Augmented Global Protein Acetylation Diminishes Cell Growth and Migration of Cholangiocarcinoma Cells.

We have previously shown that the overexpression of acetyl-CoA carboxylase 1 (ACC1) was associated with the poor prognosis of cholangiocarcinoma (CCA) patients, and suppression of its expression in CCA cell lines deteriorated cell growth. The present study explored the mechanism by which ACC1 inhibition affects global protein acetylation, using genetic knockdown and pharmacological inhibition with an ACC1 inhibitor ND-646 as models. Both ACC1 knockdown and ACC1-inhibitor-treated cells displayed the hyperacetylation of proteins, accompanied by impaired growth and migration. The immunoprecipitation of hyperacetylated proteins using the anti-acetylated lysine antibody, followed by tandem mass spectrometry, identified three potential verification candidates, namely POTE ankyrin domain family member E, peroxisomal biogenesis factor 1, and heat shock protein 90 beta (HSP90B). HSP90 acetylation was the candidate selected for the verification of protein acetylation. To establish the effects of protein hyperacetylation, treatment with suberoylanilide hydroxamic acid (SAHA), a lysine deacetylase inhibitor, was conducted, and this served as an independent model. Decreased tumor growth but increased acetylated protein levels were observed in ACC1-KD xenograft tumors. Hyperacetylated-alleviated cell growth and migration were consistently observed in the SAHA-treated models. The molecular linkage between protein hyperacetylation and the AKT/GSK3ß/Snail pathway was demonstrated. This study highlighted the importance of protein acetylation in CCA progression, suggesting that ACC1 and KDAC are potential targets for CCA treatment.

CO2-Free On-Stage Incubator for Live Cell Imaging of Cholangiocarcinoma Cell Migration on Microfluidic Device.

Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO2 content, and humidity. In this study, we present a CO2-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as "whole-colony migration", "novel type of collective cell migration" and "colony formation during cell and colony migration" are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis.

Involvement of interleukin-1ß in high glucose-activated proliferation of cholangiocarcinoma.

Background: Diabetes mellitus (DM) is associated with the increased risk of development and the advancement of cholangiocarcinoma (CCA). High glucose levels were previously shown for upregulating interleukin-1ß (IL-1ß) in CCA cells with unclear functions. The present study, thus, aimed to investigate molecular mechanisms linking DM to CCA progression, with IL-1ß hypothesized as a communicating cytokine. Methods: CCA cells were cultured in media with normal (5.6 mM) or high (25 mM) glucose, resembling euglycemia and hyperglycemia, respectively. Expressions of IL-1ß and IL-1 receptor (IL-1R) in CCA tissues from patients with and without DM were examined using immunohistochemistry. Functional analyses of IL-1ß were performed using siRNA and recombinant human IL-1R antagonist (rhIL-1RA), in which Western blots investigated the knockdown efficacy. BALB/c Rag-2-/- Jak3-/- (BRJ) mice were implanted with CCA xenografts to investigate hyperglycemia's effects on CCA growth and the anti-tumor effects of IL-1RA. Results: CCA tumors from patients with hyperglycemia showed significantly higher IL-1ß expression than those from non-DM patients, while IL-1ß was positively correlated with fasting blood glucose (FBG) levels. CCA cells cultured in high glucose showed increased IL-1ß expression, resulting in increased proliferation rates. Suppressing IL-1ß signaling by si-IL-1ß or rhIL-1RA significantly reduced CCA cell proliferation in vitro. Anakinra, a synthetic IL-1RA, also exerted significant anti-tumor effects in vivo and significantly reversed the effects of hyperglycemia-induced growth in CCA xenografts. Conclusions: IL-1ß plays a crucial role in CCA progression in a high-glucose environment. Targeting IL-1ß might, then, help improve therapeutic outcomes of CCA in patients with DM and hyperglycemia.

Overcoming treatment resistance in cholangiocarcinoma: current strategies, challenges, and prospects.

