Intrahepatic Cholangiocarcinoma Samples Research Articles

A novel protein encoded by circFOXP1 enhances ferroptosis and inhibits tumor recurrence in intrahepatic cholangiocarcinoma

CircRNAs participates in the development and occurrence of multiple tumor types. However, the specific effects and underlying mechanisms of circRNA in intrahepatic cholangiocarcinoma (ICC) progression and recurrence remain poorly understood. CircRNA sequencing was performed to screen circRNAs related to ICC recurrence after surgery using 53 ICC frozen tumor specimens. We found that compared with patients who experienced postsurgical recurrence, circFOXP1 had high expression in tumor tissues from patients with no postoperative recurrence. Functional experiments revealed that circFOXP1 inhibited ICC progression in vitro and in vivo. We then found that circFOXP1 inhibited ICC progression via encoding a novel protein, circFOXP1-231aa. Mechanistically, circFOXP1-231aa directly interacted with OTUD4, which regulates NCOA4 protein stability via deubiquitination modification, and thereby enhances ferroptosis of ICC cells. Examination of clinical ICC samples found positive correlations between circFOXP1 expression levels and levels of OTUD4 and NCOA4. These three factors are predictors of prognosis in patients with ICC. Collectively, we identified circFOXP1 encoded circFOXP1-231aa, which interacted with OTUD4 to suppress ubiquitination of NCOA4 and, thereby, promoted ferroptosis and inhibited ICC recurrence.

Mobilization and activation of tumor-infiltrating dendritic cells inhibits lymph node metastasis in intrahepatic cholangiocarcinoma

Lymph node metastasis (LNM) facilitates distant tumor colonization and leads to the high mortality in patients with intrahepatic cholangiocarcinoma (ICC). However, it remains elusive how ICC cells subvert immune surveillance within the primary tumor immune microenvironment (TIME) and subsequently metastasize to lymph nodes (LNs). In this study, scRNA-seq and bulk RNA-seq analyses identified decreased infiltration of dendritic cells (DCs) into primary tumor sites of ICC with LNM, which was further validated via dual-color immunofluorescence staining of 219 surgically resected ICC samples. Tumor-infiltrating DCs correlated with increased CD8+ T cell infiltration and better prognoses in ICC patients. Mechanistically, β-catenin-mediated CXCL12 suppression accounted for the impaired DC recruitment in ICC with LNM. Two mouse ICC cell lines MuCCA1 and mIC-23 cells were established from AKT/NICD or AKT/YAP-induced murine ICCs respectively and were utilized to construct the footpad tumor LNM model. We found that expansion and activation of conventional DCs (cDCs) by combined Flt3L and poly(I:C) (FL-pIC) therapy markedly suppressed the metastasis of mIC-23 cells to popliteal LNs. Moreover, β-catenin inhibition restored the defective DC infiltration into primary tumor sites and reduced the incidence of LNM in ICC. Collectively, our findings identify tumor cell intrinsic β-catenin activation as a key mechanism for subverting DC-mediated anti-tumor immunity in ICC with LNM. FL-pIC therapy or β-catenin inhibitor could merit exploration as a potential regimen for mitigating ICC cell metastasis to LNs and achieving effective tumor immune control.

Integrin β5 is an independent prognostic marker for intrahepatic cholangiocarcinoma in a Chinese population.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver tumor and a major cause of cancer mortality worldwide. Integrin β5 (ITGB5) is considered to be involved in the intercellular signal transduction and regulation of tumorigenesis and development. The present study investigated the association between ITGB5 expression levels and the prognosis of ICC, as well as the effects of ITGB5 on the proliferation and invasion of ICC cells. RNA-sequencing transcriptomic profiling data of ICC samples were retrieved from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Tissue specimens from patients with ICC treated at Taizhou People's Hospital were collected and the ITGB5 expression levels were evaluated using immunohistochemical staining. The biological function of ITGB5 in ICC was investigated using Gene Ontology (GO), Gene Set Enrichment Analysis (GSEA) and in vitro experiments using HuCCT1 cells. After knocking down ITGB5 expression, cell proliferation was detected using Cell Counting Kit-8 assay, while cell invasion was assessed using Transwell assays. According to TCGA dataset, ITGB5 was highly expressed in ICC; however, there was no significant difference in prognosis between patients with high and low ITGB5 expression levels. High expression of ITGB5 was present in the tissues of patients with ICC from the GEO database, which was associated with poor prognosis. Survival analyses of the clinical data obtained in the present study revealed that high expression levels of ITGB5 in patients with ICC were associated with a reduced overall survival. GO and GSEA indicated that genes associated with ITGB5 were enriched in the extracellular matrix-receptor interaction and focal adhesion signaling pathways. Silencing ITGB5 inhibited the proliferation and invasion of ICC cells. In conclusion, ITGB5 may act as an essential regulator of ICC development and progression by influencing the proliferation and invasion of ICC cells. However, future studies with larger sample sizes are required to validate the role of ITGB5 in the prognosis of patients with ICC.

