Background: Interleukin-6 (IL-6) massively released by cancer-associated fibroblast (CAFs) has been shown to associate with the malignant behavior of cholangiocarcinoma (CCA). In vitro studies demonstrated the ability of CAFs-derived IL-6 to inhibit autophagy in CCA cells thus promoting their proliferation and invasiveness potential. Here, we aimed to validate with clinical and molecular data the hypothesis that CAFs infiltration and release of IL-6 predict poor prognosis in CCA patients following dysregulation of autophagy in cancer cells.Methods: Stromal IL-6 and cancer cell-associated autophagy proteins LC3 and p62 were assayed by Tissue MicroArray immunohistochemistry and their expression correlated with overall survival (OS) in a cohort of 70 CCA patients. Additionally, copy number and mRNA expression data of BECN1, MAP1-LC3B, p62/SQSTM1 and IL6 were extracted from a CCA database in TCGA and correlated with OS. 5-FU Cytotoxicity in CCA cells was assessed by cell counting, clonogenic assay, cytofluorometry and western blotting and immunofluorescence of apoptotic-related proteins. Results: We show that patients bearing a CCA with low production of stromal IL-6 and active autophagy flux in the cancer cells have the best prognosis and this correlates with a more effective response to post-operative chemotherapy. Similar trend was observed in CCA patients from TCGA database. In vitro experiments with primary CAFs isolated from human CCA and epigenetic manipulations showed that IL-6 plays a pivotal role in determining the autophagy-associated apoptotic response to chemotherapeutic drug in cultured human CCA cells. Conclusions: Our data support a therapeutic strategy that includes autophagy-enhancing drugs along with adjuvants limiting the stromal inflammation (i.e., the secretion of IL-6) to improve the survival of CCA patients.

Background: Cholangiocarcinoma (CCA) has been categorized based on tumor location as intrahepatic (ICCA), perihilar (PCCA) or distal (DCCA), and based on the morphology of the tumor of the bile duct as mass forming (MF), periductal infiltrating (PI) or intraductal (ID). To date, there is limited evidence available regarding the survival of CCA among these different anatomical and morphological classifications. This study aimed to evaluate the survival rate and median survival time after curative surgery among CCA patients according to their anatomical and morphological classifications, and to determine the association between these classifications and survival. Methods: This study included CCA patients who underwent curative surgery with a pathological diagnosis from the Cholangiocarcinoma Screening and Care Program (CASCAP), Northeast Thailand. Survival rates of CCA and median survival time since the date of CCA surgery and 95% confidence intervals (CI) were calculated. Multiple cox regression was performed to evaluate factors associated with survival which were quantified by hazard ratios (HR) and their 95% CIs. Results: Of the 746 CCA patients, 514 had died at the completion of the study which constituted 15,643.6 person-months of data recordings. The mortality rate was 3.3 per 100 patients per month (95% CI: 3.0-3.6), with median survival time of 17.8 months (95% CI: 15.4-20.2), and 5-year survival rate of 24.6% (95% CI: 20.7­28.6). The longest median survival time was 21.8 months (95% CI: 16.3-27.3) while the highest 5-year survival rate of 34.8% (95% CI: 23.8-46.0) occurred in the DCCA group. A combination of anatomical and morphological classifications, PCCA+ID, was associated with the longest median survival time of 40.5 months (95% CI: 17.9-63.0) and the highest 5-year survival rate of 42.6% (95% CI: 25.4-58.9). The ICCA+MF combination was associated with survival (adjusted HR: 1.45; 95% CI: 1.01-2.09; P = 0.013) compared to ICCA+ID patients. Conclusions: Among patients receiving surgical treatment, those with PCCA+ID had the highest 5-year survival rate, which was higher than in groups classified by only anatomical characteristics. Additionally, the patients with ICCA+MF showed the highest survival association. Therefore, further investigations into CCA imaging should focus on patients with a combination of anatomical and morphological classifications.

