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Intrahepatic cholangiocarcinoma (iCCA) denotes a rare, highly malignant, and heterogeneous class of primary liver adenocarcinomas exhibiting phenotypic characteristics of cholangiocyte differentiation. Among the distinctive pathological features of iCCA, one that differentiates the most common macroscopic subtype (eg, mass-forming type) of this hepatic tumor from conventional hepatocellular carcinoma, is a prominent desmoplastic reaction manifested as a dense fibro-collagenous-enriched tumor stroma. Cancer-associated fibroblasts (CAFs) represent the most abundant mesenchymal cell type in the desmoplastic reaction. Although the protumor effects of CAFs in iCCA have been increasingly recognized, more recent cell lineage tracing studies, advanced single-cell RNA sequencing, and expanded biomarker analyses have provided new awareness into their ontogeny, as well as underscored their biological complexity as reflected by the presence of multiple subtypes. In addition, evidence has been described to support CAFs' potential to display cancer-restrictive roles, including immunosuppression. However, CAFs also play important roles in facilitating metastasis, as exemplified by lymph node metastasis and peritoneal carcinomatosis, which are common in iCCA. Herein, the authors provide a timely appraisal of the origins and phenotypic and functional complexity of CAFs in iCCA, together with providing mechanistic insights into lymphangiogenesis and peritoneal metastasis relevant to this lethal human cancer.
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BACKGROUND & AIMS: Mechanisms and clinical impact of portal microthrombosis featuring severe COVID-19 are unknown. Intrapulmonary vascular dilation (IPVD)-related hypoxia has been described in severe liver diseases. We hypothesized that portal microthrombosis is associated with IPVD and fatal respiratory failure in COVID-19. METHODS: Ninety-three patients who died from COVID-19, were analysed for portal microvascular damage (histology), IPVD (histology and chest-computed tomography, CT), and hypoxemia (arterial blood gas). Seventeen patients who died from COVID-19-unrelated pneumonia served as controls. Vascular lesions and microthrombi were phenotyped for endothelial (vWF) and pericyte (αSMA/PDGFR-ß) markers, tissue factor (TF), viral spike-protein and nucleoprotein (SP, NP), fibrinogen, platelets (CD41a). Viral particles in vascular cells were assessed by transmission electron microscopy (TEM). Cultured pericytes were infected with SARS-CoV-2 to measure TF expression and tubulisation of human pulmonary microvascular endothelial cells (HPMEC) was assessed upon vWF treatment. RESULTS: IPVD was present in 16/66 COVID-19 patients with both liver and lung histology, with a younger age (62 vs 78yo), longer illness (25 vs 14 days), worsening hypoxemia (PaO2/FiO2 from 209 to 89), and more ventilatory support (63 vs 22%) compared to COVID-19/Non-IPVD. IPVD, absent in controls, were confirmed by chest-CT. COVID-19/IPVD liver histology showed portal microthrombosis in >82.5% of portal areas, with a thicker wall of αSMA/PDGFR-ß+/ SP+/NP+ pericytes compared with COVID-19/Non-IPVD. Thrombosed portal venules correlated with αSMA+ area, whereas infected SP+/NP+ pericytes expressed TF. SARS-CoV-2 viral particles were observed in portal pericytes. In-vitro SARS-CoV-2 infection of pericytes up-regulated TF and induced endothelial cells to overexpress vWF, which expanded HPMEC tubules. CONCLUSIONS: SARS-CoV-2 infection of liver pericytes elicits a local procoagulant response associated with extensive portal microthrombosis, IPVD and worsening respiratory failure in fatal COVID-19. IMPACT AND IMPLICATIONS: Vascular involvement of the liver represents a serious complication of COVID-19 infection that must be considered in the work-up of patients with long-lasting and progressively worsening respiratory failure, as it may associate with the development of intrapulmonary vascular dilations. This clinical picture is associated with a pro-coagulant phenotype of portal venule pericytes, which is induced by SARS-CoV-2 infection of pericytes. Both observations provide a model that may apply, at least in part, to other vascular disorders of the liver, featuring obliterative portal venopathy, similarly characterized at the clinical level by development of hypoxemia and at the histological level, by phlebosclerosis and reduced caliber of the portal vein branches in the absence of cirrhosis. Moreover, our findings bring light to an as yet overlooked player of thrombosis pathophysiology, i.e. pericytes, which may provide novel therapeutic tools to halt prothrombotic mechanisms.
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BACKGROUND AND AIMS: We have previously shown in a model of hepatic ischaemia/reperfusion injury that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) restores reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), an inverse modulator of metalloproteases (MMPs) and inhibitor of the sheddases ADAM10 and ADAM17 involved in inflammation and fibrogenesis. Here, the effects of FXR agonists OCA and INT-787 on hepatic levels of RECK, MMPs, ADAM10 and ADAM17 were compared in a diet-induced ob/ob mouse model of non-alcoholic steatohepatitis (NASH). METHODS: Lep ob/ob NASH mice fed a high-fat diet (HFD) or control diet (CD) for 9 weeks (wks) were treated with OCA or INT-787 0.05% dosed via HFD admixture (30 mg/kg/day) or HFD for further 12 wks. Serum alanine transaminase (ALT) and inflammatory cytokines, liver RECK, MMP-2 and MMP-9 activity as well as ADAM10, ADAM17, collagen deposition (Sirius red), hepatic stellate cell activation (α-SMA) and pCK+ reactive biliary cells were quantified. RESULTS: Only INT-787 significantly reduced serum ALT, IL-1ß and TGF-ß. A downregulation of RECK expression and protein levels observed in HFD groups (at 9 and 21 wks) was counteracted by both OCA and INT-787. HFD induced a significant increase in liver MMP-2 and MMP-9; OCA administration reduced both MMP-2 and MMP-9 while INT-787 markedly reduced MMP-2 expression. OCA and INT-787 reduced both ADAM10 and ADAM17 expression and number of pCK+ cells. INT-787 was superior to OCA in decreasing collagen deposition and α-SMA levels. CONCLUSION: INT-787 is superior to OCA in controlling specific cell types and clinically relevant anti-inflammatory and antifibrotic molecular mechanisms in NASH.
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Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/farmacología , Hígado/metabolismo , Ácido Quenodesoxicólico/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Dieta Alta en Grasa/efectos adversos , Colágeno/metabolismo , Colágeno/farmacologíaRESUMEN
Cholangiocarcinoma remains an aggressive and deadly malignancy that is often diagnosed late. Intrinsic tumour characteristics and the growth pattern of cancer cells contribute to the challenges of diagnosis and chemoresistance. However, establishing an early and accurate diagnosis, and in some instances identifying targetable changes, has the potential to impact survival. Primary sclerosing cholangitis, a chronic cholangiopathy prodromal to the development of a minority of cholangiocarcinomas, poses a particular diagnostic challenge. We present our diagnostic and theranostic approach to the initial evaluation of cholangiocarcinomas, focusing on extrahepatic cholