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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.

Background The presence of hepatic vein tumor thrombus (HVTT) is a major determinant of survival outcomes in hepatocellular carcinoma (HCC) patients. This study compared survival outcomes between liver resection (LR) and intensity-modulated radiation therapy (IMRT) in HCC patients with HVTT. Methods Data from patients who underwent LR or IMRT for HCC with HVTT at the Eastern Hepatobiliary Surgery Hospital were retrospectively analyzed. Their survival outcomes were compared before and after propensity score matching (PSM). Results Three hundred and seven HCC patients with HVTT who underwent either LR (n=140) or IMRT (n=167) were enrolled. PSM matched 82 pairs of patients. The overall survival (OS) and recurrence-free survival (RFS) rates were significantly higher for patients in the LR group than those in the IMRT group. On subgroup analysis, significantly better survival outcomes were obtained after LR than IMRT in patients with peripheral type of HVTT (pHVTT) and major type of HVTT (mHVTT). However, similar survival outcomes were obtained after LR and IMRT when the HVTT had developed into inferior vena cava tumor thrombus (IVCTT). Conclusions LR resulted in significantly better survival outcomes in HCC patients with HVTT when compared to IMRT. Once the HVTT had developed IVCTT, LR and IMRT resulted in similarly bad survival outcomes.

IntroductionTo investigate the effects and mechanisms of all-trans retinoic acid (ATRA) with and without oxaliplatin (OXA) on chemotherapy-resistant hepatocellular carcinoma cell lines.Material and methodsOXA-resistant cell lines (CSQT-2-R and Hep3b-R) and subcutaneous xenograft model were used in this study. MTT assay, flow cytometry, crystal violet assay, transwell assay and western-blotting were conducted to evaluate the effects of co-treatment with ATRA and OXA on OXA-resistant HCC in vivo and in vitro. The differences between two groups were analyzed using ANOVA. All statistical tests in the study were two-sided, and statistical significance was set at PResultsWe established two oxaliplatin-Resistant HCC cell lines (CSQT-2-R and Hep3b-R). The drug resistance ability can be increased up to 100% than their parental cells(CSQT-2 and Hep3b) in certain concentration of OXA. ATRA alone could not inhibited the viability of CSQT-2-R and Hep3b-R, but it can enhance the ability of OXA on apoptosis than OXA alone (75% vs 35%, pConclusionsATRA combined with OXA can elicit cell cycle arrest of CSQT-2-R and Hep3b-R at G2/M phase, thereby inhibiting the proliferation of resistant HCC cell, which provides a new treatment for chemotherapy-resistant HCC.

Background Microvascular invasion (MVI) is a major determinant of survival outcome for hepatocellular carcinoma (HCC). This study aimed to investigate the efficacy of postoperative adjuvant Sorafenib (PA-Sorafenib) in HCC patients with MVI after R0 liver resection (LR). Methods The data of patients who underwent R0 LR for HCC with histologically confirmed MVI at the Eastern Hepatobiliary Surgery Hospital were retrospectively analyzed. The survival outcomes for patients who underwent PA-Sorafenib were compared with those who underwent R0 LR alone. Propensity score matching (PSM) analysis was performed. Results 728 HCC patients had MVI in the resected specimens after R0 resection, with 581 who underwent LR alone and 147 patients who received in additional adjuvant sorafenib. PSM matched 113 patients in each of these two groups. The overall survival (OS) and recurrence free survival (RFS) were significantly better for patients in the PA-sorafenib group (for OS: before PSM, P = 0.003; after PSM, P = 0.007), (for RFS: before PSM, P = 0.029; after PSM, P = 0.001), respectively. Similar results were obtained in patients with BCLC 0-A, BCLC B and Child-Pugh A stages of disease. Conclusions PA-Sorafenib was associated with significantly better survival outcomes than LR alone for HCC patients with MVI.

The benefits of adjuvant transarterial chemoembolization (TACE) in hepatocellular carcinoma (HCC) patients with microvascular invasion (MVI) remain controversial. We compared the efficacy and safety of adjuvant TACE and hepatic resection (HR) alone for HCC patients with MVI.The PubMed, EMBASE, Cochrane Library, VIP, Wan Fang, and Sino Med databases were systematically searched to compare adjuvant TACE and HR alone for the treatment of HCC with MVI from inception to January 1, 2019. The study outcomes, including overall survival (OS) and disease-free survival (DFS), were extracted independently by two authors.12 trials involving 2190 patients were evaluated. A meta-analysis of 11 studies suggested that the 1-, 3-, and 5-year overall survival (OS) rates (OR = 0.33, P 0.001; OR = 0.49, P 0.001; and OR = 0.59, P 0.01; respectively), favored adjuvant TACE over HR alone. 11 studies were included in the meta-analysis of DFS, and adjuvant TACE showed better 1-, 3-, and 5-DFS (OR = 0.45, P 0.001; OR = 0.50, P 0.001; and OR = 0.58, P 0.001; respectively) compared to HR alone. Subgroup analysis demonstrated that adjuvant TACE could benefit HCC patients with MVI with tumor diameter5 cm or multinodular tumors.Adjuvant TACE may improve OS and DFS for HCC patients with MVI compared to HR alone and should be recommended for selected HCC patients with MVI. However, these results need to be validated through further high-quality clinical studies.The benefits of adjuvant TACE in HCC patients with microvascular invasion remain controversial. Twelve studies involving 2190 patients were include in our meta-analysis. Adjuvant TACE may improve OS and DFS for HCC patients with MVI compared to HR alone and should be recommended for selected HCC patients with MVI.