Background: Sulfasalazine (SSZ) is widely known as an xCT inhibitor suppressing CD44v9 expressed cancer stem like cells (CSCs) being related to redox regulation. Cholangiocarcinoma (CCA) has a high recurrence rate and no effective chemotherapy. A recent report revealed high levels of CD44v9 positive cells in CCA patients. Therefore, a combination of drugs could prove a suitable strategy for CCA treatment via individual metabolic profiling. Methods: We examined the effect of xCT-targeted CD44v9-CSCs using sulfasalazine combined with cisplatin (CIS) or gemcitabine in CCA in vitro and in vivo model and did NMR-based metabolomics of xenograft mice tumor tissues. Results: Our findings suggest that combined SSZ and CIS leads to a higher inhibition of cell proliferation and induction of cell death than CIS alone in both in vitro and in vivo models. Xenograft mice showed that the CD44v9-CSC marker and CK-19-CCA proliferative marker were reduced in the combination treatment. Interestingly, different metabolic signatures and the significant metabolites were observed in the drug treated group compared with the control group that revealed the cancer suppression mechanisms. Conclusions: Taken together, SSZ could improve CCA therapy by sensitization to CIS through killing CD44v9-positive cells and modifying the metabolic pathways, in particular tryptophan degradation (i.e. kynurenine pathway, serotonin pathway) and nucleic acid metabolism.

Background Control and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate such a surveillance and control program. In this study, we evaluated the performance of Mini Parasep SF stool concentrator kit (stool kit) in comparison with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and coexisted parasitic infections. Methods A cross-sectional survey of parasitic infection of residents age <15 years old in a community in Kalasin province, northeast Thailand was conducted in 2018. Fecal samples were collected and screened by KK method and a subset of samples was examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections and diagnostic performances of methods for qualitative and quantitative detection of helminthiasis. Results The initial survey of parasitic infection determined by the KK method (n=567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53%, and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n=150), the detection rates of O. viverrini by the stool kit, FECT, and KK methods were 27.3%, 30.7%, and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis were similar for FECT (75.4%) and KK (70.5%) and was lower for the stool kit (67.2%, p<0.05). For other parasitic infections, FECT and stool kit performed better than KK methods particularly in detecting minute intestinal fluke (MIF), S. stercoralis, and coinfections. The intensity of O. viverrini infection (fecal egg counts) by the stool kit had significant positive correlations with KK as well as FECT (p<0.05). Conclusions As the stool kit is simple to use and showed a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis.

Background Phenotypic diversity in urinary metabolomes of different geographical populations has been recognized recently. In this study, urinary metabolic signatures from Western (United Kingdom) and South-East Asian (Thai) cholangiocarcinoma patients were characterized to understand spectral variability due to host carcinogenic processes and/or exogenous differences (nutritional, environmental and pharmaceutical). Methods Urinary liquid chromatography mass spectroscopy (LC-MS) spectral profiles from Thai (healthy=20 and cholangiocarcinoma=14) and UK cohorts (healthy=22 and cholangiocarcinoma=10) were obtained and modelled using chemometric data analysis. Results Healthy metabolome disparities between the two distinct populations were primarily related to differences in dietary practices and body composition. Metabolites excreted due to drug treatment were dominant in urine specimens from cholangiocarcinoma patients, particularly in Western individuals. Urine from participants with sporadic (UK) cholangiocarcinoma contained greater levels of a nucleotide metabolite (uridine/pseudouridine). Higher relative concentrations of 7-methylguanine were observed in urine specimens from Thai cholangiocarcinoma patients. The urinary excretion of hippurate and methyladenine (gut microbial-host co-metabolites) showed a similar pattern of lower levels in patients with malignant biliary tumours from both countries.Conclusion Intrinsic (body weight and body composition) and extrinsic (xenobiotic metabolism) factors were the main causes of disparities between the two populations. Regardless of the underlying aetiology, biological perturbations associated with cholangiocarcinoma urine metabolome signatures appeared to be influenced by gut microbial community metabolism. Dysregulation in nucleotide metabolism was associated with sporadic cholangiocarcinoma, possibly indicating differences in mitochondrial energy production pathways between cholangiocarcinoma tumour subtypes. Mapping population-specific metabolic disparities may aid in interpretation of disease processes and identification of candidate biomarkers.