Extracellular Vesicles in Hepatobiliary Malignancies

Primary hepatobiliary malignancies include a heterogeneous group of cancers with dismal prognosis, among which hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatoblastoma (HB) stand out. These tumors mainly arise from the malignant transformation of hepatocytes, cholangiocytes (bile duct epithelial cells) or hepatoblasts (embryonic liver progenitor cells), respectively. Early diagnosis, prognosis prediction and effective therapies are still a utopia for these diseases. Extracellular vesicles (EVs) are small membrane-enclosed spheres secreted by cells and present in biological fluids. They contain multiple types of biomolecules, such as proteins, RNA, DNA, metabolites and lipids, which make them a potential source of biomarkers as well as regulators of human pathobiology. In this review, the role of EVs in the pathogenesis of hepatobiliary cancers and their potential usefulness as disease biomarkers are highlighted. Moreover, the therapeutic value of EV regulation is discussed and future directions on basic and clinical research are indicated

Amphiregulin contributes to the transformed phenotype of human hepatocellular carcinoma cells

Hepatocellular carcinoma is a major cause of cancer-related deaths. Current treatments are not effective, and the identification of relevant pathways and novel therapeutic targets are much needed. Increasing evidences point to the activation of the epidermal growth factor receptor (EGFR) as an important mechanism in the development of hepatocarcinoma. We previously described that amphiregulin (AR), a ligand of the EGFR, is not expressed in healthy liver but is up-regulated during chronic liver injury, the background on which most liver tumors develop. Now, we have studied the expression and role of AR in human hepatocarcinoma. AR expression and function was studied in human liver tumors and cell lines. AR is expressed in human hepatocellular carcinoma tissues and cell lines and behaves as a mitogenic and antiapoptotic growth factor for hepatocarcinoma cells. We provide several lines of evidence, including AR silencing by small interfering RNAs and inhibition of amphiregulin by neutralizing antibodies, showing the existence of an AR-mediated autocrine loop that contributes to the transformed phenotype. Indeed, interference with endogenous AR production resulted in reduced constitutive EGFR signaling, inhibition of cell proliferation, anchorage-independent growth, and enhanced apoptosis. Moreover, knockdown of AR potentiated transforming growth factor-beta and doxorubicin-induced apoptosis. Conversely, overexpression of AR in SK-Hep1 cells enhanced their proliferation rate, anchorage-independent growth, drug resistance, and in vivo tumorigenic potential. These observations suggest that AR is involved in the acquisition of neoplastic traits in the liver and thus constitutes a novel therapeutic target in human hepatocarcinoma

The search for novel diagnostic and prognostic biomarkers in cholangiocarcinoma

[EN] The poor prognosis of cholangiocarcinoma (CCA) is in part due to late diagnosis, which is currently achieved by a combination of clinical, radiological and histological approaches. Available biomarkers determined in serum and biopsy samples to assist in CCA diagnosis are not sufficiently sensitive and specific. Therefore, the identification of new biomarkers, preferably those obtained by minimally invasive methods, such as liquid biopsy, is important. The development of innovative technologies has permitted to identify a significant number of genetic, epigenetic, proteomic and metabolomic CCA features with potential clinical usefulness in early diagnosis, prognosis or prediction of treatment response. Potential new candidates must be rigorously evaluated prior to entering routine clinical application. Unfortunately, to date, no such biomarker has achieved validation for these purposes. This review is an up-to-date of currently used biomarkers and the candidates with promising characteristics that could be included in the clinical practice in the next future. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen

The epidermal growth factor receptor ligand amphiregulin participates in the development of mouse liver fibrosis

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TREM-2 plays a protective role in cholestasis by acting as a negative regulator of inflammation

