Intrahepatic Cholangiocarcinoma Tissues Research Articles

Radiomics of Intrahepatic Cholangiocarcinoma and Peritumoral Tissue Predicts Postoperative Survival: Development of a CT-Based Clinical-Radiomic Model.

For many tumors, radiomics provided a relevant prognostic contribution. This study tested whether the computed tomography (CT)-based textural features of intrahepatic cholangiocarcinoma (ICC) and peritumoral tissue improve the prediction of survival after resection compared with the standard clinical indices. All consecutive patients affected by ICC who underwent hepatectomy at six high-volume centers (2009-2019) were considered for the study. The arterial and portal phases of CT performed fewer than 60days before surgery were analyzed. A manual segmentation of the tumor was performed (Tumor-VOI). A 5-mm volume expansion then was applied to identify the peritumoral tissue (Margin-VOI). The study enrolled 215 patients. After a median follow-up period of 28months, the overall survival (OS) rate was 57.0%, and the progression-free survival (PFS) rate was 34.9% at 3years. The clinical predictive model of OS had a C-index of 0.681. The addition of radiomic features led to a progressive improvement of performances (C-index of 0.71, including the portal Tumor-VOI, C-index of 0.752 including the portal Tumor- and Margin-VOI, C-index of 0.764, including all VOIs of the portal and arterial phases). The latter model combined clinical variables (CA19-9 and tumor pattern), tumor indices (density, homogeneity), margin data (kurtosis, compacity, shape), and GLRLM indices. The model had performance equivalent to that of the postoperative clinical model including the pathology data (C-index of 0.765). The same results were observed for PFS. The radiomics of ICC and peritumoral tissue extracted from preoperative CT improves the prediction of survival. Both the portal and arterial phases should be considered. Radiomic and clinical data are complementary and achieve a preoperative estimation of prognosis equivalent to that achieved in the postoperative setting.

Expression of Fascin-1 and its diagnostic value in liver cancer

Although some studies have reported on the expression and clinical significance of Fascin-1 (FSCN1) in liver cancer, the clinical application and differential diagnosis value of FSCN1 in liver cancer are still unclear. The aim of this study was to analyze the expression level of FSCN1 protein in liver cancer tissues and explore its diagnostic and application value in differentiating between hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). The immunehistochemical analysis was used to detect the expression of FSCN1 in 108 cases of HCC, 26 cases of ICC, 23 cases of liver cirrhosis, and 11 cases of normal liver tissues. The differences in the positive expression rate and strong positive expression rate of FSCN1 among different groups were analyzed. The positive rate of FSCN1 in normal liver tissues, liver cirrhosis, HCC, and ICC tissues was 0.0% (0/11), 0.0% (0/23), 13.9% (15/108), and 92.3% (24/26), respectively, while the strong positive rate was 0.0% (0/11), 0.0% (0/23), 0.9% (1/108), and 69.2% (18/26), respectively. Both the positive rate and strong positive rate of FSCN1 in ICC tissues were significantly higher than those in HCC, liver cirrhosis, and normal liver tissues. Additionally, the positive rate of FSCN1 in moderately to poorly differentiated HCC tissues was 18.8% (15/80), significantly higher than in well-differentiated HCC (0.0%, 0/28) (P = 0.031). In liver cancer, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FSCN1 positive prediction for ICC were 92.3%, 86.1%, 61.5%, and 97.9%, respectively, whereas the sensitivity, specificity, PPV, and NPV of FSCN1 strong positive prediction for ICC were 69.2%, 99.1%, 94.7%, and 93.0%, respectively. These results suggest that FSCN1 may play an important role in the occurrence and progression of liver cancer, and it can be used as a novel diagnostic marker for ICC.

YBX1 promotes stemness and cisplatin insensitivity in intrahepatic cholangiocarcinoma via the AKT/β-catenin axis.

