Oleuropein, a Component of Extra Virgin Olive Oil, Improves Liver Steatosis and Lobular Inflammation by Lipopolysaccharides-TLR4 Axis Downregulation.

Gut-dysbiosis-induced lipopolysaccharides (LPS) translocation into systemic circulation has been suggested to be implicated in nonalcoholic fatty liver disease (NAFLD) pathogenesis. This study aimed to assess if oleuropein (OLE), a component of extra virgin olive oil, lowers high-fat-diet (HFD)-induced endotoxemia and, eventually, liver steatosis. An immunohistochemistry analysis of the intestine and liver was performed in (i) control mice (CTR; n = 15), (ii) high-fat-diet fed (HFD) mice (HFD; n = 16), and (iii) HFD mice treated with 6 µg/day of OLE for 30 days (HFD + OLE, n = 13). The HFD mice developed significant liver steatosis compared to the controls, an effect that was significantly reduced in the HFD + OLE-treated mice. The amount of hepatocyte LPS localization and the number of TLR4+ macrophages were higher in the HFD mice in the than controls and were lowered in the HFD + OLE-treated mice. The number of CD42b+ platelets was increased in the liver sinusoids of the HFD mice compared to the controls and decreased in the HFD + OLE-treated mice. Compared to the controls, the HFD-treated mice showed a high percentage of intestine PAS+ goblet cells, an increased length of intestinal crypts, LPS localization and TLR4+ expression, and occludin downregulation, an effect counteracted in the HFD + OLE-treated mice. The HFD-fed animals displayed increased systemic levels of LPS and zonulin, but they were reduced in the HFD + OLE-treated animals. It can be seen that OLE administration improves liver steatosis and inflammation in association with decreased LPS translocation into the systemic circulation, hepatocyte localization of LPS and TLR4 downregulation in HFD-induced mouse model of NAFLD.

Recommendations on maximising the clinical value of tissue in the management of patients with intrahepatic cholangiocarcinoma.

Background & Aims: Patients with intrahepatic cholangiocarcinoma can now be managed with targeted therapies directed against specific molecular alterations. Consequently, tissue samples submitted to the pathology department must produce molecular information in addition to a diagnosis or, for resection specimens, staging information. The pathologist's role when evaluating these specimens has therefore changed to accommodate such personalised approaches. Methods: We developed recommendations and guidance for pathologists by conducting a systematic review of existing guidance to generate candidate statements followed by an international Delphi process. Fifty-nine pathologists from 28 countries in six continents rated statements mapped to all elements of the specimen pathway from receipt in the pathology department to authorisation of the final written report. A separate survey of 'end-users' of the report including surgeons, oncologists, and gastroenterologists was undertaken to evaluate what information should be included in the written report to enable appropriate patient management. Results: Forty-eight statements reached consensus for inclusion in the guidance including 10 statements about the content of the written report that also reached consensus by end-user participants. A reporting proforma to allow easy inclusion of the recommended data points was developed. Conclusions: These guiding principles and recommendations provide a framework to allow pathologists reporting on patients with intrahepatic cholangiocarcinoma to maximise the informational yield of specimens required for personalised patient management. Impact and Implications: Biopsy or resection lesional tissue from intrahepatic cholangiocarcinoma must yield information about the molecular abnormalities within the tumour that define suitability for personalised therapies in addition to a diagnosis and staging information. Here, we have developed international consensus guidance for pathologists that report such cases using a Delphi process that sought the views of both pathologists and 'end-users of pathology reports. The guide highlights the need to report cases in a way that preserves tissue for molecular testing and emphasises that reporting requires interpretation of histological characteristics within the broader clinical and radiological context. The guide will allow pathologists to report cases of intrahepatic cholangiocarcinoma in a uniform manner that maximises the value of the tissue received to facilitate optimal multidisciplinary patient management.

Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFß Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.

Molecular portraits of patients with intrahepatic cholangiocarcinoma who diverge as rapid progressors or long survivors on chemotherapy.

OBJECTIVE: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. DESIGN: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. RESULTS: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. CONCLUSIONS: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

Association between gut-derived endotoxins and porto-sinusoidal vascular disorder with portal hypertension.