Background & Aims: Inflammation, particularly that mediated by bacterial components translocating from the gut to the liver and binding to toll-like receptors (TLRs), is central to cholestatic liver injury. The triggering receptor expressed on myeloid cells-2 (TREM-2) inhibits TLR-mediated signaling and exerts a protective role in hepatocellular injury and carcinogenesis. This study aims to evaluate the role of TREM-2 in cholestasis.Methods: TREM-2 expression was analyzed in the livers of pa-tients with primary biliary cholangitis (PBC) or primary scle-rosing cholangitis (PSC), and in mouse models of cholestasis. Wild-type (WT) and Trem-2 deficient (Trem-2-/-) mice were subjected to experimental cholestasis and gut sterilization. Pri-mary cultured Kupffer cells were incubated with lipopolysac-charide and/or ursodeoxycholic acid (UDCA) and inflammatory responses were analyzed.Results: TREM-2 expression was upregulated in the livers of patients with PBC or PSC, and in murine models of cholestasis. Compared to WT, the response to bile duct ligation (BDL)-induced obstructive cholestasis or alpha-naphtylisothiocyanate (ANIT)-induced cholestasis was exacerbated in Trem-2-/-mice. This was characterized by enhanced necroptotic cell death, in-flammatory responses and biliary expansion. Antibiotic treat-ment partially abrogated the effects observed in Trem-2-/-mice after BDL. Experimental overexpression of TREM-2 in the liver of WT mice downregulated ANIT-induced IL-33 expression and neutrophil recruitment. UDCA regulated Trem-1 and Trem-2 expression in primary cultured mouse Kupffer cells and damp-ened inflammatory gene transcription via a TREM-2-dependent mechanism.Conclusions: TREM-2 acts as a negative regulator of inflamma-tion during cholestasis, representing a novel potential thera-peutic target.Lay summary: Cholestasis (the reduction or cessation of bile flow) causes liver injury. This injury is exacerbated when gut-derived bacterial components interact with receptors (spe-cifically Toll-like receptors or TLRs) on liver-resident immune cells, promoting inflammation. Herein, we show that the anti-inflammatory receptor TREM-2 dampens TLR-mediated signaling and hence protects against cholestasis-induced liver injury. Thus, TREM-2 could be a potential therapeutic target in cholestasis.Spanish Carlos III Health Institute (ISCIII) [J.M. Banales (FIS PI18/01075, PI21/00922 and Miguel Servet Program CPII19/00008); M.J. Perugorria (FIS PI14/00399, PI17/00022 and PI20/00186); J.J.G. Marin (FIS PI16/00598 and PI19/00819); P.M. Rodrigues (Sara Borrell CD19/00254)] cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER); “Instituto de Salud Carlos III” [CIBERehd: M.J. Monte, J.J.G. Marin, J.M. Banales, M.J. Perugorria, P. Aspichueta, P.M. Rodrigues and L. Bujanda], Spain; “Diputación Foral de Gipuzkoa” (M.J. Perugorria: DFG18/114), Department of Health of the Basque Country (M.J. Perugorria: 2019111024, 2015111100 and J.M. Banales: 2021111021), “Euskadi RIS3” (J.M. Banales: 2019222054, 2020333010, 2021333003), and Department of Industry of the Basque Country (J.M. Banales: Elkartek: KK-2020/00008); “Junta de Castilla y Leon” (J.J.G. Marin: SA063P17). La Caixa Scientific Foundation (J.M. Banales: HR17-00601). “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientific Foundation, to J.M. Banales and J.J.G. Marin); “Centro Internacional sobre el Envejecimiento” (J.J.G. Marin: OLD-HEPAMARKER, 0348_CIE_6_E); Fundació Marato TV3 (J.J.G. Marin: Ref. 201916-31). O Sharif was funded by the Austrian Science Fund (FWF-P35168). Work in the lab of T. Luedde was funded by the European Research Council (ERC) (Grant Agreement 771083), the German Research Foundation (DFG – LU 1360/3-2 (279874820), LU 1360/4-(1461704932) and SFB-CRC 1382-Project A01) and the German Ministry of Health (BMG – DEEP LIVER 2520DAT111). Contributions of M. Marzioni were funded by the Università Politecnica delle Marche PSA2017_UNIVPM grant. Contributions of DAM were supported by programme grants from CRUK (C18342/A23390) and MRC (MR/K0019494/1 and MR/R023026/1). MJ Perugorria was funded by the Spanish Ministry of Economy and Competitiveness (MINECO: “Ramón y Cajal” Programme RYC-2015-17755), I. Labiano, A. Agirre-Lizaso, P. Olaizola, A. Echebarria and F. González-Romero by the Basque Government (PRE_2016_1_0152, PRE_2018_1_0184, PRE_2016_1_0269 PRE_2020_1_0080, PRE_2018_1_0120, respectively), I. Olaizola by the Ministry of Universities (FPU 19/03327) and A. Esparza-Baquer by the University of the Basque Country (PIF2014/11). The funding sources had no involvement in study design, data collection and analysis, decision to publish, or preparation of the article

