Role of Heavy Water in Modified University of Wisconsin Solution for Extended Cold Storage of Rat Liver.

To resolve the critical donor shortage worldwide, enlarging the potential donor pool to include expanded criteria donors is necessary. Despite numerous attempts to establish new preservation solutions, no dramatic innovation has occurred since University of Wisconsin (UW) solution displaced Euro Collins' solution; UW solution remains the global gold standard. We previously developed a heavy water (D2O)-containing organ storage solution, Dsol, which is effective for livers subjected to extended cold storage (CS), and reported its effectiveness. Dsol is a modified UW solution; however, the substances or conditions that exhibit a synergistic or additive effect with D2O are unclear. Here we made UWD solution by removing hydroxyethyl starch (HES) from and adding 30%-D2O to UW solution, and compared the effects of these solutions. After 48 hours of CS, the livers were reperfused at 37 °C on an isolated perfused rat liver apparatus, and their perfusion kinetics, functions, and injuries were compared. In the UW group, portal vein resistance significantly increased and the oxygen consumption rate and bile production decreased; in contrast, these changes were suppressed in the UWD group. Organ expansion and liver damage progressed in both groups. These results confirmed that the removal of HES from and addition of D2O to the UW solution reduced CS-induced cellular function impairments and microcirculatory disorders. However, to reduce injury during reperfusion after CS, it is necessary to provide conditions that inhibit injury progression after reperfusion.

Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver.

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

Expression Analysis of Early Metastatic Seeding of Colorectal Cancer.

BACKGROUND: Distant metastasis is the leading cause of death in patients with colorectal cancer (CRC). Tumor dissemination for metastasis formation occurs in advanced cancers and also during early stages of tumorigenesis. Here, we investigated the genes involved in early metastatic seeding of CRC using gene expression analysis. PATIENTS AND METHODS: We performed a cDNA microarray using specimens resected from stages I-II CRC with and without metachronous metastatic recurrence. For the candidate genes, we immunohistochemically validated protein expression using a tissue microarray of stages I-III CRC. RESULTS: The expression of TROP2, VWCE, and BMP7 was upregulated in the recurrence group rather than in the non-recurrence group. Protein expression analysis revealed significant association of these genes with distant metastatic recurrence. The specimens with high expression of BMP7 showed worse recurrence-free survival (RFS; p = 0.02). Those with high expression of TROP2 and VWCE showed worse overall survival (OS) and RFS (TROP2: p = 0.01 and p = 0.03; VWCE: p < 0.05 and p < 0.001, respectively). In the multivariate analysis, high expression of VWCE and BMP7 was an independent predictor of recurrence [VWCE: hazard ratio (HR) 3.41, p < 0.001; BMP7: HR 2.93, p = 0.005]. In contrast, TROP2 was an independent prognostic factor for OS (HR 4.58, p = 0.03). CONCLUSIONS: Gene expression analysis revealed that TROP2, VWCE, and BMP7 were involved in early metastatic seeding. The high expression of these genes may warrant careful surveillance or adjuvant therapy, even in stages I-II CRC cases.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers.

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Hypothermic Machine Perfusion with Hydrogen Gas Reduces Focal Injury in Rat Livers but Fails to Restore Organ Function.

BACKGROUND: We have previously reported the efficacy of post-reperfusion H2 gas treatment in cold storage (CS) and subsequent reperfusion of the rat liver. The present study aimed to evaluate the effect of H2 gas treatment during hypothermic machine perfusion (HMP) in rat livers retrieved from donation after circulatory death (DCD) and elucidate the mechanism of action of H2 gas. METHODS: Liver grafts were procured from rats after 30 min of cardiopulmonary arrest. The graft was subjected to HMP for 3 hours at 7°C using Belzer MPS with or without dissolved H2 gas. The graft was reperfused using an isolated perfused rat liver apparatus at 37°C for 90 minutes. Perfusion kinetics, liver damage, function, apoptosis, and ultrastructure were evaluated. RESULTS: Portal venous resistance, bile production, and oxygen consumption rates were identical in the CS, MP, and MP-H2 groups. Liver enzyme leakage was suppressed by MP (vs control), whereas H2 treatment did not show a combination effect. Histopathology revealed poorly stained areas with a structural deformity just below the liver surface in the CS and MP groups, whereas these findings disappeared in the MP-H2 group. The apoptotic index in the CS and MP groups was high but decreased in the MP-H2 group. Mitochondrial cristae were damaged in the CS group but preserved in the MP and MP-H2 groups. CONCLUSIONS: In conclusion, HMP and H2 gas treatment are partly effective in DCD rat livers but insufficient. Hypothermic machine perfusion can improve focal microcirculation and preserve mitochondrial ultrastructure.

Quantification of DNA methylation for carcinogenic risk estimation in patients with non-alcoholic steatohepatitis.

BACKGROUND: In recent years, non-alcoholic steatohepatitis (NASH) has become the main cause of hepatocellular carcinoma (HCC). As a means of improving the treatment of NASH-related HCCs based on early detection, this study investigated the feasibility of carcinogenic risk estimation in patients with NASH. RESULTS: Normal liver tissue (NLT), non-cancerous liver tissue showing histological findings compatible with non-alcoholic fatty liver from patients without HCC (NAFL-O), non-cancerous liver tissue showing NASH from patients without HCC (NASH-O), non-cancerous liver tissue showing non-alcoholic fatty liver from patients with HCC (NAFL-W), non-cancerous liver tissue showing NASH from patients with HCC (NASH-W) and NASH-related HCC were analyzed. An initial cohort of 171 tissue samples and a validation cohort of 55 tissue samples were used. Genome-wide DNA methylation screening using the Infinium HumanMethylation450 BeadChip and DNA methylation quantification using high-performance liquid chromatography (HPLC) with a newly developed anion-exchange column were performed. Based on the Infinium assay, 4050 CpG sites showed alterations of DNA methylation in NASH-W samples relative to NLT samples. Such alterations at the precancerous NASH stage were inherited by or strengthened in HCC samples. Receiver operating characteristic curve analysis identified 415 CpG sites discriminating NASH-W from NLT samples with area under the curve values of more than 0.95. Among them, we focused on 21 CpG sites showing more than 85% specificity, even for discrimination of NASH-W from NASH-O samples. The DNA methylation status of these 21 CpG sites was able to predict the coincidence of HCC independently from histopathological findings such as ballooning and fibrosis stage. The methylation status of 5 candidate marker CpG sites was assessed using a HPLC-based system, and for 3 of them sufficient sensitivity and specificity were successfully validated in the validation cohort. By combining these 3 CpG sites including the ZC3H3 gene, NAFL-W and NASH-W samples from which HCCs had already arisen were confirmed to show carcinogenic risk with 95% sensitivity in the validation cohort. CONCLUSIONS: After a further prospective validation study using a larger cohort, carcinogenic risk estimation in liver biopsy specimens of patients with NASH may become clinically applicable using this HPLC-based system for quantification of DNA methylation.

Versican Secreted by Cancer-Associated Fibroblasts is a Poor Prognostic Factor in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is highly recurrent. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, promote malignancy; however, the mechanisms underlying their actions are obscure. We aimed to identify CAF-specific proteins in HCC and determine whether they could be potential therapeutic targets. METHODS: Using comprehensive proteomic analysis of CAFs and noncancerous fibroblasts (NFs) primary-cultured from resected HCC specimens from the same patients, CAF-specific proteins were identified. Immunohistochemistry for versican (VCAN) was performed on cancerous tissues obtained from 239 patients with HCC. Conditioned medium from CAFs transfected with siRNA for VCAN was analyzed in vitro. RESULTS: CAFs significantly promoted HCC cell proliferation, migration, and invasion (p < 0.01, 0.01, and 0.01, respectively) compared with NFs. VCAN was upregulated in CAFs, and its stromal level correlated with poor differentiation (p = 0.009) and positive vascular invasion (p = 0.003). Stromal VCAN level was also associated with significantly lower overall (p = 0.002) and relapse-free (p < 0.001) survival rates. It also independently predicted prognosis and recurrence. VCAN-knockdown CAFs significantly suppressed HCC cell migration and invasion compared with negative control. CONCLUSIONS: VCAN secreted from CAFs promoted malignant transformation of HCC cells and has potential as a new therapeutic target in HCC.

The Phenolic Antioxidant 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) Prevents Enterocyte Cell Death under Oxygen-Dissolving Cold Conditions through Polyphyletic Antioxidant Actions.