Aims To investigate the impact of postoperative intensity modulated radiation therapy (IMRT) in patients with hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT) after partial hepatectomy +/− thrombectomy. Methods From July 2013 to June 2016, consecutive patients with HCC and PVTT who underwent partial hepatectomy were randomly assigned to 2 groups: a control group and an adjuvant IMRT group. Survival outcomes in the 2 groups were studied. Results The 52 patients in this study were equally randomized into 2 groups with comparable clinicopathological data. The median disease-free survival (DFS) and overall survival (OS) were 9.1 ± 1.6 months, 18.9 ± 1.8 months for the adjuvant RT group and 4.1 ± 0.5 months, 10.8 ± 1.3 months for the control group, respectively. The 1-, 2- and 3-years overall survival rates were significantly better in the adjuvant IMRT group (76.9%, 19.2%, and 11.5%; respectively) than the control group (26.9%, 11.5% and 0%, respectively; p = 0.005). Conclusions Postoperative IMRT significantly improved the overall survival outcomes in patients with HCC and PVTT after partial hepatectomy +/− thrombectomy.

Background The effect of microvascular invasion (MVI) on the postoperative long-term prognosis of solitary small hepatocellular carcinoma remains controversial. We compared the long-term outcomes of MVI-positive and MVI-negative groups of patients with solitary small hepatocellular carcinoma. Methods The PubMed, EMBASE, Cochrane Library, VIP, Wan Fang, and Sino Med databases were systematically searched to compare the long-term outcomes of MVI-positive and MVI-negative groups of patients with solitary small hepatocellular carcinoma from inception to November 1, 2018. The study outcomes, including overall survival (OS) and disease-free survival (DFS), were extracted independently by two authors. Results Fourteen studies involving 3033 patients were evaluated. A meta-analysis of all 14 studies suggested that the OS of the MVI-positive group was significantly worse than that of the MVI-negative group (HR = 2.39, 95% CI = 2.02–2.84, I2 = 22.8%; P Conclusion Microvascular invasion was a risk factor for poorer prognosis for solitary small hepatocellular carcinoma.

Microvascular invasion (MVI) predicts poor prognosis in patients with hepatocellular carcinoma (HCC). HCC patients with hypercoagulability are prone to develop thrombosis; however, the relationship between preoperative coagulability state, as reflected by the international normalized ratio (INR) level, and MVI remains unclear. From January 2009 to December 2012, HCC patients who underwent R0 liver resection (LR) from four cancer centers entered into this study. The overall survival (OS) and recurrence-free survival (RFS) rates were compared using the Kaplan–Meier method and Cox regression analysis. Of the 2509 HCC patients who were included into this study, 1104 were found to have MVI in the resected specimens. These patients were divided into the low (n = 151), normal (n = 796), and high (n = 157) INR subgroups based on the preoperative INR levels. The low INR subgroup had a significantly higher incidence of MVI than the normal or high INR subgroups (61.6% vs. 41.6% vs. 44.6%; p

Due to the high incidence of liver cancer, chemoradiotherapy and prognosis of liver cancer are a primary focus of medical research. microRNAs (miRNAs/miRs) serve crucial roles in resistance to chemotherapy and radiotherapy. The aim of the present study was to investigate the effects of miR-101 on the chemotherapeutic efficacy of cisplatin (CDDP) in liver cancer. First, human liver cancer cells (HepG2) were transfected with a miR-101 mimic or miR-101 inhibitor to bidirectionally regulate the expression of miR-101. Cell proliferation, apoptosis, intracellular reactive oxygen species and comet assay results indicated that the upregulation of miR-101 sensitized HepG2 cells to CDDP, and downregulation of miR-101 reduced chemosensitivity. A xenograft mouse model further confirmed that miR-101 overexpression increased CDDP sensitivity in liver cancer. Luciferase reporter and western blotting assays demonstrated that transfection of the miR-101 mimic markedly reduced activity of the DNA-dependent protein kinase catalytic subunit/protein kinase B/mammalian target of rapamycin (DNA-PKcs/Akt/mTOR) pathway and increased expression of apoptotic protein caspase 3, which is induced by CDDP treatment. By contrast, miR-101 inhibitors partially reversed these changes. Moreover, the miR-101 mimic suppressed activity of the nuclear factor-κB (NF-κB) pathway, leading to increased susceptibility of HepG2 cells to chemotherapeutic agents. In conclusion, miR-101 overexpression augmented cytotoxicity and reduced chemoresistance to CDDP in HepG2 cells, and this was associated with negative regulation of DNA-PKcs/Akt/NF-κB signaling.

