Accuracy of brush cytology in biliopancreatic strictures: a single-center cohort study.

OBJECTIVE: False positive and negative results are associated with biliary tract cell brushing cytology during endoscopic retrograde cholangiopancreatography (ERCP). The causes are uncertain. The purpose of this study was to evaluate the accuracy of diagnoses made via cell brushing in our center, and to explore the factors influencing diagnosis. METHODS: The clinical data of patients who underwent cell brushing at our center from January 2016 to August 2019 were retrospectively analyzed. These included age, gender, stricture location, thickness of the bile duct wall in the narrow segment, maximum diameter of the biliary duct above the stricture, number of cell brush smears, carbohydrate antigen 19-9, and carcinoembryonic antigen. Positive brush cytology results were compared with results of surgical histology or tumor biopsy as well as with the patient's clinical course. RESULTS: Of the 48 patients who underwent cell brushing cytology, 27 (56.3%) had positive results. The sensitivity and specificity of biliary duct cell brushing was 79.4%, and 85.7%, respectively. None of the above-mentioned factors were associated with positive cytology brushing results. CONCLUSIONS: Cell brushing cytology remains a reliable method for diagnosis of pancreaticobiliary malignancies.

Macrovascular Endothelial Cells Enhance the Motility of Liver Cancer Cells by Up-regulation of MMP-3, Activation of Integrin/FAK Signaling Pathway and Induction of Non-classical Epithelial-mesenchymal Transition.

Background: Liver cancer with portal vein tumor thrombus (PVTT) indicates a serious prognosis. The molecular mechanism of PVTT formation is not totally clarified, the invasion of blood vessels by liver cancer cells is the key step and portal vein endothelial cells plays critical role. Methods: Conditioned medium (CM) of human umbilical vein endothelial cells (HUVEC) were used to culture liver cancer cells and prostate cancer cells for cell motility and viability analysis for the purpose of simulating the role of macrovascular endothelial cells in the development of liver cancer. Results: HUVEC-CM caused long spindle-shaped changes in liver cancer cells; the invasion and migration ability of Bel-7402 and MHCC-LM3 (cultured in HUVEC-CM) increased significantly. Integrins/FAK (focal adhesion kinase) signaling pathway was activated and MMP-3 was up-regulated. However, classical epithelial-mesenchymal transition (EMT) did not involve. HUVEC-CM caused a decrease of cell population in G1- and S-phase of Bel-7402, it also caused an accumulation of cell population in G1 phase and a decrease of cell population in S-phase of MHCC-LM3, MHCC-97L and DU-145. HUVEC-CM promotes apoptosis of Bel-7402 and MHCC-97L and the nude mouse tumorigenic experiment did not find that the HUVEC-CM increase the tumorigenic ability of liver cancer cells. Conclusion: HUVEC may provide an easy-to-adhere roadbed for liver cancer cells invasion of blood vessels by altering extracellular matrix (ECM), activating integrins/FAK pathway and inducing non-classical EMT. The effect of HUVEC-CM on cell viability was cancer cell type dependent. It is a meaningful glance at the mechsanism of PVTT.

MRC-5 Cancer-associated Fibroblasts Influence Production of Cancer Stem Cell Markers and Inflammation-associated Cell Surface Molecules, in Liver Cancer Cell Lines.

Background: Current opinion suggests that expansion of cancer stem cells (CSCs) and activation of pro-tumoral inflammation cascade correlate with cancer progression. Materials and methods: We explored the possible contributions of MRC-5 cancer-associated fibroblasts to the expression profiles of CSC markers and inflammation-associated cell surface molecules. The liver cancer cell lines Bel-7402, SMMC-7721, MHCC-LM3, and HepG2 cultured in conditioned medium (CM) from MRC-5 served as test groups, whereas the liver cancer cell lines cultured in normal medium served as control groups. Results: Flow cytometry revealed that the proportions of CD90+ cells were significantly higher in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, and moderately higher in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, than in controls. The CD90+/CD45- proportions were elevated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, but reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, as compared to controls. Western blotting indicated that Nanog was downregulated in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls; that POU5F1 (OCT4/3) was downregulated in MHCC-LM3-(MRC-5)-CM, but upregulated in Bel-7402-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls, and that CK19 was upregulated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, compared to controls. Proportions of cells expressing Toll-like receptor-1+ (TLR1) and TLR4 were significantly higher in MHCC-LM3-(MRC-5)-CM cells, and moderately higher in HepG2-(MRC-5)-CM cells, than controls. However, the TLR1+ and TLR4+ proportions were lower in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells than controls. Proportions of CD25+ cells were reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, but elevated in MHCC-LM3-(MRC-5)-CM and Bel-7402-(MRC-5)-CM cells, compared to controls. Proportion of CD61+ cells was higher in liver cancer cells cultured in MRC-5-CM than in controls. Proportion of CD14+ cells was lower in HCC cells cultured in MRC-5-CM than in controls. Conclusion: MRC-5 extensively affected the production of CSC markers and inflammation-associated cell surface molecules. Tumor-targeting molecular therapies should consider these findings.

