Type I IFNs repolarized a CD169+ macrophage population with anti-tumor potentials in hepatocellular carcinoma.

Macrophages constitute a major component in human hepatocellular carcinoma (HCC) and perform various functions to facilitate disease progression. Reprogramming or reconstituting the tumor surveillance phenotypes of macrophages represents an attractive immunotherapeutic strategy in cancer treatments. The current study identified CD169 as a potential target for macrophage repolarization since it signified a population of macrophages positively correlated with an activated immune signature and better prognosis of patients with HCC. In vitro experiments revealed that a low dose of type I interferon (IFN) could effectively reprogram human monocyte-derived macrophages to upregulate CD169 expression, and such induced CD169+ macrophages exhibited significantly enhanced phagocytotic and CD8+ T cell-activating capacities compared to controls. A low dose of IFNα also inhibited hepatoma growth in mice in vivo, presumably through polarizing the CD169+ macrophage population and enhancing CD8+ T cell activities. Notably, IFNα also induced substantial PD-L1 expression on macrophages in vivo, and thus blockade of PD-L1 could further increase the anti-tumor efficacy of IFNα in the treatment of HCC. We propose a low dose of IFNα in combination with a PD-L1 blocking agent as a potential anti-tumor therapeutic strategy via its effects on macrophage polarization.

Myeloid signature reveals immune contexture and predicts the prognosis of hepatocellular carcinoma.

BACKGROUNDDespite an increasing appreciation of the roles that myeloid cells play in tumor progression and therapy, challenges remain in interpreting the tumor-associated myeloid response balance and its translational value. We aimed to construct a simple and reliable myeloid signature for hepatocellular carcinoma (HCC).METHODSUsing in situ immunohistochemistry, we assessed the distribution of major myeloid subtypes in both peri- and intratumoral regions of HCC. A 2-feature-based, myeloid-specific prognostic signature, named the myeloid response score (MRS), was constructed using an L1-penalized Cox regression model based on data from a training subset (n = 244), a test subset (n = 244), and an independent internal (n = 341) and 2 external (n = 94; n = 254) cohorts.RESULTSThe MRS and the MRS-based nomograms displayed remarkable discriminatory power, accuracy, and clinical usefulness for predicting recurrence and patient survival, superior to current staging algorithms. Moreover, an increase in MRS was associated with a shift in the myeloid response balance from antitumor to protumor activities, accompanied by enhanced CD8+ T cell exhaustion patterns. Additionally, we provide evidence that the MRS was associated with the efficacy of sorafenib treatment for recurrent HCC.CONCLUSIONWe identified and validated a simple myeloid signature for HCC that showed remarkable prognostic potential and may serve as a basis for the stratification of HCC immune subtypes.FUNDINGThis work was supported by the National Science and Technology Major Project of China, the National Natural Science Foundation of China, the Science and Information Technology of Guangzhou, the Fundamental Research Funds for the Central Universities, the Guangdong Basic and Applied Basic Research Foundation, and the China Postdoctoral Science Foundation.

Targeting adenosinergic pathway enhances the anti-tumor efficacy of sorafenib in hepatocellular carcinoma.