Vascular invasion is a major determinant of survival outcomes after curative resection for hepatocellular carcinoma (HCC) patients. This study was designed to investigate the efficacy of postoperative adjuvant transarterial chemoembolization (PA-TACE) in patients with HCC with hepatic vein tumor thrombus (HVTT). Data from patients who underwent LR for HCC with HVTT at the Eastern Hepatobiliary Surgery Hospital were retrospectively analyzed. The survival outcomes for patients who underwent PA-TACE after LR were compared with those who underwent LR alone. Propensity score matching (PSM) analysis was performed to match patients in a ratio of 1:1. All included 319 patients who underwent LR for HCC with HVTT, 134 underwent LR alone (the LR group), and 185 patients underwent in adjuvant TACE (the PA-TACE group). PSM matched 107 patients in two groups. The overall survival (OS) and recurrence-free survival (RFS) were significantly better for patients in the PA-TACE group than the LR group (for OS: before PSM, P

Portal vein tumor thrombus (PVTT) is one of the most serious forms of hepatocellular carcinoma (HCC) vessel metastasis and has a poor survival rate. However, the molecular mechanism of PVTT has not yet been elucidated. In this study, the molecular mechanism of AXL expressed in tumor-derived endothelial cells (TECs) in vessel metastasis was investigated. High AXL expression was observed in TECs, but not in the tumor cells of HCC patients with PVTT and this was associated with poor overall survival (OS) and disease-free survival (DFS). AXL overexpression was positively associated with CD 31 expression both in vitro and in vivo. AXL promoted the cell proliferation, tube formation, and migration of both TECs and normal endothelial cells (NECs). High expression of AXL in TECs promoted the cell migration, but not the proliferation of HCC cells. Further studies demonstrated that AXL promoted cell migration and tube formation through activation of the PI3K/AKT/SOX2/DKK-1 axis. AXL overexpression in HUVECs promoted tumor growth and liver or vessel metastasis of HCC in xenograft nude mice, which could be counteracted by treatment with R428, an AXL inhibitor. R428 reduced tumor growth and CD 31 expression in HCC in PDX xenograft nude mice. Therefore, AXL over-expression in TECs promotes vessel metastasis of HCC, which indicates that AXL in TECs could be a potential therapeutic target in HCC patients with PVTT.

BACKGROUND: Hepatocellular carcinoma (HCC) commonly occurs in patients with splenomegaly. This study aimed to investigate the impact of splenomegaly with or without splenectomy on long-term survival of HCC patients with portal vein tumor thrombus (PVTT) treated with liver resection (LR). METHODS: HCC patients with PVTT who underwent LR from 2005 to 2012 from 6 hospitals were retrospectively studied. The long-term overall survival (OS) and recurrence-free survival (RFS) were compared between patients with or without splenomegaly, and between patients who did or did not undergo splenectomy for splenomegaly. Propensity score matching (PSM) analysis was performed to match patients in a 1:1 ratio. RESULTS: Of 716 HCC patients with PVTT who underwent LR, 140 patients had splenomegaly (SM group) and 576 patients had no splenomegaly (non-SM group). The SM group was further subdivided into 49 patients who underwent splenectomy (SPT group), and 91 patients who did not received splenectomy (non-SPT group). PSM matched 140 patients in the SM group, and 49 patients in the SPT group. Splenomegaly was an independent risk factor of poor RFS and OS. The OS and RFS rates were significantly better for patients in the non-SM group than the SM group (OS: P

BACKGROUND: The effect of bile duct tumor thrombus (BDTT) on the postoperative long-term prognosis of hepatocellular carcinoma (HCC) patients is still under debate. METHODS: The PubMed, Embase, Cochrane Library, Web of Science databases were systematically searched to collect the clinicopathologic characteristics, perioperative indices, and postoperative survival outcomes in the BDTT and non-BDTT groups of HCC patients from inception to February 1, 2020. The study outcomes were extracted by two independent investigators. RESULTS: A total of 15 studies involving 6,484 patients were included. The meta-analysis revealed that the levels of serum total bilirubin and alkaline phosphatase were notably higher in patients with HCC and BDTT than those without BDTT. Meanwhile, HCC patients with BDTT had more aggressive biological characteristics, such as poor tumor differentiation, macrovascular invasion, and lymph node metastasis, as compared to patients without BDTT. The 1-year [odds ratio (OR) 0.39, 95% confidence interval (CI): 0.31–0.48, P

Background: Hepatic vein tumor thrombus (HVTT) is a significant poor risk factor for survival outcomes in hepatocellular carcinoma (HCC) patients. Currently, the widely used international staging systems for HCC are not refined enough to evaluate prognosis for these patients. A new classification for macroscopic HVTT was established, aiming to better predict prognosis. Methods: This study included 437 consecutive HCC patients with HVTT who underwent different treatments. Overall survival (OS) and time-dependent receiver operating characteristic (ROC) curve area analysis were used to determine the prognostic capacities of the new classification when compared with the different currently used staging systems. Results: The new HVTT classification was defined as: type I, tumor thrombosis involving hepatic vein (HV), including microvascular invasion; type II, tumor thrombosis involving the retrohepatic segment of inferior vena cava; and type III, tumor thrombosis involving the supradiaphragmatic segment of inferior vena cava. The numbers (percentages) of patients with types I, II, and III HVTT in the new classification were 146 (33.4%), 143 (32.7%), and 148 (33.9%), respectively. The 1-, 2-, and 3-year OS rates for types I to III HVTT were 79.5%, 58.6%, and 29.1%; 54.8%, 23.3%, and 13.8%; and 24.0%, 10.0%, and 2.1%, respectively. The time-dependent-ROC curve area analysis demonstrated that the predicting capacity of the new HVTT classification was significantly better than any other staging systems. Conclusions: A new HVTT classification was established to predict prognosis of HCC patients with HVTT who underwent different treatments. This classification was superior to, and it may serve as a supplement to, the commonly used staging systems.