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy characterized by a poor prognosis and closely linked to tumor stemness. However, the key molecules that regulate ICC stemness remain elusive. Although Y-box binding protein 1 (YBX1) negatively affects prognosis in various cancers by enhancing stemness and chemoresistance, its effect on stemness and cisplatin sensitivity in ICC remains unclear. Three bulk and single-cell RNA-seq datasets were analyzed to investigate YBX1 expression in ICC and its association with stemness. Clinical samples and colony/sphere formation assays validated the role of YBX1 in stemness and sensitivity to cisplatin. AZD5363 and KYA1979K explored the interaction of YBX1 with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) and WNT/β-catenin pathways. YBX1 was significantly upregulated in ICC, correlated with worse overall survival and shorter postoperative recurrence time, and was higher in chemotherapy-non-responsive ICC tissues. The YBX1-high group exhibited significantly elevated stemness scores, and genes linked to YBX1 upregulation were enriched in multiple stemness-related pathways. Moreover, YBX1 expression is significantly correlated with several stemness-related genes (SOX9, OCT4, CD133, CD44 and EPCAM). Additionally, YBX1 overexpression significantly enhanced the colony- and spheroid-forming abilities of ICC cells, accelerated tumor growth in vivo and reduced their sensitivity to cisplatin. Conversely, the downregulation of YBX1 exerted the opposite effect. The transcriptomic analysis highlighted the link between YBX1 and the PI3K/AKT and WNT/β-catenin pathways. Further, AZD5363 and KYA1979K were used to clarify that YBX1 promoted ICC stemness through the regulation of the AKT/β-catenin axis. YBX1 is upregulated in ICC and promotes stemness and cisplatin insensitivity via the AKT/β-catenin axis. Our study describes a novel potential therapeutic target for improving ICC prognosis.

N6-methyladenosine-modified circSLCO1B3 promotes intrahepatic cholangiocarcinoma progression via regulating HOXC8 and PD-L1.

Refractoriness to surgical resection and chemotherapy makes intrahepatic cholangiocarcinoma (ICC) a fatal cancer of the digestive system with high mortality and poor prognosis. Important function invests circRNAs with tremendous potential in biomarkers and therapeutic targets. Nevertheless, it is still unknown how circRNAs contribute to the evolution of ICC. CircRNAs in paired ICC and adjacent tissues were screened by circRNAs sequencing. To explore the impact of circRNAs on ICC development, experiments involving gain and loss of function were conducted. Various experimental techniques, including quantitative real-time PCR (qPCR), western blotting, RNA immunoprecipitation (RIP), luciferase reporter assays, RNA pull-down, chromatin immunoprecipitation (ChIP), ubiquitination assays and so on were employed to identify the molecular regulatory role of circRNAs. Herein, we reported a new circRNA, which originates from exon 9 to exon 15 of the SLCO1B3 gene (named circSLCO1B3), orchestrated ICC progression by promoting tumor proliferation, metastasis and immune evasion. We found that the circSLCO1B3 gene was highly overexpressed in ICC tissues and related to lymphatic metastasis, tumor sizes, and tumor differentiation. Mechanically, circSLCO1B3 not only promoted ICC proliferation and metastasis via miR-502-5p/HOXC8/SMAD3 axis, but also eradicated anti-tumor immunity via suppressing ubiquitin-proteasome-dependent degradation of PD-L1 by E3 ubiquitin ligase SPOP. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circSLCO1B3 and stabilizes its expression. Our findings indicate that circSLCO1B3 is a potential prognostic marker and therapeutic target in ICC patients. Taken together, m6A-modified circSLCO1B3 was correlated with poor prognosis in ICC and promoted ICC progression not only by enhancing proliferation and metastasis via potentiating HOXC8 expression, but also by inducing immune evasion via antagonizing PD-L1 degradation. These results suggest that circSLCO1B3 is a potential prognostic marker and therapeutic target for ICC.

Prediction of prognosis of patients with radical resection of intrahepatic cholangiocarcinoma based on single cell omics

