FASN-mediated fatty acid biosynthesis remodels immune environment in Clonorchis sinensis infection-related intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA. METHODS: We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis-related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA. RESULTS: We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis-related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis-related iCCAs than non-C. sinensis-related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis. CONCLUSIONS: We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis-related iCCAs. IMPACT AND IMPLICATIONS: Clonorchis sinensis (C. sinensis)-infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non-C. sinensis-infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis-related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis-related iCCAs.

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.

Liver Flukes: Clonorchis and Opisthorchis.

Clonorchis sinensis, Opisthorchis viverrini and O. felineus are liver flukes of human and animal pathogens occurring across much of Europe and Asia. Nevertheless, they are often underestimated compared to other, better known neglected diseases in spite of the fact that many millions of people are infected and hundreds of millions are at risk. This is possibly because of the chronic nature of the infection and disease and that it takes several decades prior to a life-threatening pathology to develop. Several studies in the past decade have provided more information on the molecular biology of the liver flukes which clearly lead to better understanding of parasite biology, systematics and population genetics. Clonorchiasis and opisthorchiasis are characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. These chronic inflammations eventually lead to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in some infected individuals. In Thailand alone, opisthorchiasis-associated CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce hepatobiliary morbidity and CCA, the primary intervention measures focus on control and elimination of the liver fluke. Accurate diagnosis of liver fluke infections in both human and other mammalian, snail and fish intermediate hosts is important for achieving these goals. While the short-term goal of liver fluke control can be achieved by praziquantel chemotherapy, a comprehensive health education package targeting school children is believed to be more beneficial for a long-term goal/solution. It is recommended that transdisciplinary research or multisectoral control approach including one health and/or eco health intervention strategy should be applied to combat the liver flukes and hence contribute to reduction of CCA in endemic areas.

PTEN Deficiency Induced by Extracellular Vesicle miRNAs from Clonorchis sinensis Potentiates Cholangiocarcinoma Development by Inhibiting Ferroptosis.

The human phosphatase and tensin homolog (PTEN) is a tumor suppressor. A slight deficiency in PTEN might cause cancer susceptibility and progression. Infection by the liver fluke Clonorchis sinensis could lead to persistent loss of PTEN in cholangiocarcinoma. However, the mechanism of PTEN loss and its malignant effect on cholangiocarcinoma have not yet been elucidated. Extracellular vesicles secreted by Clonorchis sinensis (CS-EVs) are rich in microRNAs (miRNAs) and can mediate communication between hosts and parasites. Herein, we delved into the miRNAs present in CS-EVs, specifically those that potentially target PTEN and modulate the progression of cholangiocarcinoma via ferroptosis mechanisms. CS-EVs were extracted by differential ultra-centrifugation for high-throughput sequencing of miRNA. Lentiviral vectors were used to construct stably transfected cell lines. Erastin was used to construct ferroptosis induction models. Finally, 36 miRNAs were identified from CS-EVs. Among them, csi-miR-96-5p inhibited PTEN expression according to the predictions and dual luciferase assay. The CCK-8 assay, xenograft tumor assays and transwell assay showed that csi-miR-96-5p overexpression and PTEN knockout significantly increased the proliferation and migration of cholangiocarcinoma cells and co-transfection of PTEN significantly reversed the effect. In the presence of erastin, the cell proliferation and migration ability of the negative transfection control group were significantly impaired, although they did not significantly change with transfection of csi-miR-96-5p and PTEN knockout, indicating that they obtained ferroptosis resistance. Mechanistically, csi-miR-96-5p and PTEN knockout significantly inhibited ferroptosis through a decrease in ferrous ion (Fe2+) and malondialdehyde (MDA), and an increase in glutathione reductase (GSH), Solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). In conclusion, loss of PTEN promoted the progression of cholangiocarcinoma via the ferroptosis pathway and csi-miR-96-5p delivered by CS-EVs may mediate this process.

Opisthorchis viverrini Life Cycle, Distribution, Systematics, and Population Genetics.

Opisthorchis viverrini plays a key role as the carcinogenic liver fluke causing bile duct cancer in Southeast Asia. A comprehensive understanding of its life cycle, distribution, systematics, and population genetics is critically important as they underpin the effective development and establishment of future prevention and control programs that center on opisthorchiasis and cholangiocarcinoma. This chapter provides detailed information concerning the basic biology and updated information of O. viverrini related to its host life cycle, transmission route via raw, partially cooked or fermented freshwater cyprinid fish, endemic areas, and the discovery of new foci. Previous sequential studies over the last two decades on the phylogenetic and systematic relationships, genetic variation, and population genetics of O. viverrini as well as its snail intermediate host Bithynia spp. are presented and discussed, which have led to the currently known complex species level systematics and population genetics framework of this host-parasite system. Additionally, further directions for comprehensive research are suggested to provide a more complete understanding of liver fluke, O. viverrini-related cholangiocarcinoma.

Distinct antibody response in susceptible and non-susceptible hosts of the carcinogenic liver fluke Opisthorchis viverrini infection.

Opisthorchis viverrini is a carcinogenic parasite that can cause bile duct cancer called cholangiocarcinoma. A study of the immune response of this parasite in susceptible and non-susceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamsters and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between 1 and 2 weeks post-infection, whereas it was positive between 2 and 4 weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice reacts strongly with the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted with a single protein band. Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host reacts quicker and stronger than the susceptible host.

Influence of misconceptions and inappropriate eating behaviors on Opisthorchis viverrini infection among at-risk populations undergoing cholangiocarcinoma screening in Northeastern Thailand.

Raw or undercooked freshwater fish consumption contributes to persistent Opisthorchis viverrini infection in Northeast Thailand. This study aims to assess the relationship between misconceptions, unhealthy eating habits, and O. viverrini infection. Data were obtained from the Cholangiocarcinoma Screening and Care Program in Northeast Thailand from 2019 to 2021. Participants were screened for O. viverrini annually over the following 2 years using the Kato-Katz technique. Misconceptions and unhealthy eating habits were assessed through questionnaires. The relationship between these factors and O. viverrini infection was evaluated using adjusted risk ratios (ARRs) and 95% confidence intervals (CIs) from generalized estimating equations under binomial regression framework. Of 5375 participants screened for O. viverrini over 3 years, infection rates were 21.53%, 10.7%, and 4.6% each year, respectively. Out of those, 636 participants responded to questions regarding misconceptions. Results showed that participants who believed in the efficacy of putting lime or red ants in Koi pla (raw fish salad) or eating Koi pla with white whiskey to kill parasites, and early-stage cholangiocarcinoma can be cured, were 41% (ARR, 1.41; 95% CI, 1.03-1.94) and 57% (ARR, 1.57; 95% CI, 1.06-2.33), respectively, more likely to be infected with O. viverrini. Our study confirms that belief in using lime or red ants in Koi pla or eating Koi pla with white whiskey to make it cooked, or early-stage cholangiocarcinoma can be cured, increases O. viverrini infection risk in high-risk populations. Changing health beliefs and eating habits is necessary to reduce O. viverrini infection and its risk to cholangiocarcinoma.

Clonorchis sinensis excretory-secretory products increase malignant characteristics of cholangiocarcinoma cells in three-dimensional co-culture with biliary ductal plates.