G protein-coupled receptors (GPCRs) are the most frequent targets of approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we utilized a combined computational and experimental framework that integrated extensive molecular dynamics simulations, Markov state models, and site-directed mutagenesis for investigating the conformational landscape of activation of the angiotensin II (AngII) type 1 receptor (AT1R), a prototypical class A GPCR. Our findings suggested a synergistic transition mechanism of AT1R activation. Importantly, a key intermediate state was found in the activation pathway. We discovered a novel “cryptic” binding site in the intracellular region of the receptor in the intermediate state. Furthermore, a bioluminescence resonance energy transfer analysis revealed the insensitivity of the endogenous AngII octapeptide agonist in the activation of the downstream Gq signaling and β-arrestin-mediated pathways, upon mutations of the predicted cryptic binding site, thereby suggesting an allosteric regulatory mechanism. Together, these findings not only provide a deeper understanding of AT1R activation at an atomic level, but also open potential avenues for the design of allosteric AT1R modulators. Consequently, this will provide a broad range of applications for GPCR biology, biophysics, and medicinal chemistry.

Aim Because of the rarity of hepatic vein tumor thrombus (HVTT) in patients with hepatocellular carcinoma (HCC), little is known about HVTT. Thus, the survival benefit of liver resection (LR) versus transcatheter arterial chemoembolization (TACE) for HCC patients with HVTT or inferior vena cava tumor thrombus (IVCTT) remains controversial. We aimed to explore the survival benefits of LR versus TACE for the treatment of these patients. Methods From 2012 to 2016, a total of 276 patients with HVTT or IVCTT who underwent liver resection or TACE were enrolled in this study. Patients in the LR group were matched at a 1:1 ratio with patients treated with TACE as an initial treatment (TACE group). Clinical characteristics, overall survival, and disease-free survival were analyzed. Results The median survival time in the LR group was 4.7 months longer than that in the TACE group before PSM (19.4 vs. 14.7 months, P = 0.006) and 6.9 months longer than that in the TACE group after PSM (20.9 vs. 14.0 months, P = 0.019). The median disease-free survival time in the LR group was 3.2 months longer than that in the TACE group before PSM (12.3 vs. 9.1 months, P = 0.038) and 5.8 months longer than that in the TACE group after PSM (13.0 vs. 7.2 months, P = 0.011). Conclusion Liver resection provides a good prognosis for HCC patients with HVTT or IVCTT compared with patients undergoing TACE, and coexistence with portal vein tumor thrombus is the most important factor related to survival.

MicroRNA-96 (miR-96) has been revealed serve an oncogenic role in various types of cancer. However, the role of miR-96 in cholangiocarcinoma (CCA) development and progression is yet to be elucidated. Thus, the aim of the present study was to investigate the role of miR-96 in CCA. The expression pattern of miR-96 in CCA tissues and cell lines was evaluated using reverse transcription-quantitative PCR analysis. Kaplan-Meier curves and Cox regression analyses were conducted to investigate the prognostic significance of miR-96 in CCA. Cell Counting Kit-8 and Transwell assays were performed to identify the functions of miR-96. The association between miR-96 and metastasis suppressor-1 (MTSS1) was verified using a dual-luciferase assay. The results demonstrated that miR-96 expression levels were increased in CCA tissues and cell lines compared with those in adjacent normal tissues and normal human intrahepatic biliary epithelial cell lines, respectively. High expression levels of miR-96 were significantly associated with lymph node metastasis, differentiation and TNM stage. In addition, upregulated expression of miR-96 was associated with a poorer prognosis and was predicted to be a prognostic factor in patients with CCA. Overexpression of miR-96 in vitro promoted CCA cell proliferation, migration and invasion. Additionally, MTSS1 was identified as a direct target of miR-96. The results of the present study indicated the clinical and biological importance of miR-96 as an oncogene in CCA. miR-96 may represent an independent prognostic biomarker and may promote CCA cell proliferation, migration and invasion by targeting MTSS1.