Objectives: To analyze the survival benefit of intrahepatic cholangiocarcinoma (ICC) radical resection based on single cell omics. Methods: This is a retrospective case-series study. ICC single-cell sequencing was integrated from four data sets in the Gene Expression Omnibus Database, with a total of 46 patients undergoing radical resection, to explore the characteristics of the microenvironment. Microarray data of 100 ICC cases was analyzed in the EMBI database with survival data. The infiltration abundance of each epithelial cell cluster was calculated in each microarray data sample using the ssGSEA algorithm. The key epithelial cell cluster associated with poor patient outcomes was explored. The clinical value of key marker genes in this subgroup was studied. Prognostic marker genes were selected using the univariate and multivariate Cox proportional hazards(CoxPH) model. The The CoxPH model was constructed by the target genes and a nomogram was drawn. Kaplan-Meier survival analysis was used to verify the relationship between score and prognosis of patients. The predictive power of the model was evaluated by receiver operating characteristic(ROC) curves, calibration curves, and decision curve analysis (DCA). Results: Epithelial cell clusters infiltrated almost exclusively in tumor tissue. The MT2A+ epithelial cell subset was associated with a poorer prognosis for patients with a high invasion abundance and patients characterized by infiltration of this group were defined as antioxidant. After screening marker genes in this cluster using a univariate and multivariate CoxPH model, the following genes were found to be independent prognostic factors: FILPIL, NFKBIA, PEG10, SERPINB5. The CoxPH model was constructed using the four gene expression levels, and the survival rate of patients in the high-risk group was significantly lower than those in the low-risk group (all P<0.05). The constructed nomogram had good discrimination and validity. The ROC curve showed that the predicted area under the curve was 0.779, 0.844 and 0.845 at 1, 3 and 5 years, respectively. Compared to clinical indicators, the model had better predictive power using the calibration curve and the DCA test. Conclusions: The MT2A+ epithelial cell group may be associated with the prognosis of patients with ICC, and the concept of ICC tissue typing of antioxidant and non-antioxidant types is proposed. The type of antioxidant may predict the poor prognosis of the patients, and postoperative adjuvant therapy and other means could be considered to improve the survival of the patients.

Modulating PCGF4 Stability Is an Efficient Metastasis-Regulatory Strategy Used by Distinct Subtypes of Cancer-Associated Fibroblasts in Intrahepatic Cholangiocarcinoma

Intrahepatic Cholangiocarcinoma (ICC) is highly malignant neoplasm and prone to metastasis. It is unclear if cancer-associated fibroblasts (CAFs) affect the metastasis of ICC. Here, we have established ICC patient-derived CAF lines and related cancerous cell lines and analyzed the effects of CAFs on the tumor progressive properties of the ICC cancerous cells. Results demonstrated that CAFs can be classified into cancer-restraining or promoting categories (rCAFs or pCAFs) based on distinct tumorigenic effects. The RNA-Seq analyses of ICC cancerous cell lines identified B lymphoma Mo-MLV insertion region 1 (PCGF4, also known BMI1) as a potential metastasis regulator. Strikingly, the changes of PCGF4 levels in ICC cells perfectly mirrored the restraining or promoting effects of CAFs on ICC migration. Our Immunohistochemical analyses on the ICC tissue microarrays indicated that PCGF4 was negatively correlated to overall survival of ICC. We confirmed the promoting effects of PCGF4 on cell migration, drug resistance activity, stemness properties. Mechanistically, rCAFs triggered the proteasome-dependent degradation of PCGF4, while pCAFs enhanced the stability of PCGF4 via activating the IL-6/p-STAT3 pathway. In summary, our data identified roles of CAFs on ICC metastasis and revealed a new mechanism of the CAFs on ICC progression in which PCGF4 acted as the key effector by both categories of CAFs. These findings shed light on developing comprehensive therapeutic strategies for ICC.

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.

IL-33/ST2 Signaling and its Correlation with Macrophage Heterogeneity and Clinicopathologic Features in Human Intrahepatic Cholangiocarcinoma.

IL-33/ST2 signaling plays crucial roles in the development and progression of various human malignancies. However, its significance in intrahepatic cholangiocarcinoma (ICC) still remains unclear. This study aimed to investigate the expression of IL-33/ST2 signaling and its correlations with macrophage heterogeneity and ICC patients' clinicopathologic features. The expression of different phenotype macrophage markers and IL-33/ST2 signalingrelated markers was detected. The correlation between L-33/ST2 signaling and different phenotype macrophage markers as well as ICC patients' clinicopathologic data was evaluated. Massive heterogeneous cancer cells and PAS-positive cells were observed in tumor tissues. CD68-positive cells accumulated in tumor tissues and expression of both M1 phenotype markers and M2 phenotype macrophage markers was higher in tumor samples than para-carcinoma samples. However, M2 phenotype macrophages represented the dominant macrophage population in ICC tissues. Plasma levels of IL-33, ST2, and MIF were evidently enhanced in ICC patients compared to healthy controls. IL-33/ST2 signaling-related markers exhibited a massive increase in tumor samples than para-carcinoma samples. IL-33 and ST2 expression in ICC tissues was positively associated with M1 and M2 phenotype macrophages. Plasma levels of IL-33, ST2, and MIF were correlated with the diameter of tumor lesions, lymph node metastasis, TNM stage, and tumor differentiation degree. Multivariate analysis demonstrated IL-33 expression to exhibit a correlation with the diameter of tumor lesions, lymph node metastasis, and TNM stage. Additionally, there was a relationship observed between ST2, MIF expression, and diameter of tumor lesions plus TNM stage. IL-33/ST2 signaling exhibited a positive relationship with macrophage heterogeneity in ICC tissues, and upregulated levels of IL-33, ST2, and MIF were associated with aggressive clinicopathologic characteristics. These findings may provide promising diagnostic biomarkers and potential therapeutic strategies for ICC patients targeting IL-33/ST2 signaling.

