Intrahepatic Cholangiocarcinoma Cell Lines Research Articles

DCLK1 Expression and its Functional Significance in Intrahepatic Cholangiocarcinoma and Bil-IN Stage.

Although several studies in some neoplasms have reported correlation between the expression levels of Doublecortin-like kinase1(DCLK1) and carcinogenesis, its role in cholangiocarcinoma remains unknown. DCLK1 expression in normal epithelium (NE), biliary intraepithelial neoplasia (BilIN)1∼3, and intrahepatic cholangiocarcinoma (ICC) were investigated immuno-histochemically. The molecular effects of DCLK1 were investigated by gene silencing using RNAi [DCLK1-tagrgeting (siDCLK1)]. The human ICC cell lines HuCCT1 and HuH28 were transfected with these siRNAs, and used for assays in the presence or absence of DCLK1 inhibitors. The positive ratio of DCLK1 expression in ICC was higher than that in NE, and equally distributed among BilIN1∼3 (NE: BilIN1: BilIN2: BilIN3: ICC=62%: 91%: 97%: 100%: 95%, p<0.05). In the wound healing assay, the migration of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the cell invasion assay, the invasion of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the presence of the DCLK1 inhibitor, cell proliferative capacity at 24 hours was decreased in a concentration-dependent manner. DCLK1 was highly expressed in the early stage of ICC carcinogenesis. Human ICC cell growth was suppressed in vitro by siRNA silencing of DCLK1 or after treatment with the DCLK1 inhibitor, indicating DCLK1 may be molecular target for ICC therapy.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

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

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. 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 invitro co-culture system and in a mouse model of iCCA. 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. 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. 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.

CircPCNXL2 promotes tumor growth and metastasis by interacting with STRAP to regulate ERK signaling in intrahepatic cholangiocarcinoma

Backgroundcircular RNAs (circRNAs) have been reported to exert important effects in the progression of numerous cancers. However, the functions of circRNAs in intrahepatic cholangiocarcinoma (ICC) are still unclear.MethodscircPCNXL2 (has_circ_0016956) were identified in paired ICC by circRNA microarray. Then, we assessed the biological functions of circPCNXL2 by CCK8, EdU, clone formation, transwell, wound healing assays, and xenograft models. RNA pull-down, mass spectrometry, and RNA immunoprecipitation (RIP) were applied to explore the interaction between cirrcPCNXL2 and serine-threonine kinase receptor-associated protein (STRAP). RNA pull-down, RIP and luciferase reporter assays were used to investigate the sponge functions of circPCNXL2. In the end, we explore the effects of circPCNXL2 and trametinib (a MEK1/2 inhibitor) in vivo.ResultscircPCNXL2 was upregulated in ICC tissues and cell lines, which promoted the proliferation and metastasis of ICC in vitro and in vivo. In terms of the mechanisms, circPCNXL2 could directly bind to STRAP and induce the interaction between STRAP and MEK1/2, resulting in the tumor promotion in ICC by activation of ERK/MAPK pathways. Besides, circPCNXL2 could regulate the expression of SRSF1 by sponging miR-766-3p and subsequently facilitated the growth of ICC. Finally, circPCNXL2 could partially inhibit the anti-tumor activity of trametinib in vivo.ConclusioncircPCNXL2 played a crucial role in the progression of ICC by interacting with STRAP to activate the ERK signaling pathway, as well as by modulating the miR-766-3p/SRSF1 axis. These findings suggest that circPCNXL2 may be a promising biomarker and therapeutic target for ICC.