Clonorchis sinensis is a carcinogenic human liver fluke, prolonged infection which provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma (CCA). These effects are driven by direct physical damage caused by the worms, as well as chemical irritation from their excretory-secretory products (ESPs) in the bile duct and surrounding liver tissues. We investigated the C. sinensis ESP-mediated malignant features of CCA cells (HuCCT1) in a three-dimensional microfluidic culture model that mimics an in vitro tumor microenvironment. This system consisted of a type I collagen extracellular matrix, applied ESPs, GFP-labeled HuCCT1 cells and quiescent biliary ductal plates formed by normal cholangiocytes (H69 cells). HuCCT1 cells were attracted by a gradient of ESPs in a concentration-dependent manner and migrated in the direction of the ESPs. Meanwhile, single cell invasion by HuCCT1 cells increased independently of the direction of the ESP gradient. ESP treatment resulted in elevated secretion of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-ß1) by H69 cells and a cadherin switch (decrease in E-cadherin/increase in N-cadherin expression) in HuCCT1 cells, indicating an increase in epithelial-mesenchymal transition-like changes by HuCCT1 cells. Our findings suggest that C. sinensis ESPs promote the progression of CCA in a tumor microenvironment via the interaction between normal cholangiocytes and CCA cells. These observations broaden our understanding of the progression of CCA caused by liver fluke infection and suggest a new approach for the development of chemotherapeutic for this infectious cancer.

Oxysterols of helminth parasites and pathogenesis of foodborne hepatic trematodiasis caused by Opisthorchis and Fasciola species.

The foodborne trematodiases refer to a cluster of zoonotic neglected tropical diseases caused by trematodes, with transmission involving ingestion of contaminated plants, fishes, and crustaceans. Over 40 million people are infected with foodborne trematodes and 750 million are at risk of infection. From a public health point of view, important species include Clonorchis sinensis, Opisthorchis viverrini, Opisthorchis felineus, Fasciola hepatica, and Fasciola gigantica. Infection with C. sinensis and O. viverrini is classified as a group 1 biological carcinogen and a major risk factor for cholangiocarcinoma. The carcinogenic potential of the infection with O. felineus is less clear but recent biochemical and histopathological findings revealed that opisthorchiasis felinea also fits this pattern. By contrast, evidence of carcinogenic potential of infection with F. hepatica or F. gigantica, close phylogenetics relatives of Opisthorchis, is less certain. Oxysterols have been essentially described in animal model of opisthorchiasis and associated cholangiocarcinoma. Several oxysterol-like metabolites have been detected not only on developmental stages of O. viverrini and O. felineus but also on biofluids from experimentally infected hamsters as products of the activities of the liver flukes. These sterol derivatives are metabolized to active quinones that can modify host DNA. We have postulated that helminth parasite-associated sterols might induce tumor-like phenotypes in biliary epithelia, the cells of origin of liver fluke infection-associated cholangiocarcinoma, through the formation of DNA adducts, dysregulation of apoptosis, and other homeostatic pathways. Here we review, interpret, and discuss findings of oxysterol-like metabolites detected in liver flukes and their role in carcinogenesis, aiming to enhance understanding the pathogenesis of foodborne trematodiasis caused by Opisthorchis and Fasciola species. In future, further investigations will be necessary in order to comprehend relationship between liver flukes' oxysterols and their role in infection-associated diseases in humans.

Animation as Supplementary Learning Material About Carcinogenic Liver Fluke in Classes for Primary Schoolchildren.

Carcinogenic liver fluke is still an issue of great concern in some countries of Southeast Asia, particularly in Thailand, Cambodia, the Lao People's Democratic Republic, and Vietnam. The infection, caused by Opisthorchis viverrini, is associated to cholangiocarcinoma and is endemic among human populations for whom raw fish is frequently consumed. Prevention and health education are required. Therefore, this study aimed to evaluate the effectiveness of educational intervention to improve knowledge among primary schoolchildren based on animation-assisted education. In this study, 80 participants (40 participants in the experimental group and 40 participants in the comparison group) were selected in 2018. The effectiveness of an interactive animation program in improving the knowledge of students studying liver fluke was determined based on scores on tests given before and immediately after completion of a 4.29-min animated program on the liver fluke life cycle, risk factors, disease, diagnosis, treatment, prevention, and control. Data were analyzed using SPSS-22 via paired t tests and independent samples t tests at a significance level of 0.05. A marked and significant improvement was observed in the immediate posttest compared with the pretest scores. More importantly, the students who had used the animated program achieved a significantly higher score on the final test than the comparison group. The results offered in the first report show that the use of the animated program facilitated education about liver fluke. It is strongly believed that animations are good supplementary learning materials for students, particularly for learning about serious concepts.