Significant advancements in our understanding and clinical treatment of cholangiocarcinoma (CCA) have been achieved over the past 5 years. Groundbreaking studies have illuminated the immune landscape and pathological characteristics of the tumor microenvironment in CCA. The development of immune- and metabolism-based classification systems has enabled a nuanced exploration of the tumor microenvironment and the origins of CCA, facilitating a detailed understanding of tumor progression modulation. Despite these insights, targeted therapies have not yet yielded satisfactory clinical results, highlighting the urgent need for innovative therapeutic strategies. This review delineates the complexity and heterogeneity of CCA, examines the current landscape of therapeutic strategies and clinical trials, and delves into the resistance mechanisms underlying targeted therapies. Finally, from a single-cell and spatial transcriptomic perspective, we address the challenge of therapy resistance, discussing emerging mechanisms and potential strategies to overcome this barrier and enhance treatment efficacy.

Promotion of perineural invasion of cholangiocarcinoma by Schwann cells via nerve growth factor.

Background: Cholangiocarcinoma (CCA), a highly lethal tumor of the hepatobiliary system originating from bile duct epithelium, can be divided into the intrahepatic, hilar, and extrahepatic types. Due to its insidious onset and atypical early clinical symptoms, the overall prognosis is poor. One of the important factors contributing to the poor prognosis of CCA is the occurrence of perineural invasion (PNI), but the specific mechanisms regarding how it contributes to the occurrence of PNI are still unclear. The main purpose of this study is to explore the molecular mechanism leading to the occurrence of PNI and provide new ideas for clinical treatment. Methods: CCA cell lines and Schwann cells (SCs) were stimulated to observe the changes in cell behavior. SCs cocultured with tumor supernatant and SCs cultured in normal medium were subjected to transcriptome sequencing to screen the significantly upregulated genes. Following this, the two types of tumor cells were cultured with SC supernatant, and the changes in behavior of the tumor cells were observed. Nonobese diabetic-severe combined immunodeficiency disease (NOD-SCID) mice were injected with cell suspension supplemented with nerve growth factor (NGF) via the sciatic nerve. Four weeks later, the mice were euthanized and the tumor sections were removed and stained. Results: Nerve invasion by tumor cells was common in CCA tissues. SCs were observed in tumor tissues, and the number of SCs in tumor tissues and the degree of PNI were much higher than were those in normal tissues or tissues without PNI. The overall survival time was shorter in patients with CCA with PNI than in patients without PNI. SCs were enriched in CCA tissues, indicating the presence of PNI and associated with poor prognosis in CCA patients. CCA was found to promote NGF secretion from SCs in vitro. After the addition of exogenous NGF in CCA cell culture medium, the proliferation activity and migration ability of CCA cells were significantly increased, suggesting that SCs can promote the proliferation and migration of CCA through the secretion of NGF. NGF, in turn, was observed to promote epithelial-mesenchymal transition in CCA through tropomyosin receptor kinase A (TrkA), thus promoting its progression. Tumor growth in mice shows that NGF can promote PNI in CCA. Conclusions: In CCA, tumor cells can promote the secretion of NGF by SCs, which promotes the progression of CCA and PNI by binding to its high-affinity receptor TrkA, leading to poor prognosis.

Clinical Implications of a Missing Hepatic Segment of the Inferior Vena Cava: A Case Report.

Anomalies of the inferior vena cava (IVC) are often discovered incidentally, yet they pose significant challenges in both diagnosis and treatment. This study discusses a case of a 50-year-old female with situs inversus and well-differentiated cholangiocarcinoma, emphasizing the rarity and complexity involved. Imaging studies confirmed the presence of dextrocardia, abdominal situs inversus, and an interrupted IVC with continuation to the azygos vein. A thorough understanding of IVC embryology is crucial, as it helps explain the pathogenesis of such anomalies, which arise from disturbances in the embryonic venous system leading to various anatomical variations. Managing an interrupted IVC is critical due to the potential for vascular complications, highlighting the need for preoperative awareness. This study underlines the importance of recognizing and properly managing these rare vascular anomalies to improve patient care.

