functional analyses of individual GPCRs in human macrophage inflammatory pathways.

Hohenhaus DM ，Schaale K ，Le Cao KA ，Seow V ，Iyer A ，Fairlie DP ，Sweet MJ ... -  《-》

Glycolytic activation of peritumoral monocytes fosters immune privilege via the PFKFB3-PD-L1 axis in human hepatocellular carcinoma.

Programmed cell death 1 ligand 1 (PD-L1) expression on antigen-presenting cells is essential for T cell impairment, and PD-L1-expressing macrophages may mechanistically shape and therapeutically predict the clinical efficacy of PD-L1 or programmed cell death 1 blockade. We aimed to elucidate the mechanisms underlying PD-L1 upregulation in human tumor microenvironments, which remain poorly understood despite the clinical success of immune checkpoint inhibitors. 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 metabolic switching were studied with both ex vivo analyses and in vitro experiments. 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 cytotoxic T lymphocyte 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 nuclear factor kappa B 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 survival in patients with HCC. Our results reveal a mechanism by which the cellular metabolic switch regulates the pro-tumor functions of monocytes in a specific human tumor microenvironment. PFKFB3 in both cancer cells and tumor-associated monocytes is a potential therapeutic target in human HCC. Programmed cell death 1 ligand 1 (PD-L1) expressed on antigen-presenting cells, rather than tumor cells, has been reported to play an essential role 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 unveils a novel mechanism by which metabolic switching links immune activation responses to immune tolerance in the tumor milieu, identifying potential targets for future immune-based anti-cancer therapies.

Chen DP ，Ning WR ，Jiang ZZ ，Peng ZP ，Zhu LY ，Zhuang SM ，Kuang DM ，Zheng L ，Wu Y ... -  《-》

Glycolytic activation of monocytes regulates the accumulation and function of neutrophils in human hepatocellular carcinoma.

Neutrophils are one of the most abundant components in human hepatocellular carcinoma (HCC) and have been shown to play important roles in regulating disease progression. However, neutrophils are very short-lived cells in circulation, and mechanisms regulating their accumulation and functions in HCC are not yet fully understood. Monocytes were purified from non-tumor or paired tumor tissues of patients with HCC, and their production of neutrophil-attracting chemokines was evaluated. Mechanisms regulating the expression of CXCL2/8 by tumor monocytes, and the role of tumor monocyte-derived chemokines and cytokines in modulating neutrophil accumulation and functions were studied with both ex vivo analyses and in vitro experiments. Monocyte-derived CXCL2 and CXCL8 were major factors in regulating the recruitment of neutrophils into tumor milieus. These chemokines, in addition to tumor-derived soluble factors, could inhibit apoptosis and sustain survival of neutrophils, thus leading to neutrophil accumulation in tumor tissues. Moreover, monocyte-derived TNF-α acted synergistically with tumor-derived soluble factors to induce the production of the pro-metastasis factor OSM by neutrophils. Further, the glycolytic switch in tumor-infiltrating monocytes mediated their production of CXCL2 and CXCL8 via the PFKFB3-NF-κB signaling pathway. Accordingly, levels of PFKFB3, CXCL2/CXCL8 production in monocytes and infiltration of OSM-producing neutrophils were positively correlated in human HCC tissues. Our results unveiled a previously unappreciated link between monocytes and neutrophils in human HCC, identifying possible targets that could be therapeutically exploited in the future. Neutrophils constitute a major but poorly understood component of human hepatocellular carcinoma (HCC). Herein, we unveil a novel mechanism by which metabolic switching in monocytes promotes the accumulation of neutrophils in the tumors of patients with HCC. Both monocyte-produced chemokines and signals from the tumor microenvironment promote the production of the pro-metastatic factor OSM by neutrophils. These data identify potential targets for immune-based anticancer therapies for HCC.

Peng ZP ，Jiang ZZ ，Guo HF ，Zhou MM ，Huang YF ，Ning WR ，Huang JH ，Zheng L ，Wu Y ... -  《-》

Mechanism study of tyrosine phosphatase shp-1 in inhibiting hepatocellular carcinoma progression by regulating the SHP2/GM-CSF pathway in TAMs.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Macrophage-mediated innate immune responses play a crucial role in tumor development. This study revealed the mechanism of SHP-1 in regulating HCC progression. SHP-1 inhibits tumour development in vivo. Increasing SHP-1 expression in macrophages promotes the expression of p-SHP-1, SHP2, and p-SHP-2. In macrophages GM-CSF recruits SHP-2 to the GM-CSF receptor GM-CSFR induces p-SHP-2 dephosphorylation. GM-CSF recruits p-SHP-2 for dephosphorylation by up-regulating HoxA10HOXA10 activates the transcription of TGFβ2 by interacting with tandem cis-elements in the promoter thereby regulating the proliferation and migration of liver cancer cells. GM-CSF inhibits SHP-1 regulation of p-SHP-1, SHP2, and p-SHP-2 in macrophages. Detailed studies have shown that SHP-1 regulates SHP2 expression, and SHP-1 and SHP2 are involved in macrophage M2 polarisation. SHP-1 inhibits HOXA10 and TGFβ2 which in turn regulates the expression of the migration-associated proteins, MMP2/9, and the migration of hepatocellular carcinoma cells. Overexpression of SHP-1 inhibits macrophage M2 polarisation via the p-STAT3/6 signalling pathway Classical markers arginase-1, CD206, CD163 and regulate the expression of M2 polarisation cytokines IL-4 and IL-10. In addition, hypoxia-induced ROS inhibited SHP-1 regulation by suppressing the expression of p-SHP-1. The combined effect of GM-CSF and ROS significantly increased p-HOXA10/TGFβ2 and macrophage M2 polarisation, and the regulatory effect of ROS was significantly suppressed by GM-CSF knockdown. These findings suggest that increasing the expression of tyrosine phosphatase SHP-1 can inhibit hepatocellular carcinoma progression by modulating the SHP2/GM-CSF pathway in TAM and thus inhibit the progression of hepatocellular carcinoma.

Wei Q ，Luo S ，He G 《Scientific Reports》