Establishment and Characterization of a New Intrahepatic Cholangiocarcinoma Cell Line, ICC-X2.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor of the biliary tract that is prone to recurrence and metastasis and is characterized by poor sensitivity to chemotherapy and overall prognosis. For these reasons, there is an urgent need to understand its pathological mechanisms and develop effective treatments. To address this challenge, the establishment of suitable preclinical models is critical. Fresh ICC tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, and short tandem repeat (STR) analysis. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-fluorouracil (5-FU) were evaluated by CCK-8 assay. Subcutaneous injection of 1 × 106 cells to three BALB/c nude mice was conducted for xenograft studies. The hematoxylin and eosin (H&E) staining was used to detect the pathological status of the cell line. The expression of biomarkers CK7, CK19, Ki-67, E-cadherin and vimentin was determined by immunocytochemistry assay. A new ICC cell line named ICC-X2 was successfully established. Like ICC-X3 established using the same patient's metastatic tumor, the cell line has been continuously cultured in vitro for more than a year and has been passaged more than 100 times. ICC-X2 retained the typical biliary epithelial morphology. The population doubling time of ICC-X2 is 48 h. The cells demonstrated an abnormal nearly tetraploid karyotype. The STR analysis confirmed that ICC-X2 was highly consistent with the primary tumor tissue and not cross-contaminated by existing cell lines. ICC-X2 cells positively expressed CK7, CK19, E-cadherin, and vimentin, and the positive expression of Ki-67 in ICC-X2 cells was 40%. The ICC-X2 cells exhibited a strong clonogenic ability. The drug sensitivity test indicated that ICC-X2 was sensitive to oxaliplatin and paclitaxel, but naturally resistant to gemcitabine and 5-FU. ICC-X2 was rapidly able to form transplanted tumors in vivo after subcutaneous inoculation in nude mice. ICC-X2 is an excellent experimental model that can be used for studying the occurrence, development, and metastasis of ICC and investigating the mechanism of tumor drug resistance.

Ferroptosis-related gene glutathione peroxidase 4 promotes reprogramming of glucose metabolism via Akt-mTOR axis in intrahepatic cholangiocarcinoma.

The role of the ferroptosis-related gene glutathione peroxidase 4 (GPX4) in oncology has been extensively investigated. However, the clinical implications of GPX4 in patients with intrahepatic cholangiocarcinoma (ICC) remain unknown. This study aimed to evaluate the prognostic impact of GPX4 and its underlying molecular mechanisms in patients with ICC. Fifty-seven patients who underwent surgical resection for ICC between 2010 and 2017 were retrospectively analyzed. Based on the immunohistochemistry, patients were divided into GPX4 high (n = 15) and low (n = 42) groups, and clinical outcomes were assessed. Furthermore, the roles of GPX4 in cell proliferation, migration and gene expression were analyzed in ICC cell lines in vitro and in vivo. The results from clinical study showed that GPX4 high group showed significant associations with high SUVmax on 18F-fluorodeoxyglucose-positron emission tomography (≥8.0, P = 0.017), multiple tumors (P = 0.004), and showed glucose transporter 1 (GLUT1) high expression with a trend toward significance (P = 0.053). Overall and recurrence-free survival in the GPX4 high expression group were significantly worse than those in the GPX4 low expression group (P = 0.038 and P < 0.001, respectively). In the experimental study, inhibition of GPX4 attenuated cell proliferation and migration in ICC cell lines. Inhibition of GPX4 also decreased the expression of glucose metabolism-related genes, such as GLUT1 or HIF1α. Mechanistically, these molecular changes are regulated in Akt-mechanistic targets of rapamycin axis. In conclusion, this study suggested the pivotal value of GPX4 serving as a prognostic marker for patients with ICC. Furthermore, GPX4 can mediate glucose metabolism of ICC.

Aspartate β-hydroxylase (ASPH) Accelerates Intrahepatic Cholangiocarcinoma Metastasis via Upregulating SHH Signaling Pathway.

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy with a poor prognosis. Aspartate β-hydroxylase (ASPH) is an α-ketoglutarate-dependent dioxygenase involved in the post-translational hydroxylation of target proteins. ASPH has been demonstrated to be upregulated in ICC, yet its role remains to be elucidated. This study aimed to investigate the potential function of ASPH in ICC metastasis. Survival curves for the overall survival of pan-cancer data from The Cancer Genome Atlas (TCGA) database was depicted using the Kaplan-Meier method and compared using the log-rank test. The expression of ASPH, glycogen synthase kinase (GSK)-3β, phosphorylation GSK-3β (p-GSK-3β), epithelial-mesenchymal transition (EMT) biomarkers, and sonic hedgehog (SHH) signaling elements in ICC cell lines was analyzed by western blot. Wound healing and transwell assays were conducted to examine the effects of ASPH knockdown and overexpression on cell migration and invasion. An immunofluorescence assay was conducted to evaluate the expression of glioma-associated oncogene 2 (GLI2), GSK-3β and ASPH. The effect of ASPH on tumor in vivo was analyzed using a nude mouse xenograft model. Pan-cancer data showed that expressed ASPH was significantly correlated with a poor prognosis in patients. ASPH knockdown inhibited the migration and invasion of human ICC cells lines QBC939 and RBE. ASPH overexpression contributed to an increase in the N-cadherin and Vimentin, resulting in the promotion of the EMT process. The p-GSK-3β levels decreased in the presence of ASPH overexpression. The overexpression of ASPH led to an upregulation of the expression of SHH signaling elements GLI2 and SUFU. The results of in vivo experiments with a lung metastasis model in nude mice with ICC cell line RBE are consistent with these results. ASPH accelerated metastasis of ICC cells by facilitating EMT via a GSK-3β/SHH/GLI2 axis-dependent manner, in which phosphorylation of GSK-3β was downregulated and the SHH signaling pathway was activated.