Cholangiocarcinoma imaging: from diagnosis to response assessment.

Cholangiocarcinoma (CCA), a highly aggressive primary liver cancer arising from the bile duct epithelium, represents a substantial proportion of hepatobiliary malignancies, posing formidable challenges in diagnosis and treatment. Notably, the global incidence of intrahepatic CCA has seen a rise, necessitating a critical examination of diagnostic and management strategies, especially due to presence of close imaging mimics such as hepatocellular carcinoma (HCC) and combined hepatocellular carcinoma-cholangiocarcinoma (cHCC-CCA). Hence, it is imperative to understand the role of various imaging modalities such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), elucidating their strengths, and limitations in diagnostic precision and staging accuracy. Beyond conventional approaches, there is emerging significance of functional imaging tools including positron emission tomography (PET)-CT and diffusion-weighted (DW)-MRI, providing pivotal insights into diagnosis, therapeutic assessment, and prognostic evaluation. This comprehensive review explores the risk factors, classification, clinical features, and role of imaging in the holistic spectrum of diagnosis, staging, management, and restaging for CCA, hence serving as a valuable resource for radiologists evaluating CCA.

Generation of Metastatic Cholangiocarcinoma Patient-Derived Xenograft Models.

Cholangiocarcinoma (CCA) poses a substantial clinical hurdle as it is often detected at advanced metastatic stages with limited therapeutic options. To enhance our understanding of advanced CCA, it is imperative to establish preclinical models that faithfully recapitulate the disease's characteristics. Patient-derived xenograft (PDX) models have emerged as a valuable approach in cancer research, offering an avenue to reproduce and study the genomic, histologic, and molecular features of the original human tumors. By faithfully preserving the heterogeneity, microenvironmental interactions, and drug responses observed in human tumors, PDX models serve as highly relevant and predictive preclinical tools. Here, we present a comprehensive protocol that outlines the step-by-step process of generating and maintaining PDX models using biopsy samples from patients with advanced metastatic CCA. The protocol encompasses crucial aspects such as tissue processing, xenograft transplantation, and subsequent monitoring of the PDX models. By employing this protocol, we aim to establish a robust collection of PDX models that accurately reflect the genomic landscape, histologic diversity, and therapeutic responses observed in advanced CCA, thereby enabling improved translational research, drug development, and personalized treatment strategies for patients facing this challenging disease.

Circulating tumor DNA analysis guiding adjuvant treatment in resected stage III cholangiocarcinoma: a case report.

Background: Cholangiocarcinoma (CCA) is a rare and aggressive gastrointestinal cancer. Unfortunately, 60% to 70% of early-stage CCA patients experience disease recurrence after curative resection and standard adjuvant therapy. Currently, there is no reliable tool to identify CCA recurrence before radiographic detection. Longitudinal monitoring of circulating tumor DNA (ctDNA) has shown promising value in molecular identification of relapse prior to conventional surveillance in other solid tumors. However, there is a scarcity of data on ctDNA in CCA after curative surgery. Case Description: An 81-year-old male with stage 3A intrahepatic CCA achieved radiographic remission after curative resection and was started on standard adjuvant capecitabine on post-operative day (POD) 50. Tumor-informed ctDNA tested positive on two consecutive occasions, with the titer increasing from 0.16 mean tumor molecule (MTM)/mL on POD 92 to 0.80 MTM/mL on POD 183, despite being on capecitabine. carbohydrate antigen 19-9 (CA19-9) also continued to increase from 175.6 U/mL on POD 92 to 7,594.9 U/mL on POD 217. Notably, surveillance computed tomography (CT) scans showed no evidence of disease (NED) on POD 126, 186, and 211. Molecular profiling and next-generation sequencing (NGS) panels from CCA tissue revealed microsatellite instability-high (MSI-H). After extensive discussions with the patient regarding the rising ctDNA titer despite being on capecitabine for nearly 6 months, we initiated pembrolizumab on POD 224 prior to radiographic recurrence. Given the tumor is MSI-H, and the preferred toxicity profile compared to the front-line chemotherapy option for CCA, we started pembrolizumab. ctDNA became undetectable, and CA19-9 returned to the reference range with pembrolizumab. As of the last follow-up on POD 876, the patient has continued pembrolizumab without noticeable side effects, and imaging continues to show NED, with persistent negative ctDNA and normal CA19-9 levels. Conclusions: This case demonstrates the potential utility of tumor-informed ctDNA in CCA as (I) an early detection tool before radiographic recurrence; (II) a response monitoring tool as a surrogate biomarker that can guide therapy optimization; and (III) shows that early intervention with immunotherapy or potentially targeted agents based on ctDNA may lead to improved survival outcomes.