BACKGROUND: Sorafenib is the most widely used first-line treatment for patients with advanced hepatocellular carcinoma (HCC), but such treatment provides only limited survival benefits that might be related to the immune status of distinct tumor microenvironments. A fundamental understanding of the distribution and phenotypes of T lymphocytes in tumors will undoubtedly lead to the development of novel immunotherapeutic strategies that could possibly enhance the efficacy of sorafenib treatments. METHODS: Flow cytometry, immunohistochemistry and immunofluorescence analyses were performed to detect the infiltration and distribution of various leukocyte populations, and the expression of different immune checkpoint molecules in fresh HCC tumor tissues. Correlations among indicating genes were calculated in 365 patients with HCC from The Cancer Genome Atlas (TCGA) data set, and the cumulative overall survival time was calculated using the Kaplan-Meier method. Moreover, role of adenosinergic pathway on sorafenib anti-tumor efficacy was investigated using both subcutaneous and orthotopic transplantation tumor model in immune competent C57BL/6 mice. RESULTS: We revealed that levels of CD3+ and CD8+ T cells were significantly downregulated in HCC tumor tissue, so were the infiltration of CD169+ cells (a Mφ subpopulation with T cell activation capacities) and their contact with CD8+ cells in tumor milieus. Moreover, levels of PD-1 and CD39 expression were significantly upregulated in human HCC-infiltrating CD4+ and CD8+ T cells, and CD39+CD8+ T cells exhibited a CD69+PD-1+perforinlowIFNγlow "exhausted" phenotype. Levels of both CD39+ T cells infiltration and adenosine receptor ADORA2B expression in tumor tissues were negatively correlated with overall survival of patients with HCC. Accordingly, mice treated with sorafenib in combination with adenosine A2B receptor blockage reagents exhibited significantly reduced tumor progression compared with control groups. CONCLUSIONS: These results suggest that adenosinergic pathway might represent an applicable target for sorafenib-combined-therapies in human HCC.

Monocytes/Macrophages promote vascular CXCR4 expression via the ERK pathway in hepatocellular carcinoma.

We recently identified CXCR4 as a novel vascular marker for vessel sprouting in hepatocellular carcinoma (HCC) tissues. Thus, CXCR4+ endothelial cells (ECs) could serve as a potential predictor for patients who may benefit from sorafenib treatment; however, the mechanism that regulates vascular CXCR4 expression in HCC remains largely unknown. Here, we revealed a large number of monocytes/macrophages (Mo/Mϕ) to be selectively enriched in the perivascular areas of CXCR4+ vessels in HCC samples. The depletion of Mo/Mϕ with gadolinium chloride (GdCl3) or zoledronic acid (ZA) treatment significantly reduced vascular CXCR4 expression in HCC tumors. This phenomenon was also confirmed in CCR2-KO mice, which exhibited reduced infiltration of inflammatory Mo/Mϕ in tumor tissues. Mechanistic studies revealed that inflammatory cytokines derived from tumor conditioned Mo/Mϕ, especially TNF-α, could up-regulate CXCR4 expression on ECs. TNF-α-induced activation of the Raf-ERK pathway, but not Notch signaling, was responsible for the expression of CXCR4. Moreover, the combination treatment of sorafenib with ZA was associated with improved anti-tumor efficacy by significantly reducing vascular CXCR4 expression. These findings revealed that Mo/Mϕ could regulate CXCR4 expression in the tumor vasculature. Thus, the inhibition of Mo/Mϕ inflammation might enhance the treatment efficacy of sorafenib in HCC.

[Magnetic Resonance Imaging Tracing the Biodistribution of SPIO-shRNA Molecular Probe in vivo].

OBJECTIVES: To investigate the biodistribution of superparamagnetic iron oxide (SPIO)-shRNA molecular probe by magnetic resonance imaging (MRI) in vivo. METHODS: Six New Zealand white rabbits were injected intravenously with SPIO-shRNA molecular probe (9.6 mg Fe/kg) via ear edge vein. The blood samples were collected to analyse the pharmacokinetic parameters through measuring the iron content by atomic absorption spectrometry (AAS) method at 30 min before and 1 min, 3 min, 5 min, 10 min, 15 min, 30 min and at 1, 2, 3, 6, 12, and 24 h after the injection. Six Kun Ming (KM) mice were injected intravenously with SPIO-shRNA molecular probe (4.8 mg Fe/kg). The biodistribution of SPIO-shRNA molecular probe was traced by MRI in vivo. Ninety six KM mice were randomly divided into control group and experimental group: each mouse in experimental group was injected intravenously with SPIO-shRNA molecular probe (4.8 mg Fe/kg). The liver, spleen, kidney, brain and muscle of the control group and the experimental group on 1, 3, 5, 7, 9, 11 and 14 d after the injection were collected. The organ iron content were measured by AAS method and Prussian blue staining in order to observe the distribution of the SPIO-shRNA molecular probe in the main organ. RESULTS: Our results demonstrated that the pharmacokinetics of the molecular probe complied with two-compartment model, and the blood half-life was (3.692±0.196) h. The data of MRI showed that the probe were distributed in liver and spleen, and the signs were reduced in accord with the increase of probe's doses in liver and spleen. The probe's metabolism was slow, and the probe was cleared from liver and spleen at 2 weeks after the injection. The results of AAS and Prussian blue staining further testified the results of MRI. CONCLUSIONS: Our data showed the biodistribution of SPIO-shRNA molecular probe in main organs can be traced by MRI in vivo. Meanwhile, it provides important information for the effectiveness of the probe by MRI at tumor in vivo.