Cold preservation in University of Wisconsin (UW) solution is not enough to maintain the viability of the small intestine, due to the oxidative stress. The novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has dual properties to reduce oxidative stress, radical scavenging, and antioxidant protein induction, in other cells. This study was designed to determine whether DHMBA reduces cold preservation injury of enterocytes, and to identify the effector site. Enterocytes were subjected to 48-h cold preservation under atmosphere in UW solution (±DHMBA), and then returned to normal culture to replicate reperfusion of the small intestine after cold preservation. At the end of cold preservation (ECP) and at 1, 3, 6, and 72 h after rewarming (R1h, R3h, R6h, and R72h), we evaluated cell function and the injury mechanism. The results showed that DHMBA protected mitochondrial function mainly during cold preservation, and suppressed cell death after rewarming, as shown by the MTT, ATP, mitochondrial membrane potential, LDH, and lipid peroxidation assays, together with enhanced survival signals (PI3K, Akt, p70S6K) and induction of antioxidant proteins (HO-1, NQO-1, TRX-1). We found that DHMBA mitigates the cold-induced injury of enterocytes by protecting the mitochondria through direct and indirect antioxidative activities.

Myostatin as a fibroblast-activating factor impacts on postoperative outcome in patients with hepatocellular carcinoma.

AIM: In patients with liver cirrhosis, high levels of serum myostatin are associated with poor prognosis. We aimed to clarify the influence of myostatin on the prognosis of patients with non-alcoholic fatty liver disease-hepatocellular carcinoma (NAFLD-HCC) without cirrhosis and on the progression of liver fibrosis. METHODS: Serum myostatin levels were evaluated in 234 patients who underwent primary surgical resection for single HCC. To clarify the impact of myostatin on liver fibrosis, we established human primary liver fibroblasts from resected livers, and cultured them in the presence of myostatin. RESULTS: The median age was 67.4 years, the median L3 skeletal muscle mass index was 44.4 cm2 /m2 , and the median body mass index was 23.4 kg/m2 . Eighty-two (35.0%) patients had sarcopenia (L3 skeletal muscle mass index: men <42, women <38 cm2 /m2 ). The etiologies of liver disease were hepatitis B virus (n = 61), hepatitis C virus (n = 86), and non-B non-C hepatitis (n = 87) including NAFLD (n = 74). High preoperative serum myostatin and vascular invasion were independent predictors of poor overall survival (OS). High serum myostatin was associated with poor OS in patients with no sarcopenia (n = 152). In patients without advanced liver fibrosis (Fibrosis stage, 0-2; n = 58), high levels of serum myostatin were also associated with poor OS, regardless of sarcopenia. Serum myostatin levels were increased with the progression of liver fibrosis. Liver fibroblasts were activated and produced collagen following stimulation with myostatin. CONCLUSIONS: In patients with NAFLD-HCC without advanced liver fibrosis, high levels of serum myostatin were associated with poor OS. Myostatin activated primary fibroblasts and stimulated collagen production.

Establishment of a Cell Culture Model Permissive for Infection by Hepatitis B and C Viruses.

Compared with each monoinfection, coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is well known to increase the risks of developing liver cirrhosis and hepatocellular carcinoma. However, the mechanism by which HBV/HCV coinfection is established in hepatocytes is not well understood. Common cell culture models for coinfection are required to examine viral propagation. In this study, we aimed to establish a cell line permissive for both HBV and HCV infection. We first prepared a HepG2 cell line expressing sodium taurocholate cotransporting polypeptide, an HBV receptor, and then selected a cell line highly permissive for HBV infection, G2/NT18-B. After transduction with a lentivirus-encoding microRNA-122, the cell line harboring the highest level of replicon RNA was selected and then treated with anti-HCV compounds to eliminate the replicon RNA. The resulting cured cell line was transduced with a plasmid-encoding CD81. The cell line permissive for HCV infection was cloned and then designated the G2BC-C2 cell line, which exhibited permissiveness for HBV and HCV propagation. JAK inhibitor I potentiated the HCV superinfection of HBV-infected cells, and fluorescence-activated cell-sorting analysis indicated that HBV/HCV double-positive cells accounted for approximately 30% of the coinfected cells. Among several host genes tested, cyclooxygenase-2 showed synergistic induction by coinfection compared with each monoinfection. Conclusion: These data indicate that our in vitro HBV/HCV coinfection system provides an easy-to-use platform for the study of host and viral responses against coinfection and the development of antiviral agents targeting HBV and HCV.

Early administration of amatuximab, a chimeric high-affinity anti-mesothelin monoclonal antibody, suppresses liver metastasis of mesothelin-expressing pancreatic cancer cells and enhances gemcitabine sensitivity in a xenograft mouse model.