Unraveling the heterogeneity of intercellular crosstalk in intrahepatic and extrahepatic cholangiocarcinoma using single-cell and spatial transcriptome profiling.

50 Background: Extrahepatic cholangiocarcinoma (ECC) and intrahepatic cholangiocarcinoma (ICC) are two types of malignant tumors that arise from the bile duct epithelium at different locations. Currently, effective treatment options for these tumors are limited, and their microenvironmental differences remain poorly understood. Methods: We collected retrospective clinicopathological data of ECC (N=64) and ICC (N=153) patients from our hospital and integrated public database information to analyze the clinical characteristics, survival, and prognosis of ECC and ICC. We performed single-cell sequencing (scRNA-seq) and spatial transcriptomic techniques on three ECC and three ICC tumor tissues. Verification of scRNA-seq results was done using immunohistochemistry, flow cytometry, and polychromatic immunofluorescence. Additionally, we analyzed RNA and ATAC sequencing data for exhausted CD8+ T cells TEX obtained from ICC and ECC tissue samples. We used InferCNV to infer copy number variations (CNVs) of epithelial cells from ICC and ECC. Results: Our results showed that ECC and ICC differ in their clinical characteristics such as age, gender, bilirubin, CEA, and outcome. ECC had higher copy number amplification variation and exhibited inflammatory signaling and ongoing immunoediting, reflected in higher immune checkpoint expression such as PDCD1, CTLA4, and LAG3, and tumor infiltration with highly differentiated dysfunctional PDCD1+ LAG3+ CXCL13+ exhausted CD8+ T cells (TEX) and precursors of exhausted T (TPEX). RNA velocity analysis revealed different developmental trajectories of T cells in ECC and ICC, predominantly in naive/stem-like and memory T cells. We also identified a trefoil factor 3 (TFF3)-positive epithelial subpopulation in ECC. TFF3+ epithelial cells had closer interaction with CTHRC1+ fibroblasts, which induced more CD8+ T cells to TEX and TPEX. TFF3+ cells exhibited higher CNV scores and higher PD-L1 and PD-L2 expression, and co-localized with TEX and TPEX, providing one explanation for the immunosuppressive environment in ECC and suggesting a potential response to immune checkpoint therapy (ICT). Conclusions: Our study provides a comprehensive characterization of the cellular heterogeneity landscape of ECC and ICC, which can guide the optimization of personalized cancer immunotherapy. We identified potential biomarkers and targets for immunotherapies, and our findings shed light on the underlying mechanisms of tumor immune evasion and provide a foundation for further research in this area.

DAGLβ is the principal synthesizing enzyme of 2-AG and promotes aggressive phenotype of intrahepatic cholangiocarcinoma via AP-1/DAGLβ/miR4516 feedforward circuitry.

The endocannabinoid system (ECS) is dysregulated in various liver diseases. Previously, we had shown that the major endocannabinoid 2-arachidonoyl glycerol (2-AG) promoted tumorigenesis of intrahepatic cholangiocarcinoma (ICC). However, biosynthesis regulation and clinical significance of 2-AG remain elusive. In the present study, we quantified 2-AG by gas chromatography/mass spectrometry (GC/MS) and showed that 2-AG was enriched in patients with ICC samples as well as in thioacetamide-induced orthotopic rat ICC model. Moreover, we found that diacylglycerol lipase β (DAGLβ) was the principal synthesizing enzyme of 2-AG that significantly upregulated in ICC. DAGLβ promoted tumorigenesis and metastasis of ICC in vitro and in vivo and positively correlated with clinical stage and poor survival in patients with ICC. Functional studies showed that activator protein-1 (AP-1; heterodimers of c-Jun and FRA1) directly bound to the promoter and regulated transcription of DAGLβ, which can be enhanced by lipopolysaccharide (LPS). miR-4516 was identified as the tumor-suppressing miRNA of ICC that can be significantly suppressed by LPS, 2-AG, or ectopic DAGLβ overexpression. FRA1 and STAT3 were targets of miR-4516 and overexpression of miRNA-4516 significantly suppressed expression of FRA1, SATA3, and DAGLβ. Expression of miRNA-4516 was negatively correlated with FRA1, SATA3, and DAGLβ in patients with ICC samples. Our findings identify DAGLβ as the principal synthesizing enzyme of 2-AG in ICC. DAGLβ promotes oncogenesis and metastasis of ICC and is transcriptionally regulated by a novel AP-1/DAGLβ/miR4516 feedforward circuitry.NEW & NOTEWORTHY Dysregulated endocannabinoid system (ECS) had been confirmed in various liver diseases. However, regulation and function of 2-arachidonoyl glycerol (2-AG) and diacylglycerol lipase β (DAGLβ) in intrahepatic cholangiocarcinoma (ICC) remain to be elucidated. Here, we demonstrated that 2-AG was enriched in ICC, and DAGLβ was the principal synthesizing enzyme of 2-AG in ICC. DAGLβ promotes tumorigenesis and metastasis in ICC via a novel activator protein-1 (AP-1)/DAGLβ/miR4516 feedforward circuitry.