The role of cancer-associated fibroblast MRC-5 in pancreatic cancer.

Background: Our previous study showed that cancer-associated fibroblast MRC-5 promoted hepatocellular carcinoma progression by enhancing migration and invasion capability. However, few studies have explored the role of MRC-5 in pancreatic cancer (PC). In this study, we examined the exact role and associated mechanisms of MRC-5. Methods: The conditioned media for MRC-5 was used to culture PC cell lines SW1990 and PANC-1. Cell proliferation was compared based on colony formation assays of PC cells in normal media and of PC cells cultured with conditioned media of MRC-5. Cell migration and invasion were assayed by transwell chambers. The expression of EMT-related proteins and apoptosis-related proteins was evaluated using Western blot. And confocal microscopy was used to further detect the expression of EMT-related proteins. qRT-PCR was used to confirm the expression changes of related genes at the mRNA level. We also used flow cytometry to examine the cell cycle, apoptotic rate, and expression of CD3, CD4, CD14, CD25, CD45, CD61, CD90, TLR1, and TLR4. Results: MRC-5 repressed the colony formation ability of PC cells and significantly inhibited cell migration and invasion potential. MRC-5 induced S-phase cell cycle arrest but did not augment the apoptotic effects in PC cells. We hypothesized that the weakened malignant biological behavior of PC cells was correlated with MRC-5-induced altered expression of the cancer stem cell marker CD90; the immune-related cell surface molecules CD14, CD25, TLR4, and TLR1; and cell polarity complexes Par, Scribble, and Crumbs. Conclusion: MRC-5 limits the malignant activities of PC cells by suppressing cancer stem cell expansion, remolding epithelial polarity, and blocking the protumoral cascade reaction coupled to TLR4, TLR1, CD14, and CD25.

CADM1 regulates the G1/S transition and represses tumorigenicity through the Rb-E2F pathway in hepatocellular carcinoma.

BACKGROUND: Increasing evidence indicates that downregulation of cell adhesion molecule 1 (CADM1) contributes to tumorigenesis in various cancers. The present study was undertaken to investigate the CADM1 expression pattern in human hepatocellular carcinoma (HCC), and to elucidate the mechanism underlying CADM1-mediated tumor suppression. METHODS: CADM1 expression in HCC cell lines was measured by quantitative real-time PCR. The function of CADM1 in the context of tumor suppression in HCC cells was determined using proliferation assays, cell cycle analysis, EdU incorporation assays, in vitro colony formation analysis, and in vivo tumorigenicity assays. The mechanism by which CADM1 acts as a tumor suppressor gene in HCC was investigated using Western blotting analysis. RESULTS: Downregulation of CADM1 expression is frequently detected in both HCC cells and clinical samples. Restoration of CADM1 expression in HCC cell lines significantly inhibits cell growth and negatively regulates the G1/S transition. CADM1 overexpression can inhibit the tumorigenicity of HCC cells both in vitro and in vivo. Western blotting analysis revealed that ectopic expression of CADM1 in HCC cells is associated with increased expression of Retinoblastoma (Rb) protein. CONCLUSIONS: Our results showed that suppression of tumorigenesis by CADM1 may be mediated by the Rb-E2F pathway, involving upregulation of Rb protein levels. This pathway could therefore represent an attractive target for HCC therapy.

ZIP4, a novel determinant of tumor invasion in hepatocellular carcinoma, contributes to tumor recurrence after liver transplantation.