BACKGROUND: Porto-sinusoidal vascular disorder (PSVD) is characterised by lesions involving portal veins and sinusoids in absence of cirrhosis with an unclear pathophysiology. However, its association with immunodeficiency, bowel disorders and abdominal bacterial infections supports the role of altered intestinal permeability and gut-derived endotoxins. The study aimed at assessing the association between serological markers of increased intestinal permeability, pro-aggregating/procoagulant state and liver injury in PSVD and portal hypertension. METHODS: Thirty-three patients with biopsy-proven PSVD and portal hypertension and 33 healthy subjects were submitted to a venous blood sampling for the measurement of zonulin and lipopolysaccharides (LPS) as markers of intestinal permeability, of s-Glycoprotein VI, sP-selectin, ADAMTS13 and von Willebrand factor (vWF), as markers of platelet aggregation and microvascular inflammation, factor VIII and F1 + 2 as markers of hypercoagulability. In 17 PSVD patients, histomorphological and immunohistochemical study on liver biopsies was performed. RESULTS: Compared with controls, PSVD patients had higher levels of LPS, zonulin, vWF, factor VIII and sP-selectin, F1 + 2. ADAMTS13 was reduced. Serum LPS correlated with zonulin, sP-selectin, FVIII and vWF. At histological analysis, PSVD specimens had increased LPS localisation, toll-like receptor-4(TLR4)-positive macrophages and platelet number compared with samples from healthy liver donors. TLR4+ macrophage number correlated with portal inflammation and fibrosis. Sinusoid dilation and capillarisation were observed. PSVD biopsies showed signs of biliary damage and reduced ductular reaction without alteration in Sox9+ cell population. CONCLUSIONS: PSVD patients display an altered intestinal permeability and endotoxemia correlated to a pro-aggregating/procoagulant state; histologically, PSVD was associated with increased TLR4+ cell involvement and platelet clumps within sinusoids. Our study suggests that LPS-TLR4 pathway could contribute to the pathophysiological basis of PSVD with portal hypertension.

A postnatal network of co-hepato/pancreatic stem/progenitors in the biliary trees of pigs and humans.

A network of co-hepato/pancreatic stem/progenitors exists in pigs and humans in Brunner's Glands in the submucosa of the duodenum, in peribiliary glands (PBGs) of intrahepatic and extrahepatic biliary trees, and in pancreatic duct glands (PDGs) of intrapancreatic biliary trees, collectively supporting hepatic and pancreatic regeneration postnatally. The network is found in humans postnatally throughout life and, so far, has been demonstrated in pigs postnatally at least through to young adulthood. These stem/progenitors in vivo in pigs are in highest numbers in Brunner's Glands and in PDGs nearest the duodenum, and in humans are in Brunner's Glands and in PBGs in the hepato/pancreatic common duct, a duct missing postnatally in pigs. Elsewhere in PDGs in pigs and in all PDGs in humans are only committed unipotent or bipotent progenitors. Stem/progenitors have genetic signatures in liver/pancreas-related RNA-seq data based on correlation, hierarchical clustering, differential gene expression and principal component analyses (PCA). Gene expression includes representative traits of pluripotency genes (SOX2, OCT4), endodermal transcription factors (e.g. SOX9, SOX17, PDX1), other stem cell traits (e.g. NCAM, CD44, sodium iodide symporter or NIS), and proliferation biomarkers (Ki67). Hepato/pancreatic multipotentiality was demonstrated by the stem/progenitors' responses under distinct ex vivo conditions or in vivo when patch grafted as organoids onto the liver versus the pancreas. Therefore, pigs are logical hosts for translational/preclinical studies for cell therapies with these stem/progenitors for hepatic and pancreatic dysfunctions.

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential.

BACKGROUND & AIMS: Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. METHODS: Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers. RESULTS: In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. In vitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. In vivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver. CONCLUSIONS: A cell population with clonal growth and organoid formation capability, which has liver differentiation potency in vitro and in vivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver. IMPACT AND IMPLICATIONS: Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Role of ductular reaction and ductular-canalicular junctions in identifying severe primary biliary cholangitis.