Effects of external magnetic field on the transfection rate of SPIO-shRNADual functional molecular probe into ovarian carcinoma SKOV3 cells in vitro.

OBJECTIVE: To explore the transfection rate of SPIO-shRNA dual functional molecular probe into ovarian carcinoma SKOV3 cells in external magnetic field. METHODS: Dual functional molecular probe at an iron concentration of 45 mg/L was transfected into SKOV3 cells. The cells with coexisting probe and magnetic fields were set as the intervention group,the probe-transfected cells as negative control group, and normally cultured SKOV3 without any transfection as blank control group. The transfection rate was detected by flow cytometry. Cell viability was observed by CCK-8 assay. Epidermal growth factor receptor (EGFR) expression level in SKOV3 cells was determined by real-time quantitative PCR and Western blot analysis. The signal intensity was measured by magnetic resonance imaging (MRI). RESULTS: The transfection rate of the intervention group was (79.20 ± 3.31)%, which was significantly higher than that of negative control group (P=0.001). Compared with the negative control group,the cell viability of the intervention group significantly decreased (P=0.011), protein and mRNA expression levels of EGFR in the intervention group were significantly decreased (both P<0.05). The signal intensity on T2(*)WI in the intervention group also significantly decreased (P=0.0004). CONCLUSION: The external magnetic field can improve the transfection efficiency SPIO-shRNA dual functional molecular probe into ovarian carcinoma SKOV3 cells.

[Pharmacokinetics and MR imaging of SPIO-shRNA dual functional molecular probe in vivo].

In this study, we investigated the pharmacokinetics parameters of SPIO-shRNA dual functional molecular probe and observed the main organ distribution by MRI in vivo. Eighteen New Zealand white rabbits were randomly divided into three groups and injected intravenously with different doses of SPIO-shRNA molecular probe, respectively. The blood samples were collected to analyze the pharmacokinetic parameters by measuring the iron content at 30 minutes before and after the injection. Twenty-four Kun Ming (KM) mice were randomly divided into 4 groups: the control group was injected intravenously with physiological saline 200 µL per mouse via the tail vein, the other 3 groups were injected intravenously with different doses of SPIO-shRNA molecular probe. MRI observation was performed in 24 hours, and the liver, spleen, kidney, brain and muscle were collected for iron quantification with Prussian blue staining to determine distribution of the SPIO-shRNA molecular probe in the main organ in vivo. Our results suggest that the molecular probe blood half-life is more than 3 hours. The data of MRI suggest the probe was distributed in liver and spleen, and the MRI signal was reduced with the increase in probe's doses (P < 0.05). The results of Prussian blue staining confirmed the results of MRI. Most of the probe could escape the phagocytosis of mononuclear phagocyte system. Our data provide the pharmacokinetic and distribution of SPIO-shRNA molecular probe in organs. Meanwhile, it suggests the choice of the time and dose of probe for MR imaging of tumor in vivo.