Radiofrequency ablation (RFA) is one of the standards of care for early stage hepatocellular carcinoma (HCC). However, rapid progression of residual tumor after RFA has been confirmed. The aim of this study was to investigate the underlying mechanism of this phenomenon. Human HCC cell lines HCCLM3 and HepG2 were employed to establish insufficient RFA models in vivo and in vitro, respectively. The effects of insufficient RFA on metastatic potential of residual tumors were evaluated. The molecular changes after insufficient RFA were evaluated by PCR array, western blot, immunofluorescence, and immunohistochemistry. Results showed that insufficient RFA significantly promoted lung and intrahepatic residual tumor cells in vivo, and heat intervention promoted migration and invasion of hepatoma cells in vitro. PCR array revealed that the expression of integrin β3 (ITGB3) and MMP2 were up-regulated in the residual tumors of HCCLM3 xenograft model. The up-regulation of ITGB3 was confirmed by qRT-PCR, Western blot and immunohistochemistry. Knockdown ITGB3 expression in HCCLM3 cells by shRNA significantly lowered the pro-metastatic effects of insufficient RFA. Mechanism studies indicated that ITGB3 mediated the expression of MMP2 by activing FAK/PI3K/AKT signaling pathway. The up-regulation of ITGB3 contributed to enhanced metastatic potential of residual cancer in HCCLM3 model after insufficient RFA. Targeting ITGB3 expression may further improve the clinical effects of RFA.

Hepatic vein tumour thrombus (HVTT) is a major determinant of survival outcomes for patients with hepatocellular carcinoma (HCC). An Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT model was established to predict the prognosis of patients with HCC and HVTT after liver resection, in order to identify optimal candidates for liver resection.Patients with HCC and HVTT from 15 hospitals in China were included. The EHBH-HVTT model with contour plot was developed using a non-linear model in the training cohort, and subsequently validated in internal and external cohorts.Of 850 patients who met the inclusion criteria, there were 292 patients who had liver resection and 198 who did not in the training cohort, and 124 and 236 in the internal and external validation cohorts respectively. Contour plots for the EHBH-HVTT model were established to predict overall survival (OS) rates of patients visually, based on tumour diameter, number of tumours and portal vein tumour thrombus. This differentiated patients into low- and high-risk groups with distinct long-term prognoses in the liver resection cohort (median OS 34·7 versus 12·0 months; P 0·001), internal validation cohort (32·8 versus 10·4 months; P = 0·002) and external validation cohort (15·2 versus 6·5 months; P = 0·006). On subgroup analysis, the model showed the same efficacy in differentiating patients with HVTT in peripheral and major hepatic veins, the inferior vena cava, or in patients with coexisting portal vein tumour thrombus.The EHBH-HVTT model was accurate in predicting prognosis in patients with HCC and HVTT after liver resection. It identified optimal candidates for liver resection among patients with HCC and HVTT, including tumour thrombus in the inferior vena cava, or coexisting portal vein tumour thrombus.La trombosis tumoral de la vena hepática (hepatic vein tumour thrombus, HVTT) es un determinante importante de los resultados de supervivencia en pacientes con carcinoma hepatocelular (hepatocellular carcinoma, HCC). Se desarrolló el modelo llamado Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT para predecir el pronóstico de los pacientes con HCC y HVTT después de la resección hepática (liver resection, LR), con el fin de identificar los candidatos óptimos para LR entre estos pacientes. MÉTODOS: Se incluyeron pacientes con HCC y HVTT de 15 hospitales en China. El modelo EHBH-HVTT con gráfico de contorno se desarrolló utilizando un modelo no lineal en la cohorte de entrenamiento, siendo posteriormente validado en cohortes internas y externas.De 850 pacientes que cumplieron con los criterios de inclusión, hubo 292 pacientes en el grupo LR y 198 pacientes en el grupo no LR en la cohorte de entrenamiento, y 124 y 236 en las cohortes de validación interna y externa. Los gráficos de contorno del modelo EHBH-HVTT se establecieron para predecir visualmente las tasas de supervivencia global (overall survival, OS) de los pacientes, en función del diámetro del tumor, número de tumores y del trombo tumoral de la vena porta (portal vein tumour thrombus, PVTT). Esto diferenciaba a los pacientes en los grupos de alto y bajo riesgo, con distinto pronóstico a largo plazo en las 3 cohortes (34,7 versus 12,0 meses, 32,8 versus 10,4 meses y 15,2 versus 6,5 meses, P 0,001). En el análisis de subgrupos, el modelo mostró la misma eficacia en la diferenciación de pacientes con HVTT, con trombo tumoral en la vena cava inferior (inferior vena cava tumour thrombus, IVCTT) o en pacientes con PVTT coexistente. CONCLUSIÓN: El modelo EHBH-HVTT fue preciso para la predicción del pronóstico en pacientes con HCC y HVTT después de la LR. Identificó candidatos óptimos para LR en pacientes con HCC y HVTT, incluyendo IVCTT o PVTT coexistente.