Uncovering the mechanism of Kang-ai injection for treating intrahepatic cholangiocarcinoma based on network pharmacology, molecular docking, and in vitro validation

Objective: Kang-ai injection (KAI) has been a popular adjuvant treatment for solid tumors, but its anti-tumor mechanism in intrahepatic cholangiocarcinoma (ICC) remains poorly understood. This study applied a network pharmacology-based approach to unveil KAI's anti-tumor activity, key targets, and potential pharmacological mechanism in ICC by integrating molecular docking and in vitro validation. Methods: The KAI-compound-target-ICC network was constructed to depict the connections between active KAI compounds and ICC-related targets based on the available data sources. The crucial ingredients, potential targets, and signaling pathways were screened using GO, KEGG enrichment analysis, and the PPI network. Molecular docking was performed to visualize the interactions between hub targets and components. In vitro experiments were carried out to validate the findings. Results: Among the 87 active components of KAI and 80 KAI-ICC-related targets, bioinformatics analysis identified quercetin as a possible candidate. GO and KEGG enrichment analysis indicated that the PI3K-AKT signaling pathway might be essential in ICC pharmacotherapy. The PPI network and its sub-networks screened 10 core target genes, including AKT1 and IL1β. Molecular docking results showed stable binding between AKT1 and IL1β with KAI active ingredients. The in vitro experiments confirmed that KAI might suppress the proliferation of ICC cell lines by inhibiting the PI3K/AKT signaling pathway, consistent with the network pharmacology approach and molecular docking predictions. Conclusion: The study sheds light on KAI's biological activity, potential targets, and molecular mechanisms in treating ICC and provides a promising strategy for understanding the scientific basis and therapeutic mechanisms of herbal treatments for ICC. This research has important implications for developing new, targeted therapies for ICC and highlights the importance of network pharmacology-based approaches in investigating complex herbal formulations.

Knockdown of FGFR3 inhibits the proliferation, migration and invasion of intrahepatic cholangiocarcinoma.

The FGF/FGFR signaling axis deregulation of the fibroblast growth factor receptor (FGFR) family is closely related to tumorigenesis, tumor progression and drug resistance to anticancer therapy. And fibroblast growth factor receptor 3 (FGFR3) is one member of this family. In this study, we aimed to investigate the effect of siRNA-induced knockdown of FGFR3 on the biological behaviors of intrahepatic cholangiocarcinoma (ICC). The expression levels of FGFR3 were determined in three intrahepatic cholangiocarcinoma cell lines RBE, HUCCT1 and HCCC9810 cell lines by Western blot. FGFR3 expression in RBE cell line was knocked down by siRNA. Our study found that knockdown of FGFR3 inhibited the migration, invasion and proliferation of ICC cells using Wound healing assay, Transwell migration and invasion assays and Cell proliferation assay. And significantly down-regulated the protein expression levels of MMP2, cyclinD1, and NCadherin, but had no significant effect on MMP9, cyclinD3, vimentin, E-cadherin protein. In addition, we found that ERK/c-Myc presumably is its signaling pathway by bioinformatics analysis and Western blot verification. To sum up, knockdown of FGFR3 inhibited the migration, invasion and proliferation of ICC cells. It demonstrated that FGFR3 probably becomes a therapeutic target for ICC and increases the proportion of potentially curable intrahepatic cholangiocarcinoma patients treated with FGFR inhibitors.

Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis.