MCM6 promotes intrahepatic cholangiocarcinoma progression by upregulating E2F1 and enhancing epithelial-mesenchymal transition.

Minichromosome maintenance complex component 6 (MCM6), a member of the MCM family, plays a pivotal role in DNA replication initiation and genome duplication of proliferating cells. MCM6 is upregulated in multiple malignancies and is considered a novel diagnostic biomarker. However, the functional contributions and prognostic value of MCM6 in intrahepatic cholangiocarcinoma (ICC) remain unexplored. In this study, we investigated the molecular function of MCM6 in ICC. Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO, GSE107943) indicated an upregulation of MCM6 in tumor tissues. Immunohistochemical analysis performed on 115 cases of ICC samples confirmed the upregulation of MCM6 and further suggested that a high level of MCM6 expression predicted shorter overall and disease-free survival in ICC patients. Functional studies suggested that MCM6 knockdown significantly suppressed cell viability, blocked cell cycle progression and inhibited metastasis, while the enhancement of MCM6 expression promoted the proliferation and migration of ICC cells both in vitro and in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) suggested that the epithelial-mesenchymal transition (EMT) and E2F1-correlated genes were enriched in ICC tissues with high MCM6 expression. Further verification indicated that MCM6 promoted the EMT of ICC cells via upregulating E2F1. In addition, E2F1 knockdown partially blocked the pro-malignant effects of MCM6 overexpression. In summary, MCM6 was found to be a novel prognostic and predictive marker for ICC. MCM6 promoted ICC progression via activation of E2F1-mediated EMT.

Integrative analyses identify CD73 as a prognostic biomarker and immunotherapeutic target in intrahepatic cholangiocarcinoma

BackgroundCD73 promotes progression in several malignancies and is considered as a novel immune checkpoint. However, the function of CD73 in intrahepatic cholangiocarcinoma (ICC) remains uncertain. In this study, we aim to investigate the role of CD73 in ICC.MethodsMulti-omics data of 262 ICC patients from the FU-iCCA cohort were analyzed. Two single-cell datasets were downloaded to examine the expression of CD73 at baseline and in response to immunotherapy. Functional experiments were performed to explore the biological functions of CD73 in ICC. The expression of CD73 and HHLA2 and infiltrations of CD8 + , Foxp3 + , CD68 + , and CD163 + immune cells were evaluated by immunohistochemistry in 259 resected ICC samples from Zhongshan Hospital. The prognostic value of CD73 was assessed by Cox regression analysis.ResultsCD73 correlated with poor prognosis in two ICC cohorts. Single-cell atlas of ICC indicated high expression of CD73 on malignant cells. TP53 and KRAS gene mutations were more frequent in patients with high CD73 expression. CD73 promoted ICC proliferation, migration, invasion, and epithelial-mesenchymal transition. High CD73 expression was associated with a higher ratio of Foxp3 + /CD8 + tumor-infiltrating lymphocytes (TILs) and CD163 + /CD68 + tumor-associated macrophages (TAMs). A positive correlation between CD73 and CD44 was observed, and patients with high CD73 expression showed elevated expression of HHLA2. CD73 expression in malignant cells was significantly upregulated in response to immunotherapy.ConclusionsHigh expression of CD73 is associated with poor prognosis and a suppressive tumor immune microenvironment in ICC. CD73 could potentially be a novel biomarker for prognosis and immunotherapy in ICC.