GOLPH3 promotes tumor malignancy via inhibition of ferroptosis by upregulating SLC7A11 in cholangiocarcinoma.

Golgi phosphoprotein 3 (GOLPH3) has been reported as an oncogene in various tumors; however, the role and function of GOLPH3 and its relevant molecular mechanism in cholangiocarcinoma (CCA) are unclear. Herein, GOLPH3 expression in CCA tissues was observed to be significantly higher than that in paired adjacent noncancerous tissues. Clinicopathological analysis showed that GOLPH3 expression correlated positively with the tumor-node-metastasis stage. In addition, GOLPH3 expression correlated inversely with the overall survival of patients with CCA. Multivariate analysis showed that GOLPH3 was an independent prognostic factor for patients with CCA. Transcriptome analysis (RNA sequencing) of GOLPH3 knockdown cells showed that the expression levels of nine ferroptosis-related genes were significantly changed, indicating the important biological function of GOLPH3 in ferroptosis in CCA cells. Furthermore, GOLPH3 knockdown could significantly promote Erastin-induced ferroptosis in vitro and suppress tumor growth in vivo. Overexpression of GOLPH3 had the opposite effect on this phenotype. Further studies revealed that GOLPH3 knockdown was significantly associated with a decrease in cysteine content, an accumulation of the lipid peroxidation product malondialdehyde, an increase in reactive oxygen species, and sensitized CCA cells to Erastin-induced ferroptosis. Moreover, changes in GOLPH3 expression were found to be consistent with the expression of light chain subunit solute carrier family 7 member 11 (SLC7A11). Thus, our study suggested that GOLPH3 functions as an oncoprotein in CCA and may suppress ferroptosis by facilitating SLC7A11 expression, suggesting that GOLPH3 could serve as a therapeutic target for CCA treatment.

Molecular pathology for cholangiocarcinoma: a review of actionable genetic targets and their relevance to adjuvant & neoadjuvant therapy, staging, follow-up, and determination of minimal residual disease.

Cholangiocarcinoma (CCA) represents a group of epithelial cell tumors classified based on their anatomic location along the biliary tree. This rare malignancy is often diagnosed at an advanced stage and deemed unresectable. Even for those patients who are surgical candidates, recurrence rates are high and survival rates low. The mainstay of therapy for advanced CCA remains cisplatin plus gemcitabine, with a median overall survival (mOS) under 12 months, although the TOPAZ-1 trial showed a survival benefit with the addition of programmed cell death ligand 1 (PD-L1) blockade. In recent years, molecular profiling has revealed a wealth of potentially targetable genetic alterations, including fibroblast growth factor receptor (FGFR) fusions, isocitrate dehydrogenase 1 (IDH1) mutations, human epidermal growth factor receptor 2 (HER2) amplification and overexpression, and microsatellite instability (MSI). These discoveries have prompted numerous clinical trials employing drugs against these specific genetic changes. The foundation laid by early clinical studies and the landscape of ongoing trials are both summarized here. While the role of adjuvant therapy has yet to be defined in this disease, we emphasize the importance of employing targeted therapies in trials in the adjuvant and neoadjuvant spaces and discuss ways to overcome challenges due to low incidence of targetable mutations. Personalized medicine for this disease promises significant clinical benefit to patients, but further investigation is needed.

Application of AI on cholangiocarcinoma.