Background & Aims: Primary biliary cholangitis (PBC) is a chronic cholangiopathy characterised by immuno-mediated injury of interlobular bile ducts leading to intrahepatic cholestasis and progressive liver fibrosis. PBC histology is characterised by portal inflammation, progressive fibrosis, ductopenia, and the appearance of the so-called ductular reaction. The aim of the present study was to investigate the pathogenetic relevance of ductular reaction in PBC. Methods: Liver biopsies were collected from naïve people with PBC (N = 87). Clinical-serological parameters were obtained at diagnosis and after 1 year of ursodeoxycholic acid (UDCA) treatment. Histological staging was performed on all slides according to multiple scoring systems and criteria for PBC. Liver samples were obtained from Mdr2 -/- mice treated with or without UDCA. Samples were processed for histology, immunohistochemistry, and immunofluorescence. Results: Ductular reaction in people with PBC correlated with the disease stage and liver fibrosis, but not with disease activity; an extensive ductular reaction correlated with serum alkaline phosphatase levels at diagnosis, response to UDCA, and individuals' estimated survival, independently from other histological parameters, including disease stage. In people with PBC, reactive ductules were associated with the establishment of junctions with bile canaliculi and with fibrogenetic cell activation. Consistently, in a mouse model of intrahepatic cholestasis, UDCA treatment was effective in reducing ductular reaction and fibrosis and increasing ductular-canalicular junctions. Conclusions: Extensive ductular reaction outlines a severe histologic phenotype in PBC and is associated with an inadequate therapy response and a worse estimated prognosis. Lay summary: In people affected by primary biliary cholangitis (PBC), the histological appearance of extensive ductular reaction identifies individuals at risk of progressive fibrosis. Ductular reaction at diagnosis correlates with the lack of response to first-line therapy with ursodeoxycholic acid and serves to restore ductular-canalicular junctions in people with PBC. Assessing ductular reaction extension at diagnosis may add valuable information for clinicians.

Effect of Calcium-Sulphate-Bicarbonate Water in a Murine Model of Non-Alcoholic Fatty Liver Disease: A Histopathology Study.

The progression of nonalcoholic fatty liver disease (NAFLD) is associated with alterations of the gut-liver axis. The activation of toll-like receptor 4 (TLR4) pathways by endotoxins, such as lipopolysaccharide (LPS), contributes to liver injury. The aim of the present study was to evaluate the possible beneficial effects of a calcium-sulphate-bicarbonate natural mineral water on the gut-liver axis by evaluating liver and terminal ileum histopathology in a murine model of NAFLD. NAFLD was induced in mice by administrating a methionine-choline-deficient (MCD) diet. The following experimental groups were evaluated: controls (N = 10); MCD+Tap water (MCD; N = 10); MCD+Calcium-sulphate-bicarbonate water (MCD/Wcsb; N = 10). Mice were euthanised after 4 and 8 weeks. Liver and terminal ileum samples were collected. Samples were studied by histomorphology, immunohistochemistry, and immunofluorescence. In mice subjected to the MCD diet, treatment with mineral water improved inflammation and fibrosis, and was associated with a reduced number of activated hepatic stellate cells when compared to MCD mice not treated with mineral water. Moreover, MCD/Wcsb mice showed lower liver LPS localization and less activation of TLR4 pathways compared to the MCD. Finally, Wcsb treatment was associated with improved histopathology and higher occludin positivity in intestinal mucosa. In conclusion, calcium-sulphate-bicarbonate water may exert modulatory activity on the gut-liver axis in MCD mice, suggesting potential beneficial effects on NAFLD.

Patch grafting of organoids of stem/progenitors into solid organs can correct genetic-based disease states.