The impact of decreased expression of SVEP1 on abnormal neovascularization and poor prognosis in patients with intrahepatic cholangiocarcinoma.

Introduction: Intrahepatic cholangiocarcinoma (ICC) is one of the most highly heterogeneous malignant solid tumors; it is generally insensitive to clinical treatment and has a poor prognosis. Evidence suggests that abnormal neovascularization in the tumor microenvironment is an important cause of treatment resistance as well as recurrence and metastasis, but the key regulatory molecules are still largely unknown and should be identified. Method: We assessed the novel extracellular matrix protein (ECM) Sushi, von Willebrand factor type A, EGF and pentraxin containing 1 (SVEP1) expression pattern in the ICC by using immunohistochemistry. Multiplex immunofluorescence and Kaplan-Meier analysis were applied to explore the correlation between the low expression of SVEP1 and abnormal blood vessels and the clinical prognosis of ICC. Results: Our study showed that the expression of SVEP1 in most ICC samples was relatively lower than in the adjacent tissues. Statistical analysis suggested that patients with decreased SVEP1 expression always had shorter overall survival (OS) and disease-free survival (DFS). Moreover, the expression of SVEP1 was negatively correlated with the proportion of abnormal neovascularization in the tumor microenvironment of the ICC. Consistently, the key molecule of promoting vascular normalization, Ang-1, is positively correlated with the SVEP1 expression and prognosis in the ICC. In addition, the proportion of high Ki-67 expression was higher in the ICC samples with low SVEP1 expression, suggesting that the SVEP1 low expressed sample is in a malignant phenotype with high proliferation. Conclusion: This study reveals that SVEP1 is a promising prognostic biomarker for ICC and provides fresh insight into the role and potential new mechanism of abnormal neovascularization in ICC progression.

Fucosyltransferase 5 Promotes the Proliferative and Migratory Properties of Intrahepatic Cholangiocarcinoma Cells via Regulating Protein Glycosylation Profiles

Background: The incidence of intrahepatic cholangiocarcinoma (ICC) is increasing globally, and its prognosis has not improved substantially in recent years. Understanding the pathogenesis of ICC may provide a theoretical basis for its treatment. In this study, we investigated the effects and underlying mechanisms of fucosyltransferase 5 (FUT5) on the malignant progression of ICC. Methods: FUT5 expression in ICC samples and adjacent nontumor tissues was compared using quantitative real-time polymerase chain reaction and immunohistochemical assays. We performed cell counting kit-8, colony formation, and migration assays to determine whether FUT5 influenced the proliferation and mobility of ICC cells. Finally, mass spectrometry was performed to identify the glycoproteins regulated by FUT5. Results: FUT5 mRNA was significantly upregulated in most ICC samples compared with corresponding adjacent nontumor tissues. The ectopic expression of FUT5 promoted the proliferation and migration of ICC cells, whereas FUT5 knockdown significantly suppressed these cellular properties. Mechanistically, we demonstrated that FUT5 is essential for the synthesis and glycosylation of several proteins, including versican, β3 integrin, and cystatin 7, which may serve key roles in the precancer effects of FUT5. Conclusions: FUT5 is upregulated in ICC and promotes ICC development by promoting glycosylation of several proteins. Therefore, FUT5 may serve as a therapeutic target for the treatment of ICC.

Distinctive Metabolism-Associated Gene Clusters That Are Also Prognostic in Intrahepatic Cholangiocarcinoma and Hepatocellular Carcinoma.

Objective To offer new prognostic evaluations by exploring potentially distinctive genetic features of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Methods There were 12 samples for gene expression profiling processes in this study. These included three HCC lesion samples and their matched adjacent nontumor liver tissues obtained from patients with HCC, as well as three ICC samples and their controls collected similarly. In addition to the expression matrix generated on our own, profiles of other cohorts from The Cancer Genome Atlas (TCGA) program and the Gene Expression Omnibus (GEO) were also employed in later bioinformatical analyses. Differential analyses, functional analyses, protein interaction network analyses, and gene set variation analyses were used to identify key genes. To establish the prognostic models, univariate/multivariate Cox analyses and subsequent stepwise regression were applied, with the Akaike information criterion evaluating the goodness of fitness. Results The top three pathways enriched in HCC were all metabolism-related; they were fatty acid degradation, retinol metabolism, and arachidonic acid metabolism. In ICC, on the other hand, additional pathways related to fat digestion and absorption and cholesterol metabolism were identified. Consistent characteristics of such a metabolic landscape were observed across different cohorts. A prognostic risk score model for calculating HCC risk was constructed, consisting of ADH4, ADH6, CYP2C9, CYP4F2, and RDH16. This signature predicts the 3-year survival with an AUC area of 0.708 (95%CI = 0.644 to 0.772). For calculating the risk of ICC, a prognostic risk score model was built upon the expression levels of CYP26A1, NAT2, and UGT2B10. This signature predicts the 3-year survival with an AUC area of 0.806 (95% CI = 0.664 to 0.947). Conclusion HCC and ICC share commonly abrupted pathways associated with the metabolism of fatty acids, retinol, arachidonic acids, and drugs, indicating similarities in their pathogenesis as primary liver cancers. On the flip side, these two types of cancer possess distinctive promising biomarkers for predicting overall survival or potential targeted therapies.