Patients with Cholangiocarcinoma Present Specific RNA Profiles in Serum and Urine Extracellular Vesicles Mirroring the Tumor Expression: Novel Liquid Biopsy Biomarkers for Disease Diagnosis

Cholangiocarcinoma (CCA) comprises a group of heterogeneous biliary cancers with dismal prognosis. The etiologies of most CCAs are unknown, but primary sclerosing cholangitis (PSC) is a risk factor. Non-invasive diagnosis of CCA is challenging and accurate biomarkers are lacking. We aimed to characterize the transcriptomic profile of serum and urine extracellular vesicles (EVs) from patients with CCA, PSC, ulcerative colitis (UC), and healthy individuals. Serum and urine EVs were isolated by serial ultracentrifugations and characterized by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. EVs transcriptome was determined by Illumina gene expression array [messenger RNAs (mRNA) and non-coding RNAs (ncRNAs)]. Differential RNA profiles were found in serum and urine EVs from patients with CCA compared to control groups (disease and healthy), showing high diagnostic capacity. The comparison of the mRNA profiles of serum or urine EVs from patients with CCA with the transcriptome of tumor tissues from two cohorts of patients, CCA cells in vitro, and CCA cellsderived EVs, identified 105 and 39 commonly-altered transcripts, respectively. Gene ontology analysis indicated that most commonly-altered mRNAs participate in carcinogenic steps. Overall, patients with CCA present specific RNA profiles in EVs mirroring the tumor, and constituting novel promising liquid biopsy biomarkers

Retrospective cohort study: Risk of gastrointestinal cancer in a symptomatic cohort after a complete colonoscopy: Role of faecal immunochemical test

BACKGROUND: Faecal immunochemical test (FIT) has been recommended to assess symptomatic patients for colorectal cancer (CRC) detection. Nevertheless, some conditions could theoretically favour blood originating in proximal areas of the gastrointestinal tract passing through the colon unmetabolized. A positive FIT result could be related to other gastrointestinal cancers (GIC). AIM: To assess the risk of GIC detection and related death in FIT-positive symptomatic patients (threshold 10 µg Hb/g faeces) without CRC. METHODS: Post hoc cohort analysis performed within two prospective diagnostic test studies evaluating the diagnostic accuracy of different FIT analytical systems for CRC and significant colonic lesion detection. Ambulatory patients with gastrointestinal symptoms referred consecutively for colonoscopy from primary and secondary healthcare, underwent a quantitative FIT before undergoing a complete colonoscopy. Patients without CRC were divided into two groups (positive and negative FIT) using the threshold of 10 µg Hb/g of faeces and data from follow-up were retrieved from electronic medical records of the public hospitals involved in the research. We determined the cumulative risk of GIC, CRC and upper GIC. Hazard rate (HR) was calculated adjusted by age, sex and presence of significant colonic lesion. RESULTS: We included 2709 patients without CRC and a complete baseline colonoscopy, 730 (26.9%) with FIT = 10 µgr Hb/gr. During a mean time of 45.5 ± 20.0 mo, a GIC was detected in 57 (2.1%) patients: An upper GIC in 35 (1.3%) and a CRC in 14 (0.5%). Thirty-six patients (1.3%) died due to GIC: 22 (0.8%) due to an upper GIC and 9 (0.3%) due to CRC. FIT-positive subjects showed a higher CRC risk (HR 3.8, 95%CI: 1.2-11.9) with no differences in GIC (HR 1.5, 95%CI: 0.8-2.7) or upper GIC risk (HR 1.0, 95%CI: 0.5-2.2). Patients with a positive FIT had only an increased risk of CRC-related death (HR 10.8, 95%CI: 2.1-57.1) and GIC-related death (HR 2.2, 95%CI: 1.1-4.3), with no differences in upper GIC-related death (HR 1.4, 95%CI: 0.6-3.3). An upper GIC was detected in 22 (0.8%) patients during the first year. Two variables were independently associated: anaemia (OR 5.6, 95%CI: 2.2-13.9) and age = 70 years (OR 2.7, 95%CI: 1.1-7.0). CONCLUSION: Symptomatic patients without CRC have a moderate risk increase in upper GIC, regardless of the FIT result. Patients with a positive FIT have an increased risk of post-colonoscopy CRC