Amatuximab is a promising therapeutic antibody targeting mesothelin, a 40-kDa glycoprotein that is highly expressed in pancreatic cancer. We investigated the effectiveness of early amatuximab treatment, imitating an adjuvant chemotherapy setting, and combination therapy with amatuximab and gemcitabine in liver metastasis of pancreatic cancer. Liver metastasis mouse models were established in 8-week-old male BALB/c nu/nu mice using the hemisplenic injection method. Tridaily amatuximab monotherapy or combination with gemcitabine was administered to the liver metastasis mouse model before metastatic lesions had formed huge masses. Gaussia luciferase-transfected AsPC-1 was used as a mesothelin-overexpressing pancreatic cancer cell line. The amount of liver metastases and the serum luciferase activity were significantly lower in the treatment groups than those in the control IgG group. Notably, the anti-tumor activity of gemcitabine was synergically enhanced by combination therapy with amatuximab. Furthermore, western blotting revealed that the high expression of phosphorylated c-Met and AKT in liver metastatic lesions treated with gemcitabine monotherapy was canceled by its combination with amatuximab. This result indicated that the observed synergic therapeutic effect may have occurred as a result of the inhibitory effect of amatuximab on the phosphorylation of c-Met and AKT, which were promoted by exposure to GEM. In conclusion, our study revealed that early administration of amatuximab alone or in combination with GEM significantly suppressed the liver metastases of mesothelin-expressing pancreatic cancer cells. A phase II clinical trial of amatuximab as part of an adjuvant chemotherapy regimen for resected pancreatic cancer is expected.

Serum fatty acid-binding protein 5 is a significant factor in hepatocellular carcinoma progression independent of tissue expression level.

Fatty acid-binding protein 5 (FABP5) is highly expressed in hepatocellular carcinoma (HCC) tissues and is related to HCC progression. In this study, we analyzed the potential of serum FABP5 (sFABP5) as a tumor marker in HCC and its clinical significance in HCC progression. We compared the sFABP5 concentration in patients with HCC (HCC group) with that of patients with hepatitis without HCC (hepatitis group). Moreover, we measured the FABP5 expression levels in resected HCC tissues (tFABP5) and analyzed their relationship with sFABP5. We also performed cell-based assays using FABP5 knockout and overexpressing HCC cell lines to analyze the effect of extrinsic FABP5 (exFABP5) on HCC cells. We showed that sFABP5 was not a useful tumor marker for HCC, as HCC and sFABP5 were not correlated. However, sFABP5 and tFABP5 significantly correlated with survival after surgery for HCC, while sFABP5 and tFABP5 were independent of each other. In cell-based assays, exFABP5 was taken up by HCC cell lines and positively affected cell survival under glucose-depleted conditions by complementing the endogenous FABP5 function. In conclusion, sFABP5 had a significant impact on HCC progression irrespective of tFABP5 by augmenting cell viability under glucose-depleted conditions. As tFABP5 and sFABP5 are important factors that are independent of each other in HCC progression, both of them should be considered independently in improving the prognosis of patients with HCC.

Mesothelin blockage by Amatuximab suppresses cell invasiveness, enhances gemcitabine sensitivity and regulates cancer cell stemness in mesothelin-positive pancreatic cancer cells.

BACKGROUND: Mesothelin is a 40-kDa glycoprotein that is highly overexpressed in various types of cancers, however molecular mechanism of mesothelin has not been well-known. Amatuximab is a chimeric monoclonal IgG1/k antibody targeting mesothelin. We recently demonstrated that the combine therapy of Amatuximab and gemcitabine was effective for peritonitis of pancreatic cancer in mouse model. METHODS: We discover the role and potential mechanism of mesothelin blockage by Amatuximab in human pancreatic cells both expressing high or low level of mesothelin in vitro experiment and peritonitis mouse model of pancreatic cancer. RESULTS: Mesothelin blockage by Amatuximab lead to suppression of invasiveness and migration capacity in AsPC-1 and Capan-2 (high mesothelin expression) and reduce levels of pMET expression. The combination of Amatuximab and gemcitabine suppressed proliferation of AsPC-1 and Capan-2 more strongly than gemcitabine alone. These phenomena were not observed in Panc-1 and MIA Paca-2 (Mesothelin low expression). We previously demonstrated that Amatuximab reduced the peritoneal mass in mouse AsPC-1 peritonitis model and induced sherbet-like cancer cell aggregates, which were vanished by gemcitabine. In this study, we showed that the cancer stem cell related molecule such as ALDH1, CD44, c-MET, as well as proliferation related molecules, were suppressed in sherbet-like aggregates, but once sherbet-like aggregates attached to peritoneum, they expressed these molecules strongly without the morphological changes. CONCLUSIONS: Our work suggested that Amatuximab inhibits the adhesion of cancer cells to peritoneum and suppresses the stemness and viability of those, that lead to enhance the sensitivity for gemcitabine.