Developing metabolic gene signatures to predict intrahepatic cholangiocarcinoma prognosis and mining a miRNA regulatory network.

BackgroundMetabolic disturbance is closely correlated with intrahepatic cholangiocarcinoma (IHCC), and we aimed to identify metabolic gene marker for the prognosis of IHCC.MethodsWe obtained expression and clinical data from 141 patients with IHCC from public databases. Prognostic metabolic genes were selected using univariate Cox regression analysis. Unsupervised cluster analysis was applied to identify IHCC subtypes, and CIBERSORT was used for immune infiltration analysis of different subtypes. Then, the metabolic gene signature was screened using multivariate Cox regression analysis and the LASSO algorithm. The prognostic potential and regulatory network of the metabolic gene signature were further investigated.ResultsWe screened 228 prognosis‐related metabolic genes. Based on their expression levels, IHCC samples were divided into two subtypes, which showed significant differences in survival and immune cell infiltration. After LASSO analysis, eight metabolic genes including CYP19A1, SCD5, ACOT8, SRD5A3, MOGAT2, PFKFB3, PPARGC1B, and RPL17 were identified as the optimal genes for the prognosis signature. The prognostic model had excellent predictive abilities, with areas under the receiver‐operating characteristic curves over 0.8. A nomogram model was also established based on two independent prognostic clinical factors (pathologic stage and prognostic model), and the generated calibration curves and c‐indexes determined its excellent accuracy and discriminative ability to predict 1‐ and 5‐year survival status (c‐indexes>0.7). Finally, we found that miR‐26a‐5p, miR‐27a‐3p, and miR‐27b‐3p were the upstream regulators that mediate the involvement of gene signatures in metabolic pathways.ConclusionWe developed eight metabolic gene signatures to predict IHCC prognosis and proposed potential upstream regulatory axes of gene signatures.

53P FGFR2 fusion and/or rearrangement profiling in Chinese patients with intrahepatic cholangiocarcinoma

FGFR2 fusions and rearrangements occurring in 10–16% of patients with intrahepatic cholangiocarcinoma (ICC) reported in published scientific literature. Pemigatinib, a selective FGFR inhibitor, has demonstrated the high therapeutic potentials for ICC patients with FGFR2 fusions or rearrangements, according to the results of FIGHT 202 study. Whereas in Chinese population, the epidemiological data of FGFR2 fusions and rearrangements is still insufficient with limited sample sizes, and partner genes in FGFR2 fusion are still unknown. A total of 728 pathologically confirmed ICC samples (including surgical and biopsy samples from 728 patients aged over 18 years) were collected and tested FGFR2 fusion or rearrangement using fluorescence in situ hybridization (FISH) with break-apart probes. Thirty samples with known FGFR2 fusion or rearrangement were tested with next generation sequence (NGS) to identify the partner genes of FGFR2. As of October 31, 2020, 728 patients were included and their samples were tested for FGFR2 gene fusion or rearrangement, 717 samples had readout. Forty-four samples (44/717, 6.14%) were tested FGFR2 gene fusion or rearrangement positive. Regionally, the highest positive rate (10.5%) was found in Southwest China (Sichuan and Yunnan province), and the lowest positive rate (5%) was in South China (Guangdong and Guangxi province). Twenty-six different FGFR2 fusion partner genes were identified in 30 samples, 22 (84.6%) of which were unique to individual patients. The most common partner was FGFR2-WAC (3/30, 10%). Based on a large sample size, the rate of FGFR2 gene fusion or rearrangement in Chinese ICC patients was 6.14%, and the FGFR2 partner genes were highly heterogeneous.