Infection with Opisthorchis felineus induces intraepithelial neoplasia of the biliary tract in a rodent model.

The liver fluke Opisthorchis felineus is a member of the triad of epidemiologically relevant species of the trematode family Opisthorchiidae, and the causative agent of opisthorchiasis felinea over an extensive range that spans regions of Eurasia. The International Agency for Research on Cancer classifies the infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis as group 1 agents and a major risk factor for cholangiocarcinoma. However, the carcinogenic potential of the infection with O. felineus is less clear. Here, we present findings that support the inclusion of O. felineus in the Group 1 list of biological carcinogens. Two discrete lines of evidence support the notion that infection with this liver fluke is carcinogenic. First, novel oxysterol-like metabolites detected by liquid chromatography-mass spectroscopy in the egg and adult developmental stages of O. felineus, and in bile, sera, and urine of liver fluke-infected hamsters exhibited marked similarity to oxysterol-like molecules known from O. viverrini. Numerous oxysterols and related DNA-adducts detected in the liver fluke eggs and in bile from infected hamsters suggested that infection-associated oxysterols induced chromosomal lesions in host cells. Second, histological analysis of liver sections from hamsters infected with O. felineus confirmed portal area enlargement, inflammation with severe periductal fibrosis and changes in the epithelium of the biliary tract characterized as biliary intraepithelial neoplasia, BilIN. The consonance of these biochemical and histopathological changes revealed that O. felineus infection in this rodent model induced precancerous lesions conducive to malignancy.

Clonorchiasis.

On Aug 21, 1875, James McConnell published in The Lancet his findings from a post-mortem examination of a 20-year-old Chinese man--undertaken at the Medical College Hospital in Calcutta, India--in whom he found Clonorchis sinensis in the bile ducts. Now, exactly 140 years later, we have a sound understanding of the lifecycle of this liver fluke, including key clinical, diagnostic, and epidemiological features. Developments in the so-called -omics sciences have not only advanced our knowledge of the biology and pathology of the parasite, but also led to the discovery of new diagnostic, drug, and vaccine targets. C sinensis infection is primarily related to liver and biliary disorders, especially cholangiocarcinoma. Clonorchiasis mainly occurs in east Asia, as a result of the region's social-ecological systems and deeply rooted cultural habit of consuming raw freshwater fish. The Kato-Katz technique, applied on fresh stool samples, is the most widely used diagnostic approach. Praziquantel is the treatment of choice and has been considered for preventive chemotherapy. Tribendimidine showed good safety and therapeutic profiles in phase 2 trials and warrants further investigation. Still today, the precise distribution, the exact number of infected people, subtle morbidities and pathogenesis, and the global burden of clonorchiasis are unknown. Integrated control strategies, consisting of preventive chemotherapy; information, education, and communication; environmental management; and capacity building through intersectoral collaboration should be advocated.

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia.

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.

Large numbers of interleukins-22- and -17A-producing T helper cells in cholangiocarcinoma related to liver fluke infection.