The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0) and directly injected into the mass spectrometer. The unique peptides overlapped between alkaline-pH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (low-pH-MS/MS) were as low as 45%, strongly indicating that these two methods were complementary to each other. In addition, alkaline-pH-MS/MS showed identification capacity for a higher proportion of peptides with negative grand average of hydropathy (GRAVY) or high isoelectric point (pI). Compared to low-pH-MS/MS, alkaline-pH-MS/MS enabled enrichment preference toward histidine-, lysine-, methionine-, and proline-containing peptides. The complementarity of alkaline-pH-MS/MS and low-pH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating 60 min alkaline-pH-MS/MS plus 60 min low-pH-MS/MS method outperformed the conventional 120 min low-pH-MS/MS method in both the identification of amino acid variants and protein groups. Therefore, we established the alkaline-pH-MS/MS method as a simple, competitive, alternative method to low-pH-MS/MS for global proteomic analysis.

Establishment and characterization of a new intrahepatic cholangiocarcinoma cell line, ICC-X3

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer of the biliary tract that is prone to recurrence and metastasis and is characterized by poor sensitivity to chemotherapy and overall prognosis. To address this challenge, the establishment of suitable preclinical models is critical. In this study, we successfully established a new ICC cell line, named ICC-X3, from the satellite lesions of one ICC patient. The cell line was characterized with respect to phenotypic, molecular, biomarker, functional and histological properties. STR confirmed that ICC-X3 was highly consistent with primary tumor tissue. ICC-X3 cells positively expressed CK7, CK19, E-cadherin, vimentin, and Ki67. ICC-X3 was all resistant to gemcitabine, paclitaxel, 5-FU, and oxaliplatin. The cell line was able to rapidly form xenograft tumors which were highly similar to the primary tumor. The missense mutation of TP53 exon was detected in ICC-X3 cells. ICC-X3 can be used as a good experimental model to study the progression, metastasis, and drug resistance of ICC.

Establishment and characterization of a new intrahepatic cholangiocarcinoma cell line derived from a Chinese patient

Patients with intrahepatic cholangiocarcinoma (ICC) require chemotherapy due to late detection, rapid disease progression, and low surgical resection rate. Tumor cell lines are extremely important in cancer research for drug discovery and development. Here, we established and characterized a new intrahepatic cholangiocarcinoma cell line, ICC-X1. STR testing confirmed the absence of cross-contamination and high similarity to the original tissue. ICC-X1 exhibited typical epithelial morphology and formed tumor spheres in the suspension culture. The population doubling time was approximately 48 h. The cell line had a complex hypotriploid karyotype. The cell line exhibited a strong migration ability in vitro and cell inoculation into BALB/c nude mice led to the formation of xenografts. Additionally, ICC-X1 cells were sensitive to gemcitabine and paclitaxel but resistant to 5-fluorouracil and oxaliplatin. RNA sequencing revealed that the upregulated cancer-related genes were mainly enriched in several signaling pathways, including the TNF signaling pathway, NOD-like receptor signaling pathway, and NF-κB signaling pathway. The downregulated cancer-related genes were mainly enriched in the Rap1 signaling pathway and Hippo signaling pathway among other pathways. In conclusion, we have created a new ICC cell line derived from Chinese patients. This cell line can be used as a preclinical model to study ICC, specifically tumor metastasis and drug resistance mechanisms.

Glycoproteomic analysis reveals the effects of bisecting GlcNAc in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second major subtype of primary liver cancer and has caused more and more attention with increasing incidence and mortality worldwide. Our previous study found that bisecting N-glycans are commonly increased in ICC, while the effects and potential functions of bisecting GlcNAc in ICC are still largely unclear. In this study, we further confirmed that the structures of bisecting GlcNAc were significantly up-regulated in ICC compared with paracancer tissues by glycoproteomic data and lectin histochemistry. The expression of its glycosyltransferase MGAT3 was also up-regulated in ICC tissues at both mRNA and protein levels, and expression of MGAT3 is negatively correlated with overall survival explored by bioinformatic analyses and published datasets from 255 patients. Next, the silencing of MGAT3 could inhibit the growth and invasion of ICC cells, and overexpressing of MGAT3 only promoted ICC cell invasion. Further glycoproteomic analysis showed that the commonly glycoproteins modified by bisecting GlcNAc after MGAT3-overexpression in two ICC cell lines were mainly involved in cell movement-related biological processes, such as cell adhesion, integrin-related and ECM-receptor interaction. This study sheds light on the potential effects of bisecting GlcNAc in ICC cells and suggests that MGAT3 might be used as a potential target in the therapy of ICC.