DNA methylation biomarkers for diagnosis of primary liver cancer and distinguishing hepatocellular carcinoma from intrahepatic cholangiocarcinoma.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two most common pathology subtypes of primary liver cancer (PLC). Identifying DNA methylation biomarkers for diagnosis of PLC and further distinguishing HCC from ICC plays a vital role in subsequent treatment options selection. To obtain potential diagnostic DNA methylation sites for PLC, differentially methylated CpG (DMC) sites were first screened by comparing the methylation data between normal liver samples and PLC samples (ICC samples and HCC samples). A random forest algorithm was then used to select specific DMC sites with top Gini value. To avoid overfitting, another cohort was taken as an external validation for evaluating the area under curves (AUCs) of different DMC sites combination. A similar model construction strategy was applied to distinguish HCC from ICC. In addition, we identified DNA Methylation-Driven Genes in HCC and ICC via MethylMix method and performed pathway analysis by utilizing MetaCore. Finally, we not only performed methylator phenotype based on independent prognostic sites but also analyzed the correlations between methylator phenotype and clinical factors in HCC and ICC, respectively. To diagnose PLC, we developed a model based on three PLC-specific methylation sites (cg24035245, cg21072795, and cg00261162), whose sensitivity and specificity achieved 98.8%,94.8% in training set and 97.3%,81% in validation set. Then, to further divide the PLC samples into HCC and ICC, we established another mode through three methylation sites (cg17769836, cg17591574, and cg07823562), HCC accuracy and ICC accuracy achieved 95.8%, 89.8% in the training set and 96.8%,85.4% in the validation set. In HCC, the enrichment pathways were mainly related to protein folding, oxidative stress, and glutathione metabolism. While in ICC, immune response, embryonic hepatocyte maturation were the top pathways. Both in HCC and ICC, methylator phenotype correlated well with overall survival time and clinical factors involved in tumor progression. In summary, our study provides the biomarkers based on methylation sites not only for the diagnosis of PLC but also for distinguishing HCC from ICC.

Tumor-associated neutrophils and macrophages interaction contributes to intrahepatic cholangiocarcinoma progression by activating STAT3

BackgroundTumor-associated neutrophils (TANs) and macrophages (TAMs) can each influence cancer growth and metastasis, but their combined effects in intrahepatic cholangiocarcinoma (ICC) remain unclear.MethodsWe explored the distributions of TANs and TAMs...

LncRNA MT1JP plays a protective role in intrahepatic cholangiocarcinoma by regulating miR-18a-5p/FBP1 axis

BackgroundCholangiocarcinoma is a common malignant tumor of digestive system. LncRNA metallothionein 1 J, pseudogene (MT1JP) has been reported to play tumor-suppressing roles in multiple cancers. However, its effect on cholangiocarcinoma has not been evaluated.MethodsThe expression of MT1JP in intrahepatic cholangiocarcinoma specimens and paired para-carcinoma tissues were detected by real-time PCR. The overexpression plasmid and siRNA of MT1JP were transfected into intrahepatic cholangiocarcinoma cells to change the expression levels of MT1JP. CCK-8, flow cytometry and transwell assays were performed to measure proliferation, cell cycle transition, apoptosis, migration and invasion. Dual-luciferase reporter assay, real-time PCR and western blot were carried out to screen the miRNA bound by MT1JP. In addition, xenograft experiment was used to determine the tumorigenesis of cholangiocarcinoma cells in nude mice.ResultsMT1JP was downregulated in intrahepatic cholangiocarcinoma specimens, and its expression was related with TNM stage and lymph node metastasis. Overexpression of MT1JP inhibited proliferation, cell cycle transition, migration and invasion, and induced apoptosis in intrahepatic cholangiocarcinoma cells. The knockdown of MT1JP led to opposite results. MT1JP bound to miR-18a-5p to facilitate the expression of fructose-1,6-bisphosphatase 1 (FBP1). MiR-18a-5p was increased in intrahepatic cholangiocarcinoma samples, and its expression was negatively correlated with that of MT1JP. In addition, MT1JP also suppressed tumorigenesis in nude mice.ConclusionsMT1JP alleviated proliferation, migration and invasion, and induced apoptosis in cholangiocarcinoma cells by regulating miR-18a-5p/FBP1 axis. These findings may provide novel insights for clinical diagnosis and treatment of cholangiocarcinoma.

