Cholangiocarcinoma (CCA) is an aggressive malignancy with poor clinical outcomes. The current "gold standard" diagnostic approach, endoscopic retrograde cholangiopancreatography (ERCP)-obtained biopsy, has a relatively low sensitivity (i.e., ∼50%). Here, we developed a bile-based diagnostic system using transient stimulated Raman scattering (T-SRS). Except for the tolerance to autofluorescence inherited from traditional SRS spectroscopy, T-SRS features quantum-limit spectral line shapes and is further improved with self-calibration ability in this research. These advantages make the acquired Raman spectra insensitive to the drifting of the excitation parameters, facilitating long-term reliability. Based on the T-SRS spectra in the C-H stretching region from 76 bile samples accumulated over more than 1 year, we demonstrated high accuracy (i.e., 85 ± 3%) and sensitivity (i.e., 87 ± 9%) for classification between CCA and benign diseases. The T-SRS acquisition only requires ∼9-μL bile samples and features a drastically improved time cost. This study suggests that the self-calibrated T-SRS analysis of the bile sample offers a promising approach for rapid CCA diagnosis.

Exosomes are submicron-sized extracellular vesicles involved in immune regulation, tumor metastasis, and cellular communication. Their lipid composition, distinct from parental cells, plays a crucial role in diseases like cancer. However, lipidomic analysis of exosomes, particularly in complex samples like blood, requires advanced techniques. This study optimizes miniaturized flow field-flow fractionation (mFlFFF) coupled with electrospray ionization mass spectrometry (ESI-MS) for direct lipidomic analysis of exosomes in serum. The mFlFFF technique resolves exosomes for size-based lipid analysis without prior extraction. Lipidomic profiling of serum exosomes from patients with extrahepatic cholangiocarcinoma (eCCA) identified over 1000 lipid species, with 64 showing significant changes compared to healthy controls. Target lipids were analyzed by mFlFFF-ESI-MS, revealing 35 species that distinguish eCCA patients from controls, suggesting their potential as biomarkers. Elevated levels of lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (PI) were observed in the eCCA group, indicating lipid alterations linked to cancer progression and inflammation. Notably, PI 38:4, involved in the release of arachidonic acid, highlights its role in inflammatory processes associated with cancer. This study demonstrates the potential of mFlFFF-ESI-MS for lipidomic analysis of exosomes and offers a non-invasive approach for cancer diagnosis, with future implications for therapeutic targeting of lipid pathways in cholangiocarcinoma.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls.

Cholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature.

We demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1.

Our results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with cirrhosis being a major risk factor. Traditional blood markers like alpha-fetoprotein (AFP) demonstrate limited efficacy in distinguishing between HCC and cirrhosis, underscoring the need for more effective diagnostic methodologies. In this context, extracellular vesicles (EVs) have emerged as promising candidates; however, their practical diagnostic application is restricted by the current lack of label-free methods to accurately profile their molecular content. To address this gap, our study explores the potential of mid-infrared (mid-IR) spectroscopy, both alone and in combination with plasmonic nanostructures, to detect and characterize circulating EVs.

EVs were extracted from HCC and cirrhotic patients. Mid-IR spectroscopy in the Attenuated Total Reflection (ATR) mode was utilized to identify potential signatures for patient classification, highlighting significant changes in the Amide I-II region (1475-1700 cm-1). This signature demonstrated diagnostic performance comparable to AFP and surpassed it when the two markers were combined. Further investigations utilized a plasmonic metasurface suitable for ultrasensitive spectroscopy within this spectral range. This device consists of two sets of parallel rod-shaped gold nanoantennas (NAs); the longer NAs produced an intense near-field amplification in the Amide I-II bands, while the shorter NAs were utilized to provide a sharp reflectivity edge at 1800-2200 cm-1 for EV mass-sensing. A clinically relevant subpopulation of EVs was targeted by conjugating NAs with an antibody specific to Epithelial Cell Adhesion Molecule (EpCAM). This methodology enabled the detection of variations in the quantity of EpCAM-presenting EVs and revealed changes in the Amide I-II lineshape.

The presented results can positively impact the development of novel laboratory methods for the label-free characterization of EVs, based on the combination between mid-IR spectroscopy and plasmonics. Additionally, data obtained by using HCC and cirrhotic subjects as a model system, suggest that this approach could be adapted for monitoring these conditions.

Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the epithelial cells lining the bile ducts. Due to its location deep within the body and nonspecific symptoms in the early stages, it is often diagnosed at the advanced stage, thus leading to worse prognosis. Circulating tumor cells within liquid biopsies (i.e. blood) have been considered as promising biomarkers for CCA diagnosis, though current methods for profiling them are not satisfactory in terms of sensitivity and specificity. Herein we developed a new cancer cell probing and immuno-tracking assay known as "CAPTURE", which was performed on an integrated microfluidic system (IMS) to automate CCA diagnosis from bile with a sample amount of only 1 mL. The assay utilized magnetic beads surface-coated with two affinity reagents, a nucleic acid aptamer (HN16) and a glycosaminoglycan (SCH 45-mix), for capturing cancer cells in bile; the "gold standard" anti-epithelial cell adhesion molecule was used as a comparison. In a single-blind test of 54 CCA-positive (+) and 102 CCA-negative (-) clinical samples, sensitivities and specificities of 96 and 80%, respectively, were documented with the CAPTURE assay on-bench. An IMS composed of a centrifugal module for sample pretreatment and a CAPTURE module for cell capture and staining was integrated with a new "vertical integration module" for detecting cancer cells from bile without human intervention. Furthermore, a novel micro-tier structure within the centrifugal module was designed to block passage of gallbladder stones with diameters >1 mm, thereby preventing their interference during the subsequent CAPTURE assay. Improved sensitivity and specificity (100 & 83%, respectively) by using three affinity reagents were achieved on the IMS when using 26 clinical bile samples, confirming its clinical bio-applicability for CCA diagnosis. This approach could be therefore used for early-stage CCA diagnostics, ideally enabling effective treatment, as well as reducing potential for relapse.

Hepatobiliary system cancers have demonstrated an increasing incidence rate in the past years. Without the presence of early symptoms, the majority of such cancers manifest with a set of similar symptoms, such as cholestasis resulting in posthepatic icterus. Differential diagnosis of hepatobiliary cancers is required for the therapy selection, however, the similarity of the symptoms complicates diagnostics. Thus, the search for molecular markers is of high interest for such patients. Cholangiocarcinoma (CCA) is characterized by a poor prognosis due to a low resectability rate, which occurs because this disease is frequently beyond the limits of surgical therapy at the time of diagnosis. The CCA is diagnosed by the combination of clinical/biochemical features, radiological methods, and non-specific serum tumor biomarkers, although invasive examination is still needed. The main disadvantage is limited specificity and sensitivity, which complicates early diagnostics. Therefore, prognostic and predictive biomarkers are still lacking and urgently needed for early diagnosis. In contrast to serum, bile is more accessible to identify biliary disease due to its simpler composition. Moreover, bile can contain higher concentrations of tumor biomarkers due to its direct contact with the tumor. It is known that the composition of the main bile component - bile acids, may vary during different diseases of the biliary tract. This review summarizes the recent developments in the current research on the diagnostic biomarkers for CCA in serum and bile and provides an overview of the methods of bile acids analysis.

Extracellular vesicles (EVs) are membrane-derived vesicles released by a variety of cell types, including hepatocytes, hepatic stellate cells, and immune cells in normal and pathological conditions. Depending on their biogenesis, there is a complex repertoire of EVs that differ in size and origin. EVs can carry lipids, proteins, coding and non-coding RNAs, and mitochondrial DNA causing alterations to the recipient cells, functioning as intercellular mediators of cell-cell communication (auto-, para-, juxta-, or even endocrine). Nevertheless, many questions remain unanswered in relation to the function of EVs under physiological and pathological conditions. The development and optimization of methods for EV isolation are crucial for characterizing their biological functions, as well as their potential as a treatment option in the clinic. In this manuscript, we will comprehensively review the results from different studies that investigated the role of hepatic EVs during liver diseases, including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic liver disease, fibrosis, and hepatocellular carcinoma. In general, the identification of patients with early-stage liver disease leads to better therapeutic interventions and optimal management. Although more light needs to be shed on the mechanisms of EVs, their use for early diagnosis, follow-up, and prognosis has come into the focus of research as a high-potential source of 'liquid biopsies', since they can be found in almost all biological fluids. The use of EVs as new targets or nanovectors in drug delivery systems for liver disease therapy is also summarized.

Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy after hepatocellular carcinoma (HCC), with a dismal prognosis and high heterogeneity. The oncological advantages of anatomical resection (AR) and nonanatomical resection (NAR) in HCC have been studied, but surgical strategies for ICC remain controversial with insufficient investigations.

From Jan 2013 to Dec 2016, 3880 consecutive patients were retrospectively reviewed from a single center. Patients with ICC undergoing AR or NAR have been enrolled according to inclusion and exclusion criteria. Propensity score matching (PSM) analysis was performed between two groups with a 1 : 1 ratio. The primary endpoint was overall survival (OS), and the secondary endpoints included disease-free survival (DFS), intraoperative patterns, postoperative morbidity, mortality, complications and recurrence. A prognostic nomogram was developed by a multivariate Cox proportion hazard model.

After PSM, 99 paired cases were selected from 276 patients enrolled in this study. Patients in the AR group achieved better 1-, 3-, and 5-year OS (70%, 46%, and 34%, respectively) and DFS (61%, 21%, and 10%, respectively) than patients in the NAR group with statistical significance after PSM analysis. The postoperative complications and recurrence patterns were comparable between the two groups. Multivariate analysis identified NAR, tumor size >5 cm, multiple tumors, and poor differentiation as independent risk factors for OS (p < 0.05). Selected patients can benefit most from AR, according to subgroup analysis. A prognostic nomogram based on six independent risk factors for OS and factors with clinical significance was constructed to predict OS in ICC patients.