Background Microvascular invasion (MVI) is associated with poor postoperative survival outcomes in patients with hepatocellular carcinoma (HCC). An Eastern Hepatobiliary Surgery Hospital (EHBH) MVI scoring system was established to predict prognosis in patients with HCC with MVI after R0 liver resection (LR) and to supplement the most commonly used classification systems. Materials and Methods Patients with HCC with MVI who underwent R0 LR as an initial therapy were included. The EHBH-MVI score was developed from a retrospective cohort from 2003 to 2009 to form the training cohort. The variables associated with overall survival (OS) on univariate analysis were subsequently investigated using the log-rank test, and the EHBH-MVI score was developed using the Cox regression model. It was validated using an internal prospective cohort from 2011 to 2013 as well as three independent external validation cohorts. Results There were 1,033 patients in the training cohort; 322 patients in the prospective internal validation cohort; and 493, 282, and 149 patients in the three external validation cohorts, respectively. The score was developed using the following factors: α-fetoprotein level, tumor encapsulation, tumor diameter, hepatitis B e antigen positivity, hepatitis B virus DNA load, tumor number, and gastric fundal/esophageal varicosity. The score differentiated two groups of patients (≤4, >4 points) with distinct long-term prognoses outcomes (median OS, 55.8 vs. 19.6 months; p < .001). The predictive accuracy of the score was greater than the other four commonly used staging systems for HCC. Conclusion The EHBH-MVI scoring system was more accurate in predicting prognosis in patients with HCC with MVI after R0 LR than the other four commonly used staging systems. The score can be used to supplement these systems. Implications for Practice Microvascular invasion (MVI) is a major determinant of survival outcomes after curative liver resection for patients with hepatocellular carcinoma (HCC). Currently, there is no scoring system aiming to predict prognosis of patients with HCC and MVI after R0 liver resection (LR). Most of the widely used staging systems for HCC do not use MVI as an independent risk factor, and they cannot be used to predict the prognosis of patients with HCC and MVI after surgery. In this study, a new Eastern Hepatobiliary Surgery Hospital (EHBH) MVI scoring system was established to predict prognosis of patients with HCC and MVI after R0 LR. Based on the results of this study, postoperative adjuvant therapy may be recommended for patients with HCC and MVI with an EHBH-MVI score >4. This score can be used to supplement the currently used HCC classifications to predict postoperative survival outcomes in patients with HCC and MVI.

Background Among patients with hepatocellular carcinoma (HCC), 85% of patients have an advanced disease stage at diagnosis and curative therapies cannot be performed. Prognosis has been quite poor as until recently there was no proven effective chemotherapy. Our group found that all-trans-retinoic acid (ATRA) could improve the efficacy of platinum in HCC in vivo and in vitro, thus we wish to validate the efficiency of ATRA in clinical practice. Methods This is a double-blinded, 1:1 randomized, controlled, multicenter clinical trial. Three hundred and sixty-eight patients with HCC and extrahepatic metastases will receive palliative chemotherapy at the Eastern Hepatobiliary Surgery Hospital, First Hospital of Jilin University and Fujian Provincial Cancer Hospital. Subjects will be randomly assigned to one of the two arms, either ATRA + oxaliplatin + 5-fluorouracil/leucovorin (FOLFOX4) or FOLFOX4 alone. ATRA 20 mg will be given orally three times/day for 3 days prior to the initiation of FOLFOX4. ATRA will be discontinued at the end of FOLFOX4. Discussion Overall survival rate is the primary endpoint. Secondary endpoints are time to progression according to the modified response evaluation criteria in solid tumors (mRECIST) criteria, acute and chronic adverse events, and quality of life. Trial registration Chinese Clinical Trial Registry, ChiCTR-IIR-17012916. Registered on 9 October 2017. Electronic supplementary material The online version of this article (10.1186/s13063-019-3349-9) contains supplementary material, which is available to authorized users.

The aim of this study was to evaluate the effect of portal vein tumor thrombus (PVTT) on the prognosis of patients undergoing liver resection (LR) for primary liver malignancies (PLC).The recurrence-free survival (RFS) and overall survival (OS) for patients undergoing LR with and without PVTT for three primary liver malignancies, including hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and hepato-cholangio carcinoma (CHC) were compared using the Kaplan-Meier method and Cox regression analysis.In total, 3775 patients with PLC who underwent LR were included in this study. The incidence of PVTT in patients undergoing LR with HCC, IHC and CHC were 46%, 20%, and 17%, respectively. The median RFS and OS were significantly better for patients with HCC as compared to ICC or CHC (16 vs 11 vs 13 months; 21 vs 16 vs 18 months, respectively; P 0.001). However, the presence of PVTT resulted in similarly poor RFS and OS in these 3 subgroups of patients (9 vs 8 vs 8 months, P = 0.062; 14 vs 13 vs 12 months, respectively, P = 0.052).Although the prognosis of patients with PLC varied by histological subtype, once PVTT occurred, survival outcomes after LR were similarly poor across all three subgroups.

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.

BACKGROUND: The optimal surgical modality for hepatocellular carcinoma (HCC) with bile duct tumor thrombus (BDTT) remains controversial, especially regarding deciding whether to perform concurrent bile duct resection (BDR). METHODS: PubMed, EMBASE, Cochrane Library, Web of Science and Scopus databases were systematically searched from inception to February 2020, in order to compare overall survival (OS) and recurrence-free survival (RFS) rates of HCC patients with BDTT who had either received hepatectomy with extrahepatic bile duct resection (BDR group) or hepatectomy without bile duct resection (NBDR group). Relevant outcomes were extracted by two investigators. RESULTS: A total of 12 studies involving 355 patients was included. The 1-, 3- and 5-year OS rates were similar in the BDR and NBDR groups (OR =0.58, 95% CI: 0.31–1.09, P=0.09; OR =0.74, 95% CI: 0.43–1.28, P=0.28; OR =0.63, 95% CI: 0.36–1.11, P=0.11, respectively). However, the BDR group had better 1-, 3- and 5-year RFS rates than the NBDR group (OR =0.38, 95% CI: 0.22–0.65, P