Functionalized graphene oxide as a nanocarrier for multiple suppressive miRNAs to inhibit human intrahepatic cholangiocarcinoma

AbstractThe restoration of suppressed miRNAs is a promising therapeutic approach for treating tumors; however, efficient delivery remains a challenge. Herein, we aimed to develop a polyethylenimine (PEI) functionalized graphene oxide nanosheets (GO‐PEI) as a suppressive miRNA delivery system to treat human intrahepatic cholangiocarcinoma (ICC). We found that the GO‐PEI complex possessed excellent transfection efficacy and acceptable toxicity. Furthermore, using our miRNA array analysis and The Cancer Genome Atlas (TCGA) dataset, we selected four miRNAs (miR‐194‐5p, miR‐125b‐5p, miR‐122‐5p, and let‐7c‐5p) that are remarkably downregulated in ICC samples compared to that in adjacent tissues. The GO‐PEI complexed with the four miRNAs (GO‐PEI‐4miR) achieved a higher miRNA transfection efficiency than Lipo2000, as verified by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) assays, immunofluorescence, and flow cytometry analyses. In addition, in vitro experiments revealed that GO‐PEI‐4miR remarkably restored the repressed miRNAs, thereby significantly repressing tumor sphere formation, colony formation, drug resistance, and markedly inhibiting the target genes of these miRNAs and cancer stem cell‐related markers. Furthermore, both tumor weights and bioluminescent imaging analysis indicated that the GO‐PEI‐4miR significantly reduced tumor formation in vivo. Taken together, these results demonstrate that GO‐PEI could be a potential delivery system for multiple repressive miRNAs in ICC therapy.

Somatic Mutation Profiling of Intrahepatic Cholangiocarcinoma: Comparison between Primary and Metastasis Tumor Tissues.

Introduction Intrahepatic cholangiocarcinoma (ICC) exhibited increasing incidence and mortality around the world, with a 35% five-year survival rate. In this study, the genetic alteration of primary ICC and metastasis ICC was exhibited to discover novel personalized treatment strategies to improve the clinical prognosis. Methods Based on 153 primary and 49 metastasis formalin-fixed paraffin-embedded ICC samples, comprehensive genomic profiling was carried out. Results In primary tumor samples (PSs) and metastasis tumor samples (MSs), the top alteration genes were TP53 (41.8% vs 36.7%), KRAS (30.7% vs 36.7%), and ARID1A (22.2% vs 14.2%). In the top 20 most frequent alteration genes, BRAF showed lower mutation frequency in MSs as compared to PSs (0 vs 11.1%, P=0.015), while LRP1B exhibited opposed trend (22.4% vs 10.4%, P=0.032). In PSs, patients with MSI-H showed all PDL1 negative, and patients with PDL1 positive exhibited MSS both in PSs and MSs. It was found that the Notch pathway had more alteration genes in MSI-H patients (P=0.027). Furthermore, the patients with mutated immune genes in PSs were more than that in MSs (28.8% vs 8.2%, P=0.003, odd ratio = 0.2). Interestingly, the platinum drug resistance pathway was only enriched by mutated genes of MSs. Conclusions In this study, the identification of two meaningful mutated genes, BRAF and LRP1B, highly mutated immune gene harbored by primary ICC patients. Both in PSs and MSs, no patients with MSI-H showed PDL1 positive. The Notch pathway had more alteration genes in patients with MSI-H. And the enrichment of the platinum drug resistance pathway in MSs might offer reference for the novel therapeutic strategy of ICC.

Single-cell transcriptomic architecture and intercellular crosstalk of human intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy. ICC typically features remarkable cellular heterogeneity and a dense stromal reaction. Therefore, a comprehensive understanding of cellular diversity and the interplay between malignant cells and niche cells is essential to elucidate the mechanisms driving ICC progression and to develop therapeutic approaches. Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from 8 human ICCs and adjacent samples to elucidate the comprehensive transcriptomic landscape and intercellular communication network. Additionally, we applied a negative selection strategy to enrich fibroblast populations in 2 other ICC samples to investigate fibroblast diversity. The results of the analyses were validated using multiplex immunofluorescence staining, bulk transcriptomic datasets, and functional invitro and invivo experiments. We sequenced a total of 56,871 single cells derived from human ICC and adjacent tissues and identified diverse tumor, immune, and stromal cells. Malignant cells displayed a high degree of inter-tumor heterogeneity. Moreover, tumor-infiltrating CD4 regulatory T cells exhibited highly immunosuppressive characteristics. We identified 6 distinct fibroblast subsets, of which the majority were CD146-positive vascular cancer-associated fibroblasts (vCAFs), with highly expressed microvasculature signatures and high levels of interleukin (IL)-6. Functional assays indicated that IL-6 secreted by vCAFs induced significant epigenetic alterations in ICC cells, particularly upregulating enhancer of zeste homolog 2 (EZH2) and thereby enhancing malignancy. Furthermore, ICC cell-derived exosomal miR-9-5p elicited high expression of IL-6 in vCAFs to promote tumor progression. Our single-cell transcriptomic dataset delineates the inter-tumor heterogeneity of human ICCs, underlining the importance of intercellular crosstalk between ICC cells and vCAFs, and revealing potential therapeutic targets. Intrahepatic cholangiocarcinoma is an aggressive and chemoresistant malignancy. Better understanding the complex transcriptional architecture and intercellular crosstalk of these tumorswill help in the development of more effective therapies. Herein, we have identified important interactions between cancer cells and cancer-associated fibroblasts in the tumor stroma, which could have therapeutic implications.