Liquid biopsy-based protein biomarkers for risk prediction, early diagnosis, and prognostication of cholangiocarcinoma

Cholangiocarcinoma (CCA), heterogeneous biliary tumours with dismal prognosis, lacks accurate early diagnostic methods especially important for individuals at high-risk (i.e. those with primary sclerosing cholangitis [PSC]). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). EVs from patients with isolated PSC (n = 45), concomitant PSC-CCA (n = 44), PSC who developed CCA during follow-up (PSC to CCA; n = 25), CCAs from non-PSC aetiology (n = 56), and hepatocellular carcinoma (n = 34) and healthy individuals (n = 56) were characterised by mass spectrometry. Diagnostic biomarkers for PSC-CCA, non-PSC CCA, or CCAs regardless of aetiology (Pan-CCAs) were defined and validated by ELISA. Their expression was evaluated in CCA tumours at a single-cell level. Prognostic EV biomarkers for CCA were investigated. High-throughput proteomics of EVs identified diagnostic biomarkers for PSC-CCA, non-PSC CCA, or Pan-CCA, and for the differential diagnosis of intrahepatic CCA and hepatocellular carcinoma, which were cross-validated by ELISA using total serum. Machine learning-based algorithms disclosed CRP/FIBRINOGEN/FRIL for the diagnosis of PSC-CCA (local disease [LD]) vs. isolated PSC (AUC = 0.947; odds ratio [OR] =36.9) and, combined with carbohydrate antigen 19-9, overpowers carbohydrate antigen 19-9 alone. CRP/PIGR/VWF allowed the diagnosis of LD non-PSC CCAs vs. healthy individuals (AUC = 0.992; OR = 387.5). It is noteworthy that CRP/FRIL accurately diagnosed LD Pan-CCA (AUC = 0.941; OR = 89.4). Levels of CRP/FIBRINOGEN/FRIL/PIGR showed predictive capacity for CCA development in PSC before clinical evidence of malignancy. Multi-organ transcriptomic analysis revealed that serum EV biomarkers were mostly expressed in hepatobiliary tissues, and single-cell RNA sequencing and immunofluorescence analysis of CCA tumours showed their presence mainly in malignant cholangiocytes. Multivariable analysis unveiled EV prognostic biomarkers, with COMP/GNAI2/CFAI and ACTN1/MYCT1/PF4V associated negatively and positively with patients' survival, respectively. Serum EVs contain protein biomarkers for the prediction, early diagnosis, and prognostication of CCA that are detectable using total serum, representing a tumour cell-derived liquid biopsy tool for personalised medicine. The accuracy of current imaging tests and circulating tumour biomarkers for cholangiocarcinoma (CCA) diagnosis is far from satisfactory. Most CCAs are considered sporadic, although up to 20% of patients with primary sclerosing cholangitis (PSC) develop CCA during their lifetime, constituting a major cause of PSC-related death. This international study has proposed protein-based and aetiology-related logistic models with predictive, diagnostic, or prognostic capacities by combining two to four circulating protein biomarkers, moving a step forward into personalised medicine. These novel liquid biopsy tools may allow the (i) easy and non-invasive diagnosis of sporadic CCAs, (ii) identification of patients with PSC with higher risk for CCA development, (iii) establishment of cost-effective surveillance programmes for the early detection of CCA in high-risk populations (e.g. PSC), and (iv) prognostic stratification of patients with CCA, which, altogether, may increase the number of cases eligible for potentially curative options or to receive more successful treatments, decreasing CCA-related mortality.Spanish Carlos III Health Institute (ISCIII

Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease

Background and Aims Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of symptomatic biliary cysts. Current surgical and pharmacological approaches are ineffective, and liver transplantation represents the only curative option. Ursodeoxycholic acid (UDCA) and histone deacetylase 6 inhibitors (HDAC6is) have arisen as promising therapeutic strategies, but with partial benefits. Approach and Results Here, we tested an approach based on the design, synthesis, and validation of a family of UDCA synthetic conjugates with selective HDAC6i capacity (UDCA-HDAC6i). Four UDCA-HDAC6i conjugates presented selective HDAC6i activity, UDCA-HDAC6i #1 being the most promising candidate. UDCA orientation within the UDCA-HDAC6i structure was determinant for HDAC6i activity and selectivity. Treatment of polycystic rats with UDCA-HDAC6i #1 reduced their hepatomegaly and cystogenesis, increased UDCA concentration, and inhibited HDAC6 activity in liver. In cystic cholangiocytes UDCA-HDAC6i #1 restored primary cilium length and exhibited potent antiproliferative activity. UDCA-HDAC6i #1 was actively transported into cells through BA and organic cation transporters. Conclusions These UDCA-HDAC6i conjugates open a therapeutic avenue for PLDs.Supported by the Spanish Carlos III Health Institute (ISCIII; J.M. Banales: FIS PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129; M.J. Perugorria: PI14/00399, PI17/00022; J.J.G. Marin: FIS PI16/00598) cofinanced by "Fondo Europeo de Desarrollo Regional" (FEDER); CIBERehd (ISCIII): J.M. Banales, M.J. Perugorria, L. Bujanda, and J.J.G. Marin; Spanish Ministry of Economy and Competitiveness (M. J. Perugorria: Ramon y Cajal Program RYC-2015-17755); IKERBASQUE, Basque foundation for Science (M.J. Perugorria and J.M. Banales), Spain; "Junta de Castilla y Leon" (J.J.G. Marin: SA06P17); " Diputacion Foral Gipuzkoa" (J.M. Banales: DFG15/010, DFG16/004; M.J. Perugorria: DFG18/114, DFG19/081), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD to J.M. Banales), Department of Health of the Basque Country (J.M. Banales: 2017111010; M.J. Perugorria: 2019111024), and Euskadi RIS3 (J.M. Banales: 2016222001, 2017222014, and 2018222029; 2019222054); La Caixa Scientific Foundation (J.M. Banales: HR17-00601); "Fundacion Cientifica de la Asociacion Espanola Contra el Cancer" (AECC Scientific Foundation, to J.M. Banales and J.J.G. Marin); and "Centro Internacional sobre el Envejecimiento", Spain (J.J.G. Marin: OLD-HEPAMARKER, 0348-CIE-6-E). F.J. Caballero-Camino was funded by the Spanish Ministry of Science and Innovation (BES-2014-069148), A. Santos-Laso by the Basque Government (PRE_2018_2_0195), and Pui Y. Lee-Law by the European Association for the Study of the Liver (EASL; Sheila Sherlock Award). The Spanish Ministry of Science and Innovation supported F. P. Cossio: (CTQ2016-80375-P and CTQ2014-51912-REDC) as well as the Basque Government (F.P. Cossio: IT-324-07). I. Rivilla had a postdoctoral contract from the Donostia International Physics Center