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Supplementary Table S1: Primer sequence of gene used for qPCR in mouse. Supplementary Table S2: Primer sequence of gene used for qPCR in human. Supplementary Table S3: List of antibodies used for immunohistochemical staining.

Supplementary figure legends

Figure S1. Microscope pictures, and CSF1R expression of M(CSF1) cells and M(CSF2) cells. Figure S2. Microarray and qRT-PCR difference between M(LPS IFNÃŽÃ,³)/M(IL4), and between M(CSF1)/M(CSF2) cells. Figure S3. Effect of PLX3397 on the population doubling curve of the hepatoma cell lines, HUVEC, LX2, M(CSF2) cells, and T cells. Figure S4. Microvessel density expression in the vehicle and PLX3397 groups. Figure S5. TAMs of PLX3397-treated tumors were promoted towards a M1 phenotype. Figure S6. CSF2 is a key factor in between M(CSF1) and M(CSF2) cells. Figure S7. The tumor immunity environment was ameliorated after CSF1R inhibition in the allograft hepatoma model. Figure S8. CSF1R inhibition retarded HepG2 and HCCLM3 mice orthotopic xenograft growth. Figure S9. TAMs of PLX3397-treated tumors were promoted towards a M1 phenotype was also found in the xenografts after CSF1R inhibition.

Tumor-associated endothelial cells (TEC) directly facilitate tumor progression, but little is known about the mechanisms. We investigated the function of CD109 in TEC and its clinical significance in hepatocellular carcinoma (HCC). The correlation between CD109 expressed on tumor vessels and the prognosis after surgical resection of HCC was studied. The effect of human umbilical vein endothelial cells (HUVEC) with different CD109 expression on hepatoma cell proliferation, migration, and invasion was compared in co-culture assay. Associated key factors were screened by human cytokine antibody array and validated thereafter. HUVEC with different CD109 expression were co-implanted with HCCLM3 or HepG2 cells in nude mice to investigate the effect of CD109 expression on tumor growth and metastasis. Reduced expression of CD109 on tumor vessels was associated with large tumor size, microvascular invasion, and advanced tumor stage. CD109 was an independent risk factor for disease-free survival (P = 0.001) after curative resection of HCC. CD109 knockdown in HUVEC promoted hepatoma cell proliferation, migration, and invasion. Interleukin-8 (IL-8) was a key tumor-promoting factor secreted from CD109 knockdown HUVEC. CD109 knockdown upregulated IL-8 expression through activation of TGF-β/Akt/NF-κB pathway in HUVEC. Co-implantation with CD109 knockdown HUVEC accelerated tumor growth and metastasis in mice models. In conclusion, CD109 expression on tumor vessels is a potential prognostic marker for HCC, and its reduced expression on TEC promoted tumor progression by paracrine IL-8.

The S100 protein family is widely involved in the pathological process of various types of cancer. However, the prognostic value of the S100 protein family member S100A12 in hepatocellular carcinoma (HCC) remains unknown. A total of 139 patients undergoing curative surgical resection for HCC from December 2005 to June 2006 were investigated. Immunohistochemistry of S100A12 tissue was performed and expression was classified according to the total positive staining area. Co-expression of S100A12 with cluster of differentiation (CD)11B, CD15 and CD68 was evaluated using immunofluorescence. Associations between S100A12 expression and preoperative clinicopathological parameters were assessed using a χ2 test or independent sample Student's t-test. Kaplan-Meier estimator survival analysis and multivariate Cox's proportional hazard regression model were used to evaluate the prognostic value of S100A12 expression. The expression of S100A12 was restricted exclusively to stroma cells, primarily to myeloid-derived immune cells, CD15-positive neutrophils and CD68-positive macrophages in particular. A total positive staining area of 1,600 µm2 was selected as the threshold between high and low S100A12 expression. There was a statistically significant association between intratumoral S100A12 expression and tumor differentiation (P=0.010). High expression of S100A12 on intratumoral stroma cells was an independent prognostic factor for the overall (P=0.002) and disease-free survival (P=0.007) rates of HCC following curative surgical resection. No significant association was identified between peritumoral S100A12 expression and HCC prognosis. The results of the present study demonstrated that high expression of S100A12 on intratumoral stroma cells is associated with poor HCC prognosis following curative resection, which may serve as a potential target for an adjuvant therapy.

Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a well-known marker of endothelial cells and a key factor for adhesion and accumulation of platelets. CD31 plays roles in cell proliferation, apoptosis, migration, and cellular immunity. CD31 is also expressed on tumor cells, such as breast cancer cells and non-Hodgkin's lymphomas, and contributes to tumor cell invasion. Here, our experiments show that CD31 promotes metastasis by inducing the epithelial-mesenchymal transition in hepatocellular carcinoma by up-regulating integrin β1 via the FAK/Akt signaling pathway.