Cholangiocarcinoma (CCA) associated with liver fluke infection involves inflammatory and immune processes; however, whether these involve the proinflammatory cytokine IL-17A and proliferative cytokine IL-22 remains unclear. Here, numbers of IL-22- and IL-17A-producing Th cells and cytokine concentrations in 30 patients with CCA and long-term liver fluke infection, 40 patients with liver-fluke infection but not CCA, and 16 healthy controls were compared. Analyses were performed using immunohistochemistry, flow cytometry, ELISA and RT-PCR. Immunohistochemical staining showed weaker expression of IL-22 and IL-17A in patients with CCA with than in those without liver fluke infection (P < 0.01). Flow cytometry revealed significantly greater median proportions of IL-22-producing T helper cells in patients with CCA (2.2%) than in those without it (0.69%) or controls (0.4%, P < 0.001). Similar results were obtained for IL-17A-producing T helper cells. ELISA revealed plasma concentrations of IL-22 were 1.3-fold higher in patients with CCA than in those without it and 4.6-fold higher than in controls (P < 0.001). Plasma concentrations of IL-17A were 2.5-fold higher in patients with CCA than in those without it, and 21-fold higher than in controls (P < 0.001). Amounts of IL-22 and IL-17A mRNAs in blood were significantly higher in patients with CCA than in the other two groups. Proportions of CD4+ CD45RO+ T cells producing IL-22 correlated with proportions producing IL-17A (r = 0.759; P < 0.001), and plasma concentrations of IL-22 correlated with those of IL-17A (r = 0.726; P < 0.001). These results suggest that both IL-17A and IL-22 affect development of CCA related to liver fluke infection.

Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma.

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF-, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1ß, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF- and APF+) cases. Polymorphisms in genes encoding IL-1ß-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α -308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α -308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 -174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.

Significance of S100P as a biomarker in diagnosis, prognosis and therapy of opisthorchiasis-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignancy of bile duct with the difficulty in early diagnosis, poor prognosis and less alternation in therapy. S100P is a member of S100 family proteins and plays important roles in cancers. We investigated the S100P expression and its correlation with clinicopathology in 78 cases of opisthorchiasis-associated CCA, and the effects of S100P knockdown with shRNA interference on the proliferation, cell cycle, migration, apoptosis and sensitivity to anti-cancer drug. Extremely high expression of S100P mRNA was detected in the CCA tumor tissues. The increased S100P protein expression was immunohistochemically confirmed and localized in the CCA cytoplasm and/or nuclei as well as in the hyperneoplasia and dysplasia bile ducts, but not in normal bile ducts. The intensity of immunostaining was correlated with survival, tumor stage and metastasis, and the high expression could be an independent prognostic factor. High levels of S100P were detected in the serum and bile fluid of CCA patients. The shRNA-mediated knockdown of S100P expression inhibited the proliferation in vitro and in vivo, and migration of CCA cells, arrested cell cycle with the up-regulated expression of cell cycle arrest related factors, p21, p27, GADD45A, and 14-3-3 zeta. S100P knockdown also promoted CCA cell apoptosis by up-regulating expression of apoptosis related factors, DR5, TRADD, caspase 3 and BAX, and increased the sensitivity of CCA cells to the chemotherapeutic agents sunitinib and apigenin. Taken together, this study indicates that S100P might be a promising biomarker for the diagnosis, prognosis and therapy of CCA.

Suppression of trophoblast cell surface antigen 2 enhances proliferation and migration in liver fluke-associated cholangiocarcinoma.

BACKGROUND AND AIM: Trophoblast cell surface antigen 2 (TROP2) or tumor-associated calcium signal transducer 2 (TACSTD2) is a 36-kDa type I transmembrane glycoprotein and exerts dual functions as an oncogene and tumor suppressor in cancer cells. In this study, we investigated the expression and functions of TROP2 in liver fluke-associated cholangiocarcinoma (CCA). MATERIAL AND METHODS: TROP2 expression in 85 CCA tissues was detected by using immunohistochemistry. The methylation status of TROP2 promoter was studied in 15 matched pairs of normal and CCA formalin fixed paraffin embedded (FFPE) tissues using the bisulfite genomic sequencing (BGS) method. The functions of TROP2 on cancer cell behavior were investigated using siRNA in CCA cell lines. Proliferation, migration and invasion assays were performed. A PCR array was used to evaluate the impact of TROP2 knockdown on the gene expression profiles. RESULTS: TROP2 was highly expressed in all normal bile duct epithelia, but significantly down-regulated in CCA cells. Sixty percent of CCA revealed promoter hypermethylation compared to the corresponding adjacent normal tissues. TROP2 knockdown significantly enhanced the proliferation and migration in CCA cell lines, and altered the expressions of MARCK, EMP1 and FILIP1L. CONCLUSION: We provide new evidence that TROP2 is epigenetically down-regulated and operates as a negative regulator of cell proliferation and migration in liver fluke-associated CCA.