Identification of FOXP1 as a favorable prognostic biomarker and tumor suppressor in intrahepatic cholangiocarcinoma

BackgroundForkhead-box protein P1 (FOXP1) has been proposed to have both oncogenic and tumor-suppressive properties, depending on tumor heterogeneity. However, the role of FOXP1 in intrahepatic cholangiocarcinoma (ICC) has not been previously reported.MethodsImmunohistochemistry was performed to detect FOXP1 expression in ICC and normal liver tissues. The relationship between FOXP1 levels and the clinicopathological characteristics of patients with ICC was evaluated. Finally, in vitro and in vivo experiments were conducted to examine the regulatory role of FOXP1 in ICC cells.ResultsFOXP1 was significantly downregulated in the ICC compared to their peritumoral tissues (p < 0.01). The positive rates of FOXP1 were significantly lower in patients with poor differentiation, lymph node metastasis, invasion into surrounding organs, and advanced stages (p < 0.05). Notably, patients with FOXP1 positivity had better outcomes (overall survival) than those with FOXP1 negativity (p < 0.05), as revealed by Kaplan–Meier survival analysis. Moreover, Cox multivariate analysis showed that negative FOXP1 expression, advanced TNM stages, invasion, and lymph node metastasis were independent prognostic risk factors in patients with ICC. Lastly, overexpression of FOXP1 inhibited the proliferation, migration, and invasion of ICC cells and promoted apoptosis, whereas knockdown of FOXP1 had the opposite role.ConclusionOur findings suggest that FOXP1 may serve as a novel outcome predictor for ICC as well as a tumor suppressor that may contribute to cancer treatment.

Targeting PKM2 improves the gemcitabine sensitivity of intrahepatic cholangiocarcinoma cells via inhibiting β-catenin signaling pathway

Gemcitabine is considered the standard first-line chemotherapeutic agent for patients with intrahepatic cholangiocarcinoma (ICC). However, its therapeutic efficacy is hampered by the development of chemoresistance. Pyruvate kinase M2 (PKM2), a crucial mediator of the final step in glycolysis, has been implicated in the origination and advancement of diverse malignancies. Its expression is increased in many tumor types and this may correlate with increased drug sensitivity. However, the specific effect of PKM2 on the gemcitabine sensitivity in ICC remains to be elucidated. In this research, we aimed to elucidate the role and functional significance of PKM2 in ICC, as well as the heightened susceptibility of ICC cells to gemcitabine by targeting PKM2 and the underlying molecular mechanisms. Immunohistochemical and immunofluorescence analyses revealed elevated expression of PKM2 in both tumor cells and macrophages in human ICC tissues. Reducing PKM2 levels significantly restrained the proliferation of tumor cells, impeded cell cycle advance, induced programmed cell death, and suppressed metastasis. In addition, knockdown or pharmacological inhibition of PKM2 could enhance the response of ICC cells to gemcitabine in vitro. Interestingly, conditioned medium co-culture system suggested that conditioned medium from M2 macrophages increased gemcitabine sensitivity of ICC cells. However, silencing PKM2 or pharmacological inhibition of PKM2 in M2 macrophages did not ameliorate the gemcitabine resistance mediated by M2 macrophages derived conditioned medium. Mechanistically, downregulation of PKM2 repressed the expression of β-catenin and its downstream transcriptional targets, thereby hindering the propagation of β-catenin signaling cascade. Finally, the results of the subcutaneous xenograft experiment in nude mice provided compelling evidence of a synergistic interaction between PKM2-IN-1 and gemcitabine in vivo. In summary, we reported that PKM2 may function as an advantageous target for increasing the sensitivity of ICC to gemcitabine treatment. Targeting PKM2 improves the gemcitabine sensitivity of ICC cells via inhibiting β-catenin signaling pathway.