Background: Several multidisciplinary treatment strategies are now available to treat hepatocellular carcinoma (HCC) and portal vein tumour thrombus (PVTT). However, most of these studies were not powered to test the differences in overall survival (OS). Methods: The PubMed, Cochrane Library, Web of Science and Embase were searched to identify both the direct and indirect evidence for these treatments on patients with HCC and PVTT up to December 31, 2017. A Bayesian random-effects model was used to combine direct with indirect evidence. Results: After screening 1675 citations, 54 trials included 11311 participants and 8 multidisciplinary treatments. The tests of inconsistency and heterogeneity for the network meta-analysis showed the P-values of global inconsistency to range from 0.4012 to 0.6972, and the I2 values to range from 0% to 7% for the 1-, 2-, 3- and 5-year overall survival (OS) rates. After adjusting the network model by Bayesian meta-analysis with clinical variables, a stable ranking of eight treatments was achieved. The results showed that patients who received Sorafenib (SURCA=35.1) was not better than the other treatments. Comprehensive treatments based on surgery (CST) had better OS rates than patients who received other treatments (SURCA =89.6 and the odds ratio (OR) to range from 1.39 to 108.44). Conclusion: CST significantly improved OS when compared with sorafenib and other multidisciplinary treatments, especially in selected HCC patients with PVTT limited to a first-order branch or above of the main portal vein, and with good liver function. Funding Statement: This work is supported by these funding sources as following: the Key Project of Natural Science Foundation of China (No: 81730097); the grants of the Science Fund for Creative Research Groups (No: 81521091); Chang Jiang Scholars Program (2013) of the China Ministry of Education; the National Key Basic Research Program “973 project” (No: 2015CB554000); the National Natural Science Foundation of China (No: 81602523 and No: 81702335) Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: This study has been registered with PROSPERO under the number CRD42018104670.

Portal vein tumor thrombus (PVTT) is a significant poor prognostic factor for hepatocellular carcinoma (HCC). Patients with PVTT limited to a first-order branch of the main portal vein (MPV) or above could benefit from negative margin (R0) liver resection (LR). An Eastern Hepatobiliary Surgery Hospital (EHBH)/PVTT scoring system was established to predict the prognosis of HCC patients with PVTT after R0 LR and guide selection of subgroups of patients that could benefit from LR. HCC patients with PVTT limited to a first-order branch of the MPV or above who underwent R0 LR as an initial therapy were included. The EHBH-PVTT score was developed from a retrospective cohort in the training cohort using a Cox regression model and validated in a prospective internal validation cohort and three external validation cohorts. There were 432 patients in the training cohort, 285 in the prospective internal validation cohort, and 286, 189, and 135 in three external validation cohorts, respectively. The score was calculated using total bilirubin, α-fetoprotein (AFP), tumor diameter, and satellite lesions. The EHBH-PVTT score differentiated two groups of patients (≤/>3 points) with distinct long-term prognoses (median overall survival [OS], 17.0 vs. 7.9 months; P < 0.001). Predictive accuracy, as determined by the area under the time-dependent receiver operating characteristic curves (AUCs; 0.680-0.721), was greater than that of the other commonly used staging systems for HCC and PVTT. Conclusion: The EHBH-PVTT scoring system was more accurate in predicting the prognosis of HCC patients with PVTT than other staging systems after LR. It selected appropriate HCC patients with PVTT limited to a first-order branch of the MPV or above for LR. It can be used to supplement the other HCC staging systems.

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.

Rnd1, a member of Rho GTPases, was found to be downregulated in human malignancies and downregulation of Rnd1 promotes tumor invasion via various mechanisms. However, the role of Rnd1 in hepatocellular carcinoma (HCC) progression remains unclear. In this study, our results demonstrated that Rnd1 was downregulated in HCC cells and in human HCC tissues. Low expression of Rnd1 was associated with aggressive clinic-pathologic characteristics, such as vascular invasion, and poor prognosis in patients who underwent curative surgery for HCC. Overexpression of Rnd1-suppressed cell growth, migration, invasion, and EMT processes in vitro and in vivo. Furthermore, Rnd1 blocked HCC progression by restricting EMT process through inhibition of the Raf/MEK/ERK cascade, and this was correlated with a reduction in RhoA activity. Combination of Rnd1 overexpression with sorafenib, a Raf signaling pathway inhibitor, showed a more potent inhibition on HCC metastasis. Moreover, epigenetic inhibitors (5-Aza and SAHA) increased the expression of Rnd1, and potentiated sorafenib-induced toxicity in HCC cells. In a conclusion, Rnd1-suppressed EMT-mediated metastasis of HCC by reducing the activity of the RhoA/Raf/MEK/ERK signaling pathway, functioning as a favorable anti-metastasis target for HCC patients. Rnd1 overexpression in combination with sorafenib may result in enhanced anti-metastasis efficacy in HCC.