Patch grafting, a novel strategy for transplantation of stem/progenitor organoids into porcine livers, has been found successful also for organoid transplantation into other normal or diseased solid organs in pigs and mice. Each organoid contained ∼100 cells comprised of biliary tree stem cells (BTSCs), co-hepato/pancreatic stem/progenitors, and partnered with early lineage stage mesenchymal cells (ELSMCs), angioblasts and precursors to endothelia and stellate cells. Patch grafting enabled transplantation into livers or pancreases of ≥108th (pigs) or ≥106th-7th (mice) organoids/patch. Graft conditions fostered expression of multiple matrix-metalloproteinases (MMPs), especially secretory isoforms, resulting in transient loss of the organ's matrix-dictated histological features, including organ capsules, and correlated with rapid integration within a week of organoids throughout the organs and without emboli or ectopic cell distribution. Secondarily, within another week, there was clearance of graft biomaterials, followed by muted expression of MMPs, restoration of matrix-dictated histology, and maturation of donor cells to functional adult fates. The ability of patch grafts of organoids to rescue hosts from genetic-based disease states was demonstrated with grafts of BTSC/ELSMC organoids on livers, able to rescue NRG/FAH-KO mice from type I tyrosinemia, a disease caused by absence of fumaryl acetoacetate hydrolase. With the same grafts, if on pancreas, they were able to rescue NRG/Akita mice from type I diabetes, caused by a mutation in the insulin 2 gene. The potential of patch grafting for cell therapies for solid organs now requires translational studies to enable its adaptation and uses for clinical programs.

Clinical relevance of biomarkers in cholangiocarcinoma: critical revision and future directions.

Cholangiocarcinoma (CCA) is a malignant tumour arising from the biliary system. In Europe, this tumour frequently presents as a sporadic cancer in patients without defined risk factors and is usually diagnosed at advanced stages with a consequent poor prognosis. Therefore, the identification of biomarkers represents an utmost need for patients with CCA. Numerous studies proposed a wide spectrum of biomarkers at tissue and molecular levels. With the present paper, a multidisciplinary group of experts within the European Network for the Study of Cholangiocarcinoma discusses the clinical role of tissue biomarkers and provides a selection based on their current relevance and potential applications in the framework of CCA. Recent advances are proposed by dividing biomarkers based on their potential role in diagnosis, prognosis and therapy response. Limitations of current biomarkers are also identified, together with specific promising areas (ie, artificial intelligence, patient-derived organoids, targeted therapy) where research should be focused to develop future biomarkers.

Therapeutic effects of dexamethasone-loaded hyaluronan nanogels in the experimental cholestasis.

A major function of the intrahepatic biliary epithelium is bicarbonate excretion in bile. Recent reports indicate that budesonide, a corticosteroid with high receptor affinity and hepatic first pass clearance, increases the efficacy of ursodeoxycholic acid, a choleretic agent, in primary biliary cholangitis patients. We have previously reported that bile ducts isolated from rats treated with dexamethasone or budesonide showed an enhanced activity of the Na+/H+ exchanger isoform 1 (NHE1) and Cl-/HCO3- exchanger protein 2 (AE2) . Increasing the delivery of steroids to the liver may result in three beneficial effects: increase in the choleresis, treatment of the autoimmune or inflammatory liver injury and reduction of steroids' systemic harmful effects. In this study, the steroid dexamethasone was loaded into nanohydrogels (or nanogels, NHs), in order to investigate corticosteroid-induced increased activities of transport processes driving bicarbonate excretion in the biliary epithelium (NHE-1 isoform) and to evaluate the effects of dexamethasone-loaded NHs (NHs/dex) on liver injury induced by experimental cholestatis. Our results showed that NHs and NHs/dex do not reduce cell viability in vitro in human cholangiocyte cell lines. Primary and immortalized human cholangiocytes treated with NHs/dex show an increase in the functional marker expression of NHE1 cholangiocytes compared to control groups. A mouse model of cholangiopathy treated with NHs/dex shows a reduction in markers of hepatocellular injury compared to control groups (NHs, dex, or sham group). In conclusion, we believe that the NHs/dex formulation is a suitable candidate to be investigated in preclinical models of cholangiopathies.

Islet Regeneration and Pancreatic Duct Glands in Human and Experimental Diabetes.