IL-8 is a novel prometastatic chemokine in intrahepatic cholangiocarcinoma that induces CXCR2-PI3K/AKT signaling upon CD97 activation

Intrahepatic cholangiocarcinoma (ICC) is a rare but highly aggressive malignant tumor arising within the liver, with a 5-year survival rate of only 20–40% after surgery. The role of interleukin-8 (IL-8) in ICC progression remains elusive. A transcriptomic approach based on IL-8 stimulation first revealed significant upregulation of the prometastatic gene CD97 and key epithelial–mesenchymal transition (EMT) factors E-cadherin and vimentin. Immunohistochemistry of 125 ICC tissues confirmed the positive correlation between IL-8 and CD97. Multivariable Cox regression indicated that they are both independent predictors of ICC prognosis. Mechanistically, IL-8 treatment induced CD97 expression at 50 and 100 ng/ml in QBC-939 and QBE cells, respectively. Moreover, the induction of cell migration and invasion upon IL-8 treatment was attenuated by CD97 RNA interference, and the expression of EMT-associated genes was dramatically inhibited. To determine whether CXCR1 or CXCR2 are downstream effectors of IL-8, siCXCR2 was applied and shown to significantly attenuate the oncogenic effects of IL-8 by inhibiting the phosphorylation of PI3K/AKT. Finally, the induction of CD97 expression by the PI3K pathway was verified by treatment with the inhibitor LY294002. In vivo, the significant tumor growth and lung metastasis effects induced by intraperitoneal injection of IL-8 were greatly inhibited by silencing CD97 in nude mice. Collectively, the study presents a novel mechanism of the IL-8-CXCR2-PI3K/AKT axis in regulating CD97 expression, which leads to ICC metastasis mainly through EMT. The study may provide alternatives for targeting the tumor microenvironment in metastatic ICC.

COTE1 Facilitates Intrahepatic Cholangiocarcinoma Progression via Beclin1-Dependent Autophagy Inhibition.

COTE1 was recently described as an oncogene in hepatocellular carcinoma and gastric cancer. However, the roles of COTE1 in intrahepatic cholangiocarcinoma (ICC) are little known. Our study is aimed at clarifying novel functions of COTE1 in ICC progression, including proliferation, invasion, and autophagy. By using quantitative real-time PCR, immunohistochemistry staining, and western blotting, we found that COTE1 expression was frequently upregulated in ICC tissues, compared to paracarcinoma tissues. High COTE1 expression was significantly correlated with aggressive clinical features and predicted poor prognosis of ICC patients. Functional experiments revealed that ectopic COTE1 expression promoted ICC cell proliferation, colony formation, cellular invasion, migration, and in vivo tumorigenicity; in contrast, COTE1 knockdown resulted in the opposite effects. At molecular mechanism in vitro and vivo, our study revealed that COTE1 overexpression suppressed autophagy via Beclin1 transcription inhibition; conversely, COTE1 silencing facilitated autophagy through promoting Beclin1 expression. Furthermore, the suppression of COTE1 knockdown on cellular growth and invasion was rescued/aggravated by Beclin1 inhibition/accumulation. Our data, for the first time, illustrate that COTE1 is an oncogene in ICC pathogenesis, and the ectopic COTE1 expression promotes ICC proliferation and invasion via Beclin1-dependent autophagy inhibition.

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.

VIRMA facilitates intrahepatic cholangiocarcinoma progression through epigenetic augmentation of TMED2 and PARD3B mRNA stabilization.