Prognostic Significance of Circulating Tumor Cells with Mesenchymal Phenotypes in Patients with Gastric Cancer: A Prospective Study.

BACKGROUND: Circulating tumor cells (CTCs) have been shown to be heterogeneous. Focusing on the epithelial-mesenchymal transition and perioperative kinetics, we evaluated CTCs with mesenchymal phenotypes as a potential prognostic biomarker for patients with gastric cancer. METHODS: Peripheral blood was collected from 54 patients with gastric cancer before surgery and at 1 week and 1 month after surgery. CTCs were enriched using density-gradient centrifugation and magnetic-activated cell sorting (negative selection). Cell suspensions were characterized by multi-immunofluorescence staining against cytokeratin and N-cadherin, and by 4',6'-diamidino-2-phenyldole staining. RESULTS: CTCs were detected in five patients (17%) with early cancer and 14 patients (56%) with advanced cancer (p < 0.05). In our system, N-cadherin, but not cytokeratin, was expressed in the CTCs of 90% (19/21) of patients. Postoperative recurrence was detected in 10 patients, all of whom had N-cadherin+/cytokeratin-/CD45- CTCs preoperatively. Regarding perioperative kinetics, we divided patients into three risk groups: a high-risk group, with one or more preoperative CTCs and increased CTCs postoperatively; an intermediate-risk group, with one or more preoperative CTCs and decreased CTCs postoperatively; and a low-risk group, with no preoperative CTCs. Recurrence rates were 57% (4/7), 33% (4/12), and 6% (2/35), respectively. The relapse-free survival rate was lower in patients at high risk versus those at intermediate or low risk, for all patients (p = 0.00024) and in patients with advanced cancer (p = 0.00103). CONCLUSIONS: N-cadherin is a highly useful marker to detect CTCs lacking cytokeratin, and the perioperative kinetics of CTC numbers is beneficial in risk stratification for survival in patients with gastric cancer.

Analysis of the correlation between alterations in N­glycans and invasiveness in liver cancer cell lines.

The N­glycoforms of glycoproteins modify protein function and control a number of biological pathways. The aim of the present study was to investigate the correlation between alterations in N­glycans and cancer aggressiveness in terms of cancer cell invasion ability. The expression of urokinase­type plasminogen activator (uPA) and N­acetylglucosaminyltransferase V (GnT­V) in liver cancer cell lines was analyzed by western blotting. Cell invasiveness was analyzed by Matrigel invasion assays. uPA and GnT­V expression in liver cancer cell lines was knocked down by RNA interference. Furthermore, uPA was overexpressed in liver cancer cells using lentiviral vectors, and a mutant strain of HepG2 cells overexpressing uPA deficient in N­glycans was established. A glycoblotting­assisted matrix­assisted laser desorption/ionization­time­of­flight/mass spectrometry­based quantitative analysis of liver cancer cell lines was performed, in which invasiveness was altered by modifying the expression of uPA and GnT­V. N­glycan profiles were found to differ between the highly invasive liver cancer cell line HLE and the less invasive cell line HepG2. The expression of several N­glycans, including a form with m/z=1892, was changed according to invasiveness controlled by knockdown and overexpression of uPA. The invasiveness of HepG2 cells with mutant uPA did not increase regardless of the level of expression of uPA. Following GnT­V knockdown and N­glycan alteration, uPA expression did not change, whereas cell invasiveness decreased. One N­glycan (m/z=1892) was common among N­glycans in the comparative analysis between HLE and HepG2, HLE and uPA knockdown HLE, HepG2 and uPA­overexpressing HepG2, and HLE and GnT­V knockdown HLE cells and among N­glycan profiles in human uPA. Therefore, N­glycosylation is an important factor controlling invasiveness of liver cancer cells, and a specific N­glycan (m/z=1892) associated with the invasion of liver cancer cells via uPA was identified in the present study.

Adenomatous polyposis coli-binding protein end-binding 1 promotes hepatocellular carcinoma growth and metastasis.