Establishment and Characterization of a New Human Intrahepatic Cholangiocarcinoma Cell Line LIV27.

Simple SummaryCholangiocarcinoma (CCA) is usually diagnosed at a late stage and has a poor prognosis. Cell lines serve as useful models for testing scientific questions in vitro and in vivo. To aid scientific discovery for the purpose of improved early detection and treatment, we developed a CCA cell line, designated LIV27, from a surgically resected intrahepatic CCA in a Caucasian patient with primary sclerosing cholangitis (PSC). LIV27 has high tumorigenicity in nude mice and expands the availability of intrahepatic CCA cell lines.Cholangiocarcinoma (CCA) is a highly lethal cancer arising from the biliary tract epithelium. The cancer biology of this neoplasm is not well understood. To date, only a few CCA cell lines have been reported, which were mostly developed from Asian patients. In this study, we report and characterize a new intrahepatic CCA cell line, LIV27, derived from a surgically resected tumor in a 67-year-old Caucasian woman with primary sclerosing cholangitis (PSC). LIV27 cells grow well in collagen-coated flasks or plates with a doubling time of 57.8 h at passage 14. LIV27 cells have high tumorigenicity in nude mice and stain positive for CK7 and CK19, markers that differentiate CCA from hepatocellular carcinoma. Karyotype analysis showed that LIV27 is aneuploid. We established a single-locus short tandem repeat profile for the LIV27 cell line. This newly established cell line will be a useful model for studying the molecular pathogenesis of, and developing novel therapies for, cholangiocarcinoma.

Abstract PO008: Aggressive hepatocellular carcinoma and intrahepatic cholangiocarcinoma cell lines share similar response to proangiogenic priming

Abstract Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are the most common primary liver cancers. Traditionally, both neoplasms are considered distinct entities due to their different background. However, the comparison of the proliferative subgroup of iCCA with the subgroup of aggressive HCCs (overexpressing transcriptomic signature (TS) (10.1136/gutjnl-2014-308483)) showed that they share 95% of the overexpressed genes and that they hold similar severe prognosis. In TS-positive aggressive HCCs, the most expressed gene is Angiopoietin 2 (ANGPT2), which is associated with increased angiogenesis and proliferation, epithelial-mesenchymal transition (EMT), and PD1/PDL1 activation. We therefore, studied the effect of ANGPT2 and of its regulator VEGF on in vitro 3D models, with the aim of evaluating putative common carcinogenic pathways shared by aggressive HCCs and iCCA. We generated spheroids from HepG2 and HuCCT-1 cell (HCC and iCCA derived cell lines respectively), while EGI-1 cells, from extrahepatic cholangiocarcinoma (eCCA), served as control. Four days after seeding, well-shaped spheroids were stimulated with either 200 ng/mL ANGPT2 or VEGF, or with 100 ng/mL of ANGPT2/VEGF mix. After 3 and 48h, migration of adherent spheroids and expression of EMT markers (E-cadherin, N-cadherin, Vimentin, Beta-catenin) were evaluated by Western blot (WB) and immunofluorescence. Unstimulated spheroids served as negative controls. In both HepG2 and HuCCT-1, but not in EGI-1, treatment with ANGPT2, VEGF or their combination, stimulated the migration of the adherent spheroids. With respect to EMT biomarkers, in HepG2 E-cadherin expression dropped significantly starting from 3h and remaining low even at 48h. Notably, E-cadherin expression by untreated spheroids was lower at 48h than at 3h. In HuCCT-1, E-cadherin expression levels were not modified by the treatments either at 3h or at 48h. N-cadherin expression levels increased, regardless of treatment, in HepG2 from 3h, while in HUCCT-1 from 48h. Vimentin expression strikingly increased after 48h stimulation in both HepG2 and HuCCT-1, but not in controls. No differences were observed in any of the cell lines in Beta-catenin expression for any treatment. Expression of EMT markers remained unchanged in EGI-1 regardless of any treatment,. Immunofluorescence analysis of the adherent spheroids revealed a marked decrease in E-cadherin expression at the periphery of the migrating cells in both HepG2 and HuCCT-1, paralleled by an increase of N-cadherin and Vimentin expression. Taken together, our results demonstrate a similar response to proangiogenic stimulation of both HCC and iCCA cells, but not of EGI-1, thus underlying a) the substantial phenotypic differences between eCCA and iCCA, and b) the pro-invasive effects of angiogenic growth factors on both HCC or iCCA cells Supported by AIRC under IG 2020 - ID. 24858 project – P.I. Villa Erica Citation Format: Fabiola Milosa, Adriana Romanzi, Gemma Marcelli, Rosina Maria Critelli, Simone Lasagni, Massimiliano Cadamuro, Luca Fabris, Erica Villa. Aggressive hepatocellular carcinoma and intrahepatic cholangiocarcinoma cell lines share similar response to proangiogenic priming [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO008.