N6-methyladenine modification of RNA, a critical component of the regulatory role at the post-transcriptional level, has a crucial effect on tumor development and progression. vir-Like m6A methyltransferase associated (VIRMA) has been recently discovered as an N6-methyladenine methyltransferase; however, its specific role in intrahepatic cholangiocarcinoma (ICC) remains to be investigated in-depth. VIRMA expression and its association with clinicopathological characteristics were evaluated using The Cancer Genome Atlas (TCGA) dataset and tissue microarrays. In vivo and in vitro assays were performed to determine the role of VIRMA in ICC proliferation and metastasis. The underlying mechanism by which VIRMA influences ICC was clarified by RNA sequencing (RNA-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), SLAM sequencing (SLAM-seq), RNA immunoprecipitation, a luciferase reporter assay, and chromatin immunoprecipitation assay. VIRMA showed high expression in ICC tissues, and this finding predicted a dismal prognostic outcome. The high expression of VIRMA in ICC was due to the demethylation of H3K27me3 modification in the promoter region. Functionally, VIRMA is required for the endothelial-mesenchymal transition (EMT) process in ICC cells, as shown by multiple ICC models in in vitro and in vivo experiments. Mechanistically, multi-omics analysis using ICC cells demonstrated that TMED2 and PARD3B were the direct downstream target of VIRMA. The methylated TMED2 and PARD3B transcripts were directly recognized by HuR, which exerted stabilizing effects on its bound RNA. VIRMA-induced expression of TMED2 and PARD3B activated the Akt/GSK/β-catenin and MEK/ERK/Slug signaling pathways, thereby promoting ICC proliferation and metastasis. The present study showed that VIRMA plays a critical role in ICC development by stabilizing TMED2 and PARD3B expression through the m6A-HuR-mediated mechanism. Thus, demonstrating VIRMA and its pathway as candidate therapeutic targets for ICC treatment.

Hepatitis B virus-related intrahepatic cholangiocarcinoma originates from hepatocytes

BackgroundHepatitis B virus (HBV) infection is one of the most common risk factors for intrahepatic cholangiocarcinoma (ICC). However, there is no direct evidence of a causal relationship between HBV infection and ICC. In this study, we attempted to prove that ICC may originate from hepatocytes through a pathological study involving ICC tissue-derived organoids.MethodThe medical records and tumor tissue samples of 182 patients with ICC after hepatectomy were collected. The medical records of 182 patients with ICC were retrospectively analyzed to explore the prognostic factors. A microarray of 182 cases of ICC tumor tissue and 6 cases of normal liver tissue was made, and HBsAg was stained by immunohistochemistry (IHC) to explore the factors closely related to HBV infection. Fresh ICC tissues and corresponding adjacent tissues were collected to make paraffin sections and organoids. Immunofluorescence (IF) staining of factors including HBsAg, CK19, CK7, Hep-Par1 and Albumin (ALB) was performed on both fresh tissues and organoids. In addition, we collected adjacent nontumor tissues of 6 patients with HBV (+) ICC, from which biliary duct tissue and normal liver tissue were isolated and RNA was extracted respectively for quantitative PCR assay. In addition, the expression of HBV-DNA in organoid culture medium was detected by quantitative PCR and PCR electrophoresis.ResultsA total of 74 of 182 ICC patients were HBsAg positive (40.66%, 74/182). The disease-free survival (DFS) rate of HBsAg (+) ICC patients was significantly lower than that of HBsAg (−) ICC patients (p = 0.0137). IF and IHC showed that HBsAg staining was only visible in HBV (+) ICC fresh tissues and organoids, HBsAg expression was negative in bile duct cells in the portal area. Quantitative PCR assay has shown that the expression of HBs antigen and HBx in normal hepatocytes were significantly higher than that in bile duct epithelial cells. Combined with the IF and IHC staining, it was confirmed that HBV does not infect normal bile duct epithelial cells. In addition, IF also showed that the staining of bile duct markers CK19 and CK7 were only visible in ICC fresh tissue and organoids, and the staining of hepatocyte markers Hep-Par1 and ALB was only visible in normal liver tissue fresh tissue. Real-time PCR and WB had the same results. High levels of HBV-DNA were detected in the culture medium of HBV (+) organoids but not in the culture medium of HBV (−) organoids.ConclusionHBV-related ICC might be derived from hepatocytes. HBV (+) ICC patients had shorter DFS than HBV (−) ICC patients.

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.

Prognostic values of tissue-resident CD8+T cells in human hepatocellular carcinoma and intrahepatic cholangiocarcinoma