Background High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown. Methods Real-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression. Results We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3′-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/β-catenin signaling by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4 which was mediated by repressed FOXO3a. Conclusions Consistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

Flotillin-2 (Flot2) is a highly conserved and ubiquitously expressed protein that resides on the cytoplasmic side of the cell membrane within specific cholesterol rich microdomains. Some studies have reported that overexpression of Flot2 is related to cancer progression. However, the role of Flot2 in hepatocellular carcinoma (HCC) remains unclarified. In this study, we aim to explore the correlation between Flot2 expression and HCC progression and the underlying mechanism. In the present study, overexpression of Flot2 in HCC tissues and cell lines was detected, and forced overexpression of Flot2 significantly promoted the proliferation, migration, invasion and metastasis of HCC in vitro and in vivo by modulating cell cycle and inducing EMT, which was mediated via up-regulation of Twist as a result of Raf/MEK/ERK1/2 pathway activation. In contrast, silencing Flot2 expression inhibited these biological processes. Furthermore, high expression of Flot2 was significantly correlated with poor prognosis of HCC patients after curative resection and is an independent risk factor. In conclusion, Flot2 promoted tumor growth and metastasis of HCC through modulating cell cycle and inducing EMT. The expression of Flot2 may play a key role in HCC progression and may be regarded as a potential poor prognostic marker for HCC.

Colony-stimulating factor-1 (CSF-1) and its receptor, CSF-1R, regulate the differentiation and function of macrophages and play an important role in macrophage infiltration in the context of hepatocellular carcinoma. The therapeutic effects of CSF-1R blockade in hepatocellular carcinoma remain unclear. In this study, we found that CSF-1R blockade by PLX3397, a competitive inhibitor with high specificity for CSF-1R tyrosine kinase, significantly delayed tumor growth in mouse models. PLX3397 inhibited the proliferation of macrophages in vitro, but intratumoral macrophage infiltration was not decreased by PLX3397 in vivo. Gene expression profiling of tumor-associated macrophages (TAM) showed that TAMs from the PLX3397-treated tumors were polarized toward an M1-like phenotype compared with those from vehicle-treated tumors. In addition, PLX3397 treatment increased CD8+ T-cell infiltration, whereas CD4+ T-cell infiltration was decreased. Further study revealed that tumor cell–derived CSF-2 protected TAMs from being depleted by PLX3397. In conclusion, CSF-1R blockade delayed tumor growth by shifting the polarization rather than the depletion of TAMs. CSF-1R blockade warrants further investigation in the treatment of hepatocellular carcinoma. Mol Cancer Ther; 16(8); 1544–54. ©2017 AACR.

Our previous studies demonstrated that traditional Chinese herbal medicine 'Songyou Yin' inhibited the growth and invasion of hepatocellular carcinoma (HCC) cells, and altered epithelial‑mesenchymal transition (EMT) markers in oxaliplatin‑treated HCC tissues and cell lines. In the present study, we aimed to explore whether astragaloside IV (AS-IV), a component of 'Songyou Yin', can affect the growth and invasion of HCC cells and the underlying mechanism involved. Human HCC cell lines Huh7 and MHCC97-H, with low and high metastatic potential, respectively, were treated with increasing doses of AS-IV. The Cell Counting Kit-8 (CCK-8), plate clone formation, Transwell, wound healing and immunofluorescence assays were used to investigate the effects of AS-IV on HCC cell proliferation, migration and invasion. The protein expression levels were analyzed by western blotting and immunofluorescence assay. The CCK-8 and plate clone formation assays showed that AS-IV had little effect on the proliferation of HCC cells in vitro. However, the Transwell and wound healing assays demonstrated that AS-IV inhibited the migration and invasion of HCC cells in a dose-dependent manner and the morphology of HCC cells was altered from spindle into oval shaped in the AS-IV pretreated groups. The upregulation of E-cadherin and downregulation of N-cadherin, vimentin, α-SMA and Slug were also observed in the AS-IV pretreated groups. Additionally, AS-IV treatment resulted in a profound decrease in the phosphorylated forms of Akt and GSK-3β, which in turn inhibited the expression of β-catenin. Thus, we conclude that AS-IV attenuates the invasive and migratory abilities of HCC cells through the inhibition of EMT by targeting the Akt/GSK-3β/β-catenin pathway.

The original article [1] contains an error in Fig. 5a whereby the Western blot bands representing CyclinD1 have mistakenly been duplicated over the Western blot bands intended to represent SGK.

Background microRNAs (miRNAs) have been reported to modulate macrophage colony-stimulating factor (M-CSF) and macrophages. The aim of this study was to find whether miR-26a can suppress M-CSF expression and the recruitment of macrophages. Methods Hepatocellular carcinoma (HCC) cell lines with decreased or increased expression of miR-26a were established in a previous study. M-CSF expression by tumor cells was measured by enzyme-linked immunosorbent assay, and cell migration assays were used to explore the effect of HCC cell lines on macrophage recruitment in vitro. Real-time PCR measured a panel of mRNAs expressed by macrophages. Xenograft models were used to observe tumor growth. Immunohistochemistry was conducted to study the relation between miR-26a expression and M-CSF expression and macrophage recruitment in patients with HCC. Results Ectopic expression of miR-26a reduced expression of M-CSF. The conditioned medium (CM) from HepG2 cells that overexpressed miR-26a reduced the migration ability of THP-1 cells stimulated by phorbol myristate acetate (PMA) increased expression of interleukin (IL)-12b or IL-23 mRNA and decreased expression of chemokine (C-C motif) ligand (CCL)22, CCL17, and IL-10 mRNA, in comparison to the medium from the parental HepG2 cells. These effects could be interrupted by the PI3K/Akt pathway inhibitor LY294002. Ectopic expression of miR-26a in HCC cells suppressed tumor growth, M-CSF expression, and infiltration of macrophages in tumors. Similar results were also found when using HCCLM3 cells. Furthermore, the expression of miR-26a was inversely correlated with M-CSF expression and macrophage infiltration in tumor tissues from patients with HCC. Conclusions miR-26a expression reduced M-CSF expression and recruitment of macrophages in HCC.