INOS is associated with tumorigenicity as an independent prognosticator in human intrahepatic cholangiocarcinoma.

BackgroundInducible nitric oxide synthase (iNOS) has supposed to implicate in inflammation, infection, liver cirrhosis, and neoplastic diseases. This study was designed to explore the biological and clinical function of iNOS in intrahepatic cholangiocarcinoma (ICC).MethodsRT-PCR (Real-time quantitative PCR) and immunohistochemical staining were used to analyze the expression of iNOS in ICC and adjacent tissues. CCK8, transwell assays, flow cytometry were conducted to detect the proliferation, apoptosis, cell cycle. Western blotting was performed to detect the expression of target proteins. Multivariate analyses were conducted to analysis associates between clinicopathological values and survival.ResultsWe found that levels of iNOS mRNA and protein were dramatically increased in ICC samples and positively correlated with complicated bile duct stone, differentiation, pathology T, pathology M, Wip1, MMP-2, and MMP-9. iNOS expression was significantly correlated with the poor survival of ICC patients. Furthermore, iNOS was high expression in ICC cell lines (QBC-939, ICC-9810, SSP-25) compare with human normal biliary epithelium cell line (HIBEpic); both iNOS knockdown and iNOS inhibitor (1400 W) suppressed cell proliferation, invasion, and migration though nitric oxide production in ICC cells. Down-regulation of iNOS also induced G0/G1 cell cycle arrest and ICC cell apoptosis. Moreover, iNOS knockdown treatment significantly decreased Wip1, MMP-9, and MMP-2 gene expression.ConclusionLowly expressed iNOS-inhibited proliferation yet promoted apoptosis of ICC cells. Our data show targeted inhibition of iNOS in ICC may have therapeutic value.

Expression of GLS1 in intrahepatic cholangiocarcinoma and its clinical significance.

Kidney‑type glutaminase (GLS1) plays a significant role in tumor metabolism. Our recent studies demonstrated that GLS1 was aberrantly expressed in hepatocellular carcinoma (HCC) and facilitated tumor progression. However, the roles of GLS1 in intrahepatic cholangiocarcinoma (ICC) remain largely unknown. Thus, the aim of this study was to evaluate the expression and clinical significance of GLS1 in ICC. For this purpose, combined data from the Oncomine database with those of immunohistochemistry were used to determine the expression levels of GLS1 in cancerous and non‑cancerous tissues. Second, a wound‑healing assay and Transwell assay were used to observe the effects of the knockdown and overexpression of GLS1 on the invasion and migration of ICC cells. We examined the associations between the expression of GLS1 and epithelial‑mesenchymal transition (EMT)‑related markers by western blot analysis. Finally, we examined the associations between GLS1 levels and clinicopathological factors or patient prognosis. The results revealed that GLS1 was overexpressed in different digestive system tumors, including ICC, and that GLS1 expression in ICC tissue was higher than that in peritumoral tissue. The overexpression of GLS1 in RBE cells induced metastasis and invasion. Moreover, the EMT‑related markers, E‑cadherin and Vimentin, were regulated by GLS1 in ICC cells. By contrast, the knockdown of GLS1 expression in QBC939 cells yielded opposite results. Clinically, a high expression of GLS1 in ICC samples negatively correlated with E‑cadherin expression and positively correlated with Vimentin expression. GLS1 protein expression was associated with tumor differentiation (P=0.001) and lymphatic metastasis (P=0.029). Importantly, patients with a high GLS1 expression had a poorer overall survival (OS) and a shorter time to recurrence than patients with a low GLS1 expression. Multivariate analysis indicated that GLS1 expression was an independent prognostic indicator. On the whole, the findings of this study demonstrated that GLS1 is an independent prognostic biomarker of ICC. GLS1 facilitates ICC progression and may thus prove to be a therapeutic target in ICC.