KRAS mutation in intrahepatic cholangiocarcinoma: Linkage with metastasis-free survival and reduced E-cadherin expression.

Although KRAS mutations are the major driver of intrahepatic cholangiocarcinoma (ICC), their role remains unexplored. This study aimed to elucidate the prognostic effects, association with clinicopathologic characteristics and potent functions of KRAS mutations in ICC. A hundred and seven resected stage I-III ICCs were analysed for KRAS mutation status and its link with clinicopathological features. An independent validation cohort (n=138) was included. In vitro analyses using KRAS-mutant ICC cell lines were performed. KRAS mutation was significantly associated with worse overall survival in stage I-III ICCs, which was validated in an independent cohort. Recurrence-free survival did not significantly differ between cases with and without KRAS mutations, but if limited to recurrence with extrahepatic metastasis, KRAS-mutant cases showed significantly worse distant metastasis-free survival than KRAS-wild cases showed. KRAS mutations were associated with frequent tumour budding with reduced E-cadherin expression. In vitro, KRAS depletion caused marked inhibition of cell growth and migration together with E-cadherin upregulation in KRAS-mutant ICC cells. The RNA-sequencing assay revealed that KRAS depletion caused MYC pathway downregulation and interferon pathway upregulation. Our observations suggest that KRAS mutations are associated with aggressive behaviour of ICC, especially the development of extrahepatic metastasis. Mutant KRAS is likely to change the adhesive status of ICC cells, affect the responsiveness of tumour cells to interferon immune signals, and consequently promote extrahepatic metastasis. KRAS mutation status, which predicts the prognoses of patients with ICC after surgical resection, is expected to help stratify patients better for individual postoperative treatment strategies.

Novel Nitric Oxide Donor Dinitroazetidine-Coumarin Hybrids as Potent Anti-Intrahepatic Cholangiocarcinoma Agents.

Intrahepatic cholangiocarcinoma (iCC) is a serious liver cancer threatening human health. However, there are a few chemotherapeutic drugs for the treatment of iCC in the clinic. It is extremely urgent to develop new drugs for iCC. In this study, twenty dinitroazetidine and coumarin hybrids were synthesized and evaluated anti-iCC bioactivity as a new type of nitric oxide (NO) donors. Among them, compounds 2–5 and 21 showed a higher antiproliferative activity against RBE cell lines (human intrahepatic cholangiocarcinoma cell lines) and low cytotoxicity in nontumor cells (HOSEpiC and T29). The preliminary study of pharmacology mechanism indicated that compounds 2–5 and 21 could release effective concentration of NO in RBE cell lines, which leaded to inhibit the proliferation of RBE cell lines. The research results revealed that compound 3 inhibited the proliferation of RBE cell lines by inducing apoptosis and arresting cell cycle at G2/M phase. Additionally, compound 3 had acceptable metabolic stability. Therefore, compound 3 was merited to further explore for developing a desirable NO donor lead with anti-iCC activity.

The role of Tripartite motif containing 59 (TRIM59) in the proliferation and prognosis of intrahepatic cholangiocarcinoma