Robo1 is a member of the Robo immunoglobulin superfamily of proteins, and it plays an important role in angiogenesis and cancer. In this study, we investigate the role of roundabout 1 (Robo1) in tumor angiogenesis in hepatocellular carcinoma (HCC). Firstly, the relationship between Robo1 expression on tumors and patient’s survival and endothelial cells in tumor blood vessels and patient’s survival was studied. Secondly, Robo1 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). Cell proliferation, motility, and tube formation were compared in HUVEC with different Robo1 expression. Also, HUVECs with different Robo1 expression were mixed with HCCLM3 and HepG2 hepatoma cells and then implanted in a nude mouse model to examine the effects of Robo1 in endothelial cells on tumor growth and angiogenesis. Cell motility-related molecules were studied to investigate the potential mechanism how Robo1 promoted tumor angiogenesis in HCC. The disease-free survival of the patients with high Robo1 expression in tumoral endothelial cells was significantly shorter than that of those with low expression (P = 0.021). Overexpression of Robo1 in HUVECs resulted in increased proliferation, motility, and tube formation in vitro. In the implanted mixture of tumor cells and HUVECs with an increased Robo1 expression, tumor growth and microvessel density were enhanced compared with controls. Robo1 promoted cell division cycle 42 (Cdc42) expression in HUVECs, and a distorted actin cytoskeleton in HUVECs was observed when Robo1 expression was suppressed. In conclusion, Robo1 promoted angiogenesis in HCC mediated by Cdc42.

M2-polarized (alternatively activated) macrophages play an important role in the progression of hepatocellular carcinoma (HCC). Allograft inflammatory factor 1 (AIF1) is overexpressed in M2-polarized macrophages. This study explored the role of AIF1 in tumor-associated macrophages in HCC. Macrophages were stimulated with colony-stimulating factor 1 (CSF1) to characterize the regulatory pathway of AIF1 in macrophages. The chromatin immunoprecipitation and luciferase reporter gene assay were conducted to examine transcription factors associated with AIF1 expression. AIF1 was down or upregulated, and the effects on tumor progression were evaluated by using in vitro and in vivo co-culture systems. A cytokine array was performed to screen the downstream functional components of AIF1. Tumor tissue from 206 patients with HCC were used to explore the clinical significance of AIF1. AIF1 induced a M2-like phenotype of macrophages. By facilitating the binding of c-Jun to the promoter of AIF1, CSF1 secreted from hepatoma cells increased AIF1 expression through the CSF1R-MEK1/2-Erk1/2-c-Jun axis. AIF1 expressed in macrophages promoted the migration of hepatoma cells in co-culture system of RAW264.7 and Hepa1-6 and tumor growth in an animal model. The cytokine array showed that CXCL16 was increased in RAW264.7 cells with overexpressed AIF1, leading to enhanced tumor cell migration. In human HCC tissue, AIF1-positive macrophages in the adjacent microenvironment was associated with microvascular invasion and advanced TNM stages and with patients' overall and disease-free survival (p = 0.002 for both). AIF1 expression in macrophages plays a pivotal role in the interaction between macrophages and hepatoma cells.

To evaluate the value of ischemic preconditioning in clinical hepatectomy.A total of 48 unselected patients undergoing liver resection were analyzed by randomized controlled trial from December 2004 to June 2006. Forty-eight unselected patients were randomized into two groups: IP group (5 minutes of ischemia followed by 5 minutes of reperfusion) and control group (received Pringle's maneuver no and no IP was given). Postoperative days 1, 3 and 7, the liver function were checked. Perioperative mortality, morbidity and hospitalized days were compared.In IP group, ischemic times were 5 - 80 min, mean 31 min, hospitalized days were 13 - 50 days, mean 20 days. In control group, ischemic times were 10 - 60 min, mean 27 min, hospitalized days were 10 - 33 days, mean 17 days. Forty-seven patients were satisfactory with postoperative recovery, except one patient died of chronic liver dysfunction after 3 months postoperatively. Postoperative days 1, 3 and 7, the ALT, AST, TBIL, ALB levels in two groups were not statistically significant (P0.05).The clinical use of IP through 5 minutes of warm ischemia in this technique of hepatectomy does not protect the liver from hepatic injury induced by the IRI.