Cholangiocarcinoma, classified as intrahepatic, perihilar, and extrahepatic, is considered a deadly malignancy of the hepatobiliary system. Most cases of cholangiocarcinoma are asymptomatic. Therefore, early detection of cholangiocarcinoma is significant but still challenging. The routine screening of a tumor lacks specificity and accuracy. With the application of AI, high-risk patients can be easily found by analyzing their clinical characteristics, serum biomarkers, and medical images. Moreover, AI can be used to predict the prognosis including recurrence risk and metastasis. Although they have some limitations, AI algorithms will still significantly improve many aspects of cholangiocarcinoma in the medical field with the development of computing power and technology.

A dual-electrode label-free immunosensor based on in situ prepared Au-MoO3-Chi/porous graphene nanoparticles for point-of-care detection of cholangiocarcinoma.

A novel label-free electrochemical immunosensor was prepared for the detection of carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) as biomarkers of cholangiocarcinoma (CCA). A nanocomposite of gold nanoparticles, molybdenum trioxide, and chitosan (Au-MoO3-Chi) was layer-by-layer assembled on the porous graphene (PG) modified a dual screen-printed electrode using a self-assembling technique, which increased surface area and conductivity and enhanced the adsorption of immobilized antibodies. The stepwise self-assembling procedure of the modified electrode was further characterized morphologically and functionally. The electroanalytical detection of biomarkers was based on the interaction between the antibody and antigen of each marker via linear sweep voltammetry using ferrocyanide/ferricyanide as an electrochemical redox indicator. Under optimized conditions, the fabricated immunosensor showed linear relationships between current change (ΔI) and antigen concentrations in two ranges: 0.0025-0.1 U mL-1 and 0.1-1.0 U mL-1 for CA19-9, and 0.001-0.01 ng mL-1 and 0.01-1.0 ng mL-1 for CEA. The limits of detection (LOD) were 1.0 mU mL-1 for CA19-9 and 0.5 pg mL-1 for CEA. Limits of quantitation (LOQ) were 3.3 mU mL-1 for CA19-9 and 1.6 pg mL-1 for CEA. The selectivity of the developed immunosensor was tested on mixtures of antigens and was then successfully applied to determine CA19-9 and CEA in human serum samples, producing satisfactory results consistent with the clinical method.

Evaluation of the serotonin pathway as a biomarker in cholangiocarcinoma.

Background: Cholangiocarcinomas (CCAs) are rare and aggressive malignant tumors of the biliary tract. Serotonin (5HT) has tumor-promoting effects in CCA while inhibition of 5HT synthesis can decrease tumor growth. Methods: In this retrospective study, we evaluated the expression of 5HT and tryptophane hydroxylase-1 (TPH-1) in tumor specimens from patients treated with cisplatin plus gemcitabine (CisGem). We included consecutive patients ≥18 years, with locally advanced unresectable, recurrent, or metastatic CCA who were treated with CisGem and had available archival tumor tissue for immunohistochemistry. Formalin-fixed paraffin (FFPE) sections were stained for 5HT and TPH-1. Specimens were evaluated for neuroendocrine features and tumor-infiltrating lymphocytes (TILs). Serum 5HT was measured. Results: We identified 23 patients fulfilling the inclusion criteria. 5HT expression was absent in almost all tumors examined. TPH-1 expression was neither associated with stage or primary tumor location nor predictive of response to CisGem. There was a trend for improved overall survival (OS) in patients whose tumors had high TPH-1 expression. The examined tumor specimens had no neuroendocrine features. Most sections had no TILs. There was a trend for worse OS in patients with high serum 5HT concentration. Conclusions: Tumor TPH-1 expression was not predictive of response to treatment. There was a trend for improved long-term outcomes in patients with high tumor TPH expression and lower serum 5HT concentration.

Erratum: Preoperative immunological plasma markers TRAIL, CSF1, and TIE2 predict survival after resection for biliary tract cancer.

[This corrects the article DOI: 10.3389/fonc.2023.1169537.].