Contrasting evidence is present regarding the contribution of stem/progenitor cell populations to pancreatic regeneration in diabetes. Interestingly, a cell compartment with stem/progenitor cell features has been identified in the pancreatic duct glands (PDGs). The aims of the present study were to evaluate pancreatic islet injury and regeneration, and the participation of the PDG compartment in type 2 diabetic mellitus (T2DM) and in an experimental model of diabetes. Human pancreata were obtained from normal (N = 5) or T2DM (N = 10) cadaveric organ donors. Experimental diabetes was generated in mice by intraperitoneal injection of 150 mg/kg of streptozotocin (STZ, N = 10); N = 10 STZ mice also received daily intraperitoneal injections of 100 µg of human recombinant PDX1 peptide (STZ + PDX1). Samples were examined by immunohistochemistry/immunofluorescence or RT-qPCR. Serum glucose and c-peptide levels were measured in mice. Islets in T2DM patients showed ß-cell loss, signs of injury and proliferation, and a higher proportion of central islets. PDGs in T2DM patients had a higher percentage of proliferating and insulin+ or glucagon+ cells compared to controls; pancreatic islets could be observed within pancreatic duct walls of T2DM patients. STZ mice were characterized by reduced islet area compared to controls. PDX1 treatment increased islet area and the percentage of central islets compared to untreated STZ mice but did not revert diabetes. In conclusion, T2DM patients show signs of pancreatic islet regeneration and involvement of the PDG niche. PDX1 administration could support increased endocrine pancreatic regeneration in STZ. These findings contribute to defining the role and participation of stem/progenitor cell compartments within the pancreas.

Persistent biliary hypoxia and lack of regeneration are key mechanisms in the pathogenesis of posttransplant nonanastomotic strictures.

BACKGROUND AND AIMS: Nonanastomotic biliary strictures (NAS) are a major cause of morbidity after orthotopic liver transplantation (OLT). Although ischemic injury of peribiliary glands (PBGs) and peribiliary vascular plexus during OLT has been associated with the later development of NAS, the exact underlying mechanisms remain unclear. We hypothesized that bile ducts of patients with NAS suffer from ongoing biliary hypoxia and lack of regeneration from PBG stem/progenitor cells. APPROACH AND RESULTS: Forty-two patients, requiring retransplantation for either NAS (n = 18), hepatic artery thrombosis (HAT; n = 13), or nonbiliary graft failure (controls; n = 11), were included in this study. Histomorphological analysis of perihilar bile ducts was performed to assess differences in markers of cell proliferation and differentiation in PBGs, microvascular density (MVD), and hypoxia. In addition, isolated human biliary tree stem cells (hBTSCs) were used to examine exo-metabolomics during in vitro differentiation toward mature cholangiocytes. Bile ducts of patients with NAS or HAT had significantly reduced indices of PBG mass, cellular proliferation and differentiation (mucus production, secretin receptor expression, and primary cilia), reduced MVD, and increased PBG apoptosis and hypoxia marker expression, compared to controls. Metabolomics of hBTSCs during in vitro differentiation toward cholangiocytes revealed a switch from a glycolytic to oxidative metabolism, indicating the need for oxygen. CONCLUSIONS: NAS are characterized by a microscopic phenotype of chronic biliary hypoxia attributed to loss of microvasculature, resulting in reduced proliferation and differentiation of PBG stem/progenitor cells into mature cholangiocytes. These findings suggest that persistent biliary hypoxia is a key mechanism underlying the development of NAS after OLT.

Patch grafting, strategies for transplantation of organoids into solid organs such as liver.