BACKGROUND AND PURPOSE: Recently, evidence that Zinc transporter ZRT/IRT-like protein 4 (ZIP4) is involved in invasiveness and apoptosis has emerged in pancreatic cancer and prostate cancer. Our aim was to assess the role of ZIP4 in invasiveness, migration and apoptosis of hepatocellular carcinoma (HCC). The prognostic value of ZIP4 in HCC after liver transplantation was evaluated. METHODS: The role of ZIP4 in HCC was investigated by overexpressing ZIP4 in BEL7402 and HepG2 cells and inhibiting ZIP4 in HuH-7 and HepG2 cells, using overexpression and shRNA plasmids in vitro studies. Immunohistochemical analysis was used to evaluate ZIP4 expression in HCC tissues from 60 patients undergoing liver transplantation, 36 cirrhotic tissue samples, and 6 normal tissue samples. Prognostic significance was assessed using the Kaplan-Meier method and the log-rank test. RESULTS: Specific suppression of ZIP4 reduced cell migration and invasiveness, whereas ZIP4 overexpression caused increases in cell migration and invasiveness. Furthermore, overexpression of ZIP4 resulted in increased expression of pro-metastatic genes (MMP-2, MMP-9) and decreased expression of pro-apoptotic genes (caspase-3, caspase-9, Bax). In contrast, suppression of ZIP4 resulted in an opposite effect. ZIP4 was more highly expressed in tumor tissues than non-tumor tissues (P < 0.0001). ZIP4 expression was significantly associated with tumor recurrence (P = 0.002), tumor node metastasis stage (P = 0.044), Child-Turcotte-Pugh score (P = 0.042), and tumor size (P = 0.022). Univariate analysis showed that ZIP4 expression was significantly associated with overall survival (P = 0.020) and tumor-free survival (P = 0.049). Multivariate analysis revealed that ZIP4 was an independent predictor of overall survival (P = 0.037) after liver transplantation. CONCLUSIONS: ZIP4 could promote migration, invasiveness, and suppress apoptosis in hepatocellular carcinoma, and represent a novel predictor of poor prognosis and therapeutic target for patients with HCC who undergo liver transplantation.

FOXC1 contributes to microvascular invasion in primary hepatocellular carcinoma via regulating epithelial-mesenchymal transition.

The existence of microvascular invasion (MVI) formation is one of the most important risk factors predicting poor outcome in hepatocellular carcinoma (HCC) and its mechanism remains largely unknown. Epithelial-Mesenchymal Transition (EMT) has been suggested to be involved in many steps of the invasion-metastasis cascade. To elucidate the possible contribution of EMT to MVI, we initially evaluated the expression of 8 EMT-related transcription factors (TFs) in HCC patients with or without MVI and found that FOXC1 expression was significantly higher in patients with MVI than those without MVI (P < 0.05). Knockdown of FOXC1 expression in HCC cells resulted in a partial conversion of their EMT progresses, mainly regulating the mesenchymal component. Ectopic expression of snail, twist or TGF-ß1 could induce expression of FOXC1, but none of the expression of snail, twist, slug or TGF-ß was consistently down-regulated in response to FOXC1 silencing, suggesting FOXC1 might operate the downstream of other EMT regulators. In addition, knockdown of FOXC1 expression led to cytoskeleton modification accompanied by decreased ability of cell proliferation, migration, and invasion. Meanwhile, some matrix metalloproteinases (MMPs) and VEGF-A were also simultaneously down-regulated. Together, our findings demonstrate that FOXC1 is one of candidate predictive markers of MVI, and that inhibition of FOXC1 expression can partially reverse EMT program, offering a potential molecular therapeutic target for reducing tumor metastasis in HCC patients.

Aberrant methylation of the CADM1 promoter is associated with poor prognosis in hepatocellular carcinoma treated with liver transplantation.

Approximately 20-40% of hepatocellular carcinoma (HCC) patients who undergo liver transplantation (LT) experience HCC recurrence within 5 years of the operation. Current predictors cannot sufficiently differentiate patients at risk for biochemical recurrence. The aim of the present study was to investigate the methylation status and expression levels of cell adhesion molecule 1 (CADM1) in HCC; to elucidate its regulation mechanisms; and finally, to evaluate the potential predictive value for tumor recurrence. Aberrant hypermethylation of CADM1 was frequently found in HCC cell lines with decreased CADM1 mRNA by bisulfite sequencing PCR. Re-expression of CADM1 was induced by treatment with demethylating agents. The promoter region of CADM1 was identified and the basal promoter activity was located in the -226 to -146 region relative to the transcriptional start site (TSS). Site-directed mutagenesis revealed that the consensus Sp1 binding site located in the basal promoter region was important for mediating CADM1 promoter activity. Furthermore, aberrant hypermethylation of CADM1 was detected in 34 of 82 (41.5%) of HCC tissues. The recurrence rate of the patients with CADM1 methylation was higher compared to that without CADM1 methylation (70.6% versus 33.3%; P=0.001). Multivariate analysis revealed that CADM1 methylation status (HR = 2.788; 95% CI, 1.043-5.063; P=0.010) was an independent prognostic factor for disease-free survival (DFS) of HCC patients treated with LT. In conclusion, CADM1 methylation may be used as a potential predictive biomarker for tumor recurrence of HCC after LT.