AR improved the long-term survival of ICC with comparable postoperative complications and similar recurrence patterns. AR is suggested in ICC patients with sufficient remnant liver volume. In addition to surgery strategy, malignant characteristics of tumors are risk factors for ICC prognosis.

Cholangiocarcinoma (CCA) arises from the ductular epithelium of the biliary tree, either within the liver (intrahepatic CCA) or more commonly from the extrahepatic bile ducts (extrahepatic CCA). This disease has a poor prognosis and a growing worldwide prevalence. The poor outcomes of CCA are partially explained by the fact that a final diagnosis is challenging, especially the differential diagnosis between hepatocellular carcinoma and intrahepatic CCA, or distal CCA and pancreatic head adenocarcinoma. Most patients present with an advanced disease, unresectable disease, and there is a lack in non-surgical therapeutic modalities. Not least, there is an acute lack of prognostic biomarkers which further complicates disease management. Therefore, there is a dire need to find alternative diagnostic and follow-up pathways that can lead to an accurate result, either singlehandedly or combined with other methods. In the "-omics" era, this goal can be attained by various means, as it has been successfully demonstrated in other primary tumors. Numerous variants can reach a biomarker status ranging from circulating nucleic acids to proteins, metabolites, extracellular vesicles, and ultimately circulating tumor cells. However, given the relatively heterogeneous data, extracting clinical meaning from the inconsequential noise might become a tall task. The current review aims to navigate the nascent waters of the non-invasive approach to CCA and provide an evidence-based input to aid clinical decisions and provide grounds for future research.

Cholangiocarcinoma (CCA) is the second most common primary liver cancer and subsumes a heterogeneous group of malignant tumors arising from the intra- or extrahepatic biliary tract epithelium. A rising mortality from CCA has been reported worldwide during the last decade, despite significant improvement of surgical and palliative treatment. Over 50% of CCAs originate from proximal extrahepatic bile ducts and constitute the most common CCA entity in the Western world. Clinicopathological characteristics such as lymph node status and poor differentiation remain the best-studied, but imperfect prognostic factors. The identification of prognostic molecular markers as an adjunct to traditional staging systems may not only facilitate the selection of patients who would benefit the most from surgical, adjuvant or palliative treatment strategies, but may also be helpful in defining the aggressiveness of the disease and identifying patients at high-risk for tumor recurrence. The purpose of this review is to provide an overview of currently known molecular prognostic and predictive markers and their role in CCA.

Purpose: Despite considerable efforts to improve treatment modalities for cholangiocarcinoma, a common form of malignant tumor, its long-term survival rate remains poor. Hydroxychloroquine (HCQ) is a 4-aminoquinoline derivative antimalarial drug that has antimalarial and autophagy inhibition effects and exhibits comprehensive therapeutic effects on various cancers. In this study, we aimed to explore the anticancer potential and the underlying molecular mechanism of HCQ in cholangiocarcinoma treatment in vitro and in vivo. Methods: Autophagy-related genes (ARGs) were obtained from the Human Autophagy Database and Molecular Signatures Database, and the expression profiles of ARGs were downloaded from the database of The Cancer Genome Atlas. Different expression gene sets were performed using R software. The Gene Ontology and KEGG enrichment analyses were performed to reveal significantly enriched signaling pathways and to identify differentially expressed genes in cholangiocarcinoma tissues. HuCCT-1 and CCLP-1 cells were exposed to different concentrations of HCQ. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. Cell apoptosis and cycle arrest were detected by the Live/Dead cell assay and flow cytometry (FCM). The inhibition of autophagy was observed using fluorescence microscopy. The reactive oxygen species levels were assessed by fluorescence microscopy and flow cytometry. The protein levels were determined by western blot. A cholangiocarcinoma cell line xenograft model was used to evaluate the antitumor activity of HCQ in vivo. Results: Compared with normal tissues, there were 141 ARGs with an aberrant expression in cholangiocarcinoma tissues which were mainly enriched in autophagy-related processes. Inhibition of autophagy by HCQ effectively suppressed cholangiocarcinoma in vitro and in vivo. HCQ inhibited cell proliferation and induced apoptosis and cycle arrest in vitro by increasing ROS accumulation, which was involved in autophagy inhibition. The ROS scavenger reduced l-glutathione distinctly weakened HCQ-induced cell apoptosis and viability inhibition in cholangiocarcinoma cells. In addition, HCQ inhibited growth of cholangiocarcinoma cell line xenograft tumors. Conclusion: HCQ could inhibit cell proliferation and induce apoptosis in cholangiocarcinoma by triggering ROS accumulation via autophagy inhibition, which makes HCQ a potential antitumor drug candidate for cholangiocarcinoma treatment.