Epithelial cell therapies have been at an impasse because of inefficient methods of transplantation to solid organs. Patch grafting strategies were established enabling transplantation of ≥107th organoids/patch of porcine GFP+ biliary tree stem/progenitors into livers of wild type hosts. Grafts consisted of organoids embedded in soft (~100 Pa) hyaluronan hydrogels, both prepared in serum-free Kubota's Medium; placed against target sites; covered with a silk backing impregnated with more rigid hyaluronan hydrogels (~700 Pa); and use of the backing to tether grafts with sutures or glue to target sites. Hyaluronan coatings (~200-300 Pa) onto the serosal surface of the graft served to minimize adhesions with neighboring organs. The organ's clearance of hyaluronans enabled restoration of tissue-specific paracrine and systemic signaling, resulting in return of normal hepatic histology, with donor parenchymal cells uniformly integrated amidst host cells and that had differentiated to mature hepatocytes and cholangiocytes. Grafts containing donor mature hepatocytes, partnered with endothelia, and in the same graft biomaterials as for stem/progenitor organoids, did not engraft. Engraftment occurred if porcine liver-derived mesenchymal stem cells (MSCs) were co-transplanted with donor mature cells. RNA-seq analyses revealed that engraftment correlated with expression of matrix-metalloproteinases (MMPs), especially secreted isoforms that were found expressed strongly by organoids, less so by MSCs, and minimally, if at all, by adult cells. Engraftment with patch grafting strategies occurred without evidence of emboli or ectopic cell distribution. It was successful with stem/progenitor organoids or with cells with a source(s) of secreted MMP isoforms and offers significant potential for enabling cell therapies for solid organs.

Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

Molecular Landscape and Therapeutic Strategies in Cholangiocarcinoma: An Integrated Translational Approach towards Precision Medicine.

Cholangiocarcinomas (CCAs) are heterogeneous biliary tract malignancies with dismal prognosis, mainly due to tumor aggressiveness, late diagnosis, and poor response to current therapeutic options. High-throughput technologies have been used as a fundamental tool in unveiling CCA molecular landscape, and several molecular classifications have been proposed, leading to various targeted therapy trials. In this review, we aim to analyze the critical issues concerning the status of precision medicine in CCA, discussing molecular signatures and clusters, related to both anatomical classification and different etiopathogenesis, and the latest therapeutic strategies. Furthermore, we propose an integrated approach comprising the CCA molecular mechanism, pathobiology, clinical and histological findings, and treatment perspectives for the ultimate purpose of improving the methods of patient allocations in clinical trials and the response to personalized therapies.

Accuracy of Transient Elastography in Assessing Fibrosis at Diagnosis in Naïve Patients With Primary Biliary Cholangitis: A Dual Cut-Off Approach.

BACKGROUND AND AIMS: Liver fibrosis holds a relevant prognostic meaning in primary biliary cholangitis (PBC). Noninvasive fibrosis evaluation using vibration-controlled transient elastography (VCTE) is routinely performed. However, there is limited evidence on its accuracy at diagnosis in PBC. We aimed to estimate the diagnostic accuracy of VCTE in assessing advanced fibrosis (AF) at disease presentation in PBC. APPROACH AND RESULTS: We collected data from 167 consecutive treatment-naïve PBC patients who underwent liver biopsy (LB) at diagnosis at six Italian centers. VCTE examinations were completed within 12 weeks of LB. Biopsies were scored by two blinded expert pathologists, according to the Ludwig system. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROCs) for AF (Ludwig stage ≥III). Effects of biochemical and clinical parameters on liver stiffness measurement (LSM) were appraised. The derivation cohort consisted of 126 patients with valid LSM and LB; VCTE identified patients with AF with an AUROC of 0.89. LSM cutoffs ≤6.5 and >11.0 kPa enabled to exclude and confirm, respectively, AF (negative predictive value [NPV] = 0.94; positive predictive value [PPV] = 0.89; error rate = 5.6%). These values were externally validated in an independent cohort of 91 PBC patients (NPV = 0.93; PPV = 0.89; error rate = 8.6%). Multivariable analysis found that the only parameter affecting LSM was fibrosis stage. No association was found with BMI and liver biochemistry. CONCLUSIONS: In a multicenter study of treatment-naïve PBC patients, we identified two cutoffs (LSM ≤6.5 and >11.0 kPa) able to discriminate at diagnosis the absence or presence, respectively, of AF in PBC patients, with external validation. In patients with LSM between these two cutoffs, VCTE is not reliable and liver biopsy should be evaluated for accurate disease staging. BMI and liver biochemistry did not affect LSMs.

Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.