Tripartite motif containing 59 (TRIM59) is a crucial gene that is involved in the process of various types of cancer，including breast cancer, lung cancer, colorectal cancer,and so on. Its abnormal expression can affect tumor cell proliferation, metastasis, or apoptosis. In liver cancer, the incidence of intrahepatic cholangiocarcinoma (ICC) is increasing. However, However, it has not been clearly reported on TRIM59 affects the progress of intrahepatic cholangiocarcinoma cells.Firstly, we review the expression of TRIM59 in different cancers and the corresponding normal tissues，and the results preliminarily showed that TRIM59 may be abnormally expressed in many cancers. The author focuses on whether TRIM59 plays a crucial biological role in intrahepatic cholangiocarcinoma. Therefore, we have confirmed through online websites that TRIM59 is highly expressed in intrahepatic cholangiocarcinoma tissues. Furthermore we further found that TRIM59 can be used as an effective prognostic marker for the prognostic guidance of patient survival time. Next, we explore whether the expression level of TRIM59 in intrahepatic cholangiocarcinoma is related to proliferation through the CCK-8 and EDU assay in two ICC cell lines. To further explore how TRIM59 affected the molecular mechanism involved in intrahepatic cholangiocarcinoma cell growth, we found that STAT3 promotes TRIM59 transcription and TRIM59 can affect tumor progression by regulating the PI3K/AKT signaling pathway through luciferase reporter assay and Western blot experiments. In summary, we first found that TRIM59 has great research value in ICC through bioinformatic analysis, then its expression level is closely related to the prognosis through the analysis of clinicopathological indicators of patients with ICC, and the biological mechanism of TRIM59 in ICC provides precise research or therapeutic targets for future cancer treatment. The findings improve our understanding of the potential of TRIM59 in biological functions in ICC and may hold promise as markers for the diagnosis,treatment, and prognosis of ICC. Data availabilityThe raw data of this study are derived from the TCGA database, which are publicly available databases.

YTHDF1 promotes intrahepatic cholangiocarcinoma progression via regulating EGFR mRNA translation.

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive disease with the underlying mechanisms poorly understood. YTHDF1, an N6 -methyladenosine (m6 A) reader protein, has important physiological functions in regulation of tumor development. However, the effect of YTHDF1 on ICC progression remains unknown yet. The expression level of YTHDF1 in human ICC tissue was examined in The Cancer Genome Atlas database and our cohort. The role of YTHDF1 was detected using two human ICC cell lines in vitro. An ICC tumorigenesis mouse model was established via hydrodynamic transfection of AKT/YAP plasmids. m6 A sequencing, RNA immunoprecipitation sequencing, and RNA sequencing were carried out to explore the mechanism of YTHDF1 modulating ICC progression. Here, we find that YTHDF1 is upregulated in ICC and associated with shorter survival of ICC patients. Depletion of YTHDF1 inhibits cell proliferation, migration, and invasion, while overexpression of wild-type YTHDF1, but not m6 A reader domain mutant YTHDF1, significantly enhances tumor cell growth and aggressive abilities in vitro. Moreover, overexpression of YTHDF1 promotes the AKT/YAP transfection-induced orthotopic ICC tumorigenesis and progression in vivo. Mechanistically, we identify that YTHDF1 regulates the translation of epidermal growth factor receptor (EGFR) mRNA via binding m6 A sites in the 3'-UTR of EGFR transcript, thus leading to aberrant activities of downstream signal pathways that impact tumor progression. Our data uncover the oncogenic function and m6 A reader-dependent mechanism of YTHDF1 in regulation of ICC progression. Restricting abnormal oncogenic mRNA translation by targeting YTHDF1 may be a novel and promising strategy for ICC treatment.

Steroidal saponins from Trillium tschonoskii rhizome repress cancer stemness and proliferation of intrahepatic cholangiocarcinoma

A phytochemical study was carried out on the extract of Trillium tschonoskii rhizomes, resulting in the isolation of thirty-six steroidal glycosides (1–36). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence, of which 1–25 were identified as new analogues. Herein, all the isolated analogues were screened for the cytotoxicity against intrahepatic cholangiocarcinoma (ICC) cell lines of HuCCT1 and RBE through tumor colony formation and CCK-8 survival analysis, and the results demonstrated that three compounds 9, 12, and 26 significantly repressed tumor colony and sphere formation in both cell lines, respectively. Furthermore, the three analogues possessed a remarkable inhibitory role of organoid formation established from hydrodynamic induced mouse primary intrahepatic cholangiocarcinoma. Moreover, the functional assays of flow cytometry analysis, cancer stemness related gene expression, and western blotting assays all indicated that compound 26 could significantly repress cancer stem markers. Taken together, these results demonstrate that steroidal glycosides derived from T. tschonoskii rhizomes could be potentially implicated in human ICC therapy.