Wound healing and cancer progression in Opisthorchis viverrini associated cholangiocarcinoma.

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). It was shown previously that O. viverrini-secreted proteins accelerate wound resolution in human cholangiocytes. Recombinant Ov-GRN-1 (O. viverrini-derived gene encoding granulin-like growth factor) induced angiogenesis and accelerated mouse wound healing. Given the striking similarities of wound healing and cancer progression, here we discuss the major implications of this finding for an infection-induced cancer of major public health significance in the developing world.

A combination of liver fluke infection and traditional northeastern Thai foods associated with cholangiocarcinoma development.

Opisthorchis viverrini infection is one of the risk factors for cholangiocarcinoma (CCA) in northeast Thailand, a region with one of the highest reported incidence rates of CCA. The traditional practice of eating raw fish, repeated exposure to liver flukes, and consumption of nitrosamine-contaminated food are major risk factors for CCA. So far, there have been no reports about which northeastern traditional dishes may be involved in CCA development. The present study, thus, investigated the effects of traditional foods. It focused specifically on the consumption of fermented foods in combination with O. viverrini infection in hamsters. Syrian hamsters were divided into six groups: (i) normal hamsters, (ii) O. viverrini infection only and (iii)-(vi) O. viverrini infection plus fermented foods (pla som-fish fermented for 1 day), som wua-fermented beef, som phag-fermented vegetables, and pla ra-fish fermented for 6 months. Syrian hamster livers were used for analysis of histopathological changes through hematoxylin and eosin; Sirius Red; and immunohistostaining for cytokeratin-19, proliferating cell nuclear antigen, and CA19-9. Hamster sera were used for liver and kidney function tests. Results of all O. viverrini-infected groups and fermented food groups showed that histopathological changes consisted primarily of aggregations of inflammatory cells surrounding the hepatic bile duct, especially at the hilar region. However, there was a difference in virulence. Interestingly, aggregations of inflammatory cells, new bile duct formation, and fibrosis were observed in subcapsular hepatic tissue, which correlated to positive immunohistochemical staining and increased liver function test. The present study suggests that fermented food consumption can exacerbate cholangitis and cholangiofibrosis, which are risk factors for cholangiocarcinoma-associated opisthorchiasis.

Specific Features of Progression of the Parasitic Invasion, caused by Opisthorchis felineus, in Golden Hamsters.

The influence of Opisthorchis felineus invasion on the development of pathological changes in the hepatobiliary system was studied in 120 golden hamsters in a long-term experiment (42 weeks) after single infection per os in the dose of 50 metacercariae per animal. The animals were sacrificed on weeks 4, 8, 12, 16, 28 and 42. Chronic experimental infestation with O. felineus triggered a cascade of morphogenetic processes in both extrahepatic and intrahepatic biliary systems. At the early stages of the experiment, polyps and strictures of bile ducts were formed in the lobar bile ducts; in portal tracts, hyperplasia and adenomatous transformation of the newly formed epithelial structures were observed. At the later stages, third-degree biliary intraepithelial neoplasia developed in the lobar bile ducts; in the intrahepatic bile ducts, increased epitheliocyte hyperplasia and invasive growth of cell cords were observed, that impaired tissue architectonics. Progressing cell atypia can be classified as cholangiocellular cancer.