This study was performed to determine the clinical significance of adenomatous polyposis coli (APC)-binding protein end-binding 1 (EB1) in hepatocellular carcinoma (HCC) and to characterize its biochemical role in comparison with previous reports. We performed immunohistochemical staining to detect EB1 expression in tissues from 235 patients with HCC and investigated its correlations with clinicopathological features and prognosis. We also investigated the roles of EB1 in cell proliferation, migration, and tumorigenesis in vitro and in vivo by siRNA- and CRISPR/Cas9-mediated modulation of EB1 expression in human HCC cell lines. The results showed that EB1 expression was significantly correlated with several important factors associated with tumor malignancy, including histological differentiation, portal vein invasion status, and intrahepatic metastasis. Patients with high EB1 expression in HCC tissue had poorer overall survival and higher recurrence rates than patients with low EB1 expression. EB1 knockdown and knockout in HCC cells reduced cell proliferation, migration, and invasion in vitro and inhibited tumor growth in vivo. Further, genes encoding Dlk1, HAMP, and SLCO1B3 that were differentially expressed in association with EB1 were identified using RNA microarray analysis. In conclusion, elevated expression of EB1 promotes tumor growth and metastasis of HCC. EB1 may serve as a new biomarker for HCC, and genes coexpressed with EB1 may represent potential targets for therapy.

Role of Wnt5a in suppressing invasiveness of hepatocellular carcinoma via epithelial-mesenchymal transition.

Inappropriate activation of the canonical Wnt signaling pathway is associated with progression of hepatocellular carcinoma (HCC). However, the association between the non-canonical pathway activated by Wnt5a and HCC is not well known. The present study investigated the significance of Wnt5a expression in HCC. Immunohistochemical staining of Wnt5a was performed on specimens from 243 patients who underwent hepatic resection for HCC. The present study investigated whether Wnt5a expression was associated with clinical and pathological factors and prognosis. Wnt5a expression in human HCC cell lines was investigated using western blotting. The effects of overexpression or knockdown of Wnt5a were evaluated using proliferation and invasion assays. Changes in epithelial-mesenchymal transition (EMT)-related molecules were investigated using western blotting. Wnt5a negativity was significantly associated with poor tumor differentiation and positive vascular invasion. In univariate analysis, Wnt5a negativity was identified as a significant prognostic factor for overall survival (OS). Multivariate analysis of OS demonstrated that Wnt5a negativity was an independent prognostic factor. Wnt5a expression was lower in HLE and HLF cells than in HepG2 and Huh7 cells. Knockdown of Wnt5a by short hairpin RNA transfection increased the proliferation and invasiveness of Huh7 cells, and decreased the expression levels of E-cadherin. In HLF cells, overexpression of Wnt5a inhibited invasiveness and decreased the expression levels of vimentin. Wnt5a negativity was associated with poor tumor differentiation and positive vascular invasion, and was an independent poor prognostic factor in patients with HCC. Wnt5a may be a tumor suppressor involved in EMT-mediated changes in invasiveness.

Prognostic impact of CD8+ T cell distribution and its association with the HLA class I expression in intrahepatic cholangiocarcinoma.

PURPOSE: A lack of effective systemic therapy is one reason for the poor prognosis of intrahepatic cholangiocarcinoma. Newly developed immune checkpoint inhibitors function by minimizing CD8+ T cell suppression to improve tumor-specific responses. This study aimed to examine the characteristics of CD8+ T cells in intrahepatic cholangiocarcinoma. METHODS: Clinicopathological data, including the overall survival, of 69 cases of postoperative intrahepatic cholangiocarcinoma were prospectively investigated. We then immunohistochemically stained for CD8, Foxp3, CD163, PD-L1, and human leukocyte antigen (HLA) class I and counted the number of CD8+ T cells, Foxp3+ T cells, and CD163+ macrophages in different areas (outer border, interborder, and intratumor). RESULTS: A significant difference was found in the 5-year overall survival between the CD8+ T cell high group (45.5%) and low group (24.7%) in the outer border area (p = 0.0103). Furthermore, the number of CD8+ T cells and the high expression of HLA class I were positively correlated (p = 0.0341). CONCLUSION: The number of CD8+ T cells in the outer border area of the tumor correlated with the HLA class I expression of intrahepatic cholangiocarcinoma and may therefore be a prognostic factor for patients with postoperative intrahepatic cholangiocarcinoma.