BackgroundTissue-resident CD8+T cells (CD103+CD8+T cells) are the essential effector cell population of anti-tumor immune response in tissue regional immunity. And we have reported that IL-33 can promote the proliferation and effector function of tissue-resident CD103+CD8+T cells. As of now, the immunolocalization and the prognostic values of tissue-resident CD8+T cells in human hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) still remain to be illustrated.MethodsIn our present study, we used the tissue microarrays of HCC and ICC, the multicolor immunohistochemistry (mIHC), and imaging analysis to characterize the tissue-resident CD8+T cells in HCC and ICC tissues. The prognostic values and clinical associations were also analyzed. We also studied the biological functions and the cell–cell communication between tumor-infiltrating CD103+CD8+T cells and other cell types in HCC and ICC based on the published single-cell RNA sequencing (scRNA-seq) data.ResultsOur work unveiled the expressions of CD8 and CD103 and immunolocalization of tissue-resident CD8+T cells in human HCC and ICC. Elevated CD8+T cells indicated a better overall survival (OS) rate, implying that tumor-infiltrating CD8+T cells in HCC and ICC could serve as an independent prognostic factor. Moreover, the number of CD103+CD8+T cells was increased in HCC and ICC tissues compared with adjacent normal tissues. HCC patients defined as CD8highCD103high had a better OS, and the CD8lowCD103low group tended to have a poorer prognosis in ICC. Evaluation of the CD103+CD8+T-cell ratio in CD8+T cells could also be a prognostic predictor for HCC and ICC patients. A higher ratio of CD103+CD8+T cells over total CD8+T cells in HCC tissues was negatively and significantly associated with the advanced pathological stage. The percentage of higher numbers of CD103+CD8+T cells in ICC tissues was negatively and significantly associated with the advanced pathological stage. In contrast, the higher ratio of CD103+CD8+T cells over total CD8+T cells in ICC tissues was negatively and significantly associated with the advanced pathological stage. In addition, single-cell transcriptomics revealed that CD103+CD8+T cells were enriched in genes associated with T-cell activation, proliferation, cytokine function, and T-cell exhaustion.ConclusionThe CD103+ tumor-specific T cells signified an important prognostic marker with improved OS, and the evaluation of the tissue-resident CD103+CD8+T cells might be helpful in assessing the on-treatment response of liver cancer.

M6A RNA methylation-mediated upregulation of HLF promotes intrahepatic cholangiocarcinoma progression by regulating the FZD4/β-catenin signaling pathway

Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, its role in regulating intrahepatic cholangiocarcinoma (ICC) remains unclear. This study aimed to define the role of HLF in ICC progression. Here, we showed that HLF expression is upregulated in ICC and predicts the poor prognosis in patients. Mechanistically, HLF activation in ICC is mediated by METTL3-dependent m6A methylation of the HLF mRNA. As per the results from the loss- or gain-of-function experiments, HLF promoted the self-renewal, tumorigenicity, proliferation and metastasis of ICC cells. RNA-seq and CUT&Tag analyses showed that frizzled-4 (FZD4) and forkhead box Q1 (FOXQ1) are target genes of HLF. Moreover, FOXQ1 transcriptionally activates METTL3 expression, forming a positive feedback loop, which subsequently activates WNT/β-catenin signaling and downstream tumor stemness. Furthermore, HLF expression was positively correlated with METTL3, IGF2BP3, FZD4 and FOXQ1 expression in ICC tissues in a large ICC cohort. The combined IHC panels exhibited a better prognostic value for patients with ICC than any of these components alone. In conclusions, these findings demonstrated that the METTL3/HLF/FOXQ1 regulatory circuit drove FZD4-mediated WNT/β-catenin activation in ICC progression, suggesting that targeting this axis could be novel therapeutic strategy for ICC.

Circular RNA in liquid biopsy as biomarkers toward precision medicine

Circular RNA in liquid biopsy as biomarkers toward precision medicine

Proteomic and single-cell landscape reveals novel pathogenic mechanisms of HBV-infected intrahepatic cholangiocarcinoma

Despite the epidemiological association between intrahepatic cholangiocarcinoma (ICC) and hepatitis B virus (HBV) infection, little is known about the relevant oncogenic effects. A cohort of 32 HBV-infected ICC and 89 non-HBV-ICC patients were characterized using whole-exome sequencing, proteomic analysis, and single-cell RNA sequencing. Proteomic analysis revealed decreased cell-cell junction levels in HBV-ICC patients. The cell-cell junction level had an inverse relationship with the epithelial-mesenchymal transition (EMT) program in ICC patients. Analysis of the immune landscape found that more CD8 Tcells and Th2 cells were present in HBV-ICC patients. Single-cell analysis indicated that transforming growth factor beta signaling-related EMT program changes increased in tumor cells of HBV-ICC patients. Moreover, ICAM1+ tumor-associated macrophages are correlated with a poor prognosis and contributed to the EMT in HBV-ICC patients. Our findings provide new insights into the behavior of HBV-infected ICC driven by various pathogenic mechanisms involving decreased cell junction levels and increased progression of the EMT program.