IL-8 over-expression could enhance cancer metastasis. In present study, berberine hydrochloride (BER) triggered proliferative inhibition and G2/M arrest in AGS cells, down-regulated protein expression of cyclin B1, Bcl-2, up-regulated expression of p21, p53 and cleaved caspase 3, but showed no effect on protein expression of CHOP, Bip, and caspase 4. BER could down-regulate the enhanced IL-8 expression through down-regulating ERK1/2 and p38 MAPK over-activation induced by SN 38. The increased IL-8 mediated adhesive ability of AGS cells to HUVECs induced by SN 38, could be reduced by BER. Thus, BER could reduce the side-effect of SN 38 in clinic.

e14707 Background: There is an unmet need for the postoperative adjuvant therapy in hepatocellular carcinoma (HCC) patients with microvascular invasion (MVI), which is associated with early recurrence after curative resection. This study aimed to investigate the role of Sorafenib as a postoperative adjuvant therapy in HCC patients with MVI after R0 liver resection (LR). Methods: Consecutive HCC patients with pathologically confirmed MVI who underwent R0 LR between January 2009 and December 2016 at the Eastern Hepatobiliary Surgery Hospital were retrospectively reviewed. Patients in the LR+sorafenib group (n = 581) received oral sorafenib (400 mg/d) at 1 weeks after R0 LR, while patients in the LR group (n = 147) only underwent R0 LR. Propensity score matching (PSM) was performed to balance the baseline characteristics between the two groups. Overall survival (OS) and recurrence-free survival (RFS) were followed up, and multivariate Cox regression analysis was performed. Results: After PSM, there were 113 patients in each group. The LR+sorafenib group demonstrated improved OS (median, 43.3 vs. 34.9 months; 1-year, 85% vs. 65%; 3-year, 66% vs. 51%; 5-year, 57% vs. 37%; log-rank P = 0.007) and RFS (median, 32.0 vs. 15.0 months; 1-year, 72% vs. 55%; 3-year, 47% vs. 36%; 5-year, 39% vs. 19%; log-rank P = 0.001) compared with the LR group after PSM. Multivariate Cox regression analysis indicated that R0 LR with postoperative sorafenib therapy was associated with better OS and PFS ( P < 0.001). On subgroup analysis, similar survival benefits were observed for sorafenib in patients with Barcelona Clinic Liver Cancer (BCLC) 0-A, BCLC B and Child-Pugh A stages of disease. Conclusions: Sorafenib used as a postoperative adjuvant therapy in HCC patients with MVI after R0 LR was associated with significantly better survival outcomes than LR alone.

Antiangiogenic therapy, specially sorafenib, has become the standard of care for patients with advanced hepatocellular carcinoma (HCC), however, the improvement in survival time is not satisfactory. Previous studies have found that, in some circumstances, antiangiogenic therapy promoted tumor metastasis and the mechanistic studies were mainly focus on cancer-cell-autonomous manners. In two experimental metastasis models with tail-vein injection with hepatoma cells and an orthotopic HCC mouse model, we found that pretreatment with two vascular endothelial growth factor receptor (VEGFR) inhibitors, sunitinib and sorafenib, facilitated tumor cell survival in blood stream and promoted lung metastasis from tumors that were subsequently incubated after drug discontinuation, indicating that host response joined into the pro-metastatic effects. An antibody microarray identified that interleukin (IL)-12b was decreased in the peripheral blood of the mice treated with the two VEGFR inhibitors. IL-12b suppression in macrophages and dendritic cells from host organs was found to play a crucial role in treatment-induced metastasis. Supplement with recombinant mouse IL-12b or restoration of IL-12b expression in the host by zoledronic acid, which was previously reported to enhance IL-12 expression in vitro and in vivo, alleviated the metastasis-promoting effects of sunitinib and sorafenib. These studies suggest that host response to VEGFR inhibitors facilitates HCC metastasis and restoration of IL-12b expression could translate into clinical benefits.

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.

This multicenter-based retrospective study aimed to investigate the prognostic factors and report our experiences with the diagnosis and treatment of hepatic epithelioid hemangioendothelioma (HEHE), a rare malignant vascular tumor.A total of 33 patients with HEHE from two centers between 2004 and 2011 were retrospectively reviewed with respect to their clinical, radiologic, and pathologic characteristics; treatment modalities and outcomes; and potential prognostic factors.A total of 17 patients underwent liver resections (LRs) alone, 12 patients had transcatheter arterial chemoembolization (TACE) alone, three patients had LR followed by TACE, and one patient underwent liver transplantation (LT). The difference of overall survival (OS) between LR and TACE was not significant (p = 0.499). Older patients [≥47 years, n = 17; p = 0.035, hazard ratio (HR) = 7.0), those with symptoms (n = 17; p = 0.001, HR = 86.5], and those with an elevated serum CA19-9 level (37 U/ml, n = 5; p = 0.018, HR = 5.0) had a poorer OS, according to univariate analysis. The presence of symptoms was validated as a prognostic factor (p = 0.012) by multivariate analysis.Liver resection and TACE have comparable outcomes in HEHE patients. The presence of symptoms indicates a poor prognosis. Older age and elevated serum CA19-9 are potential negative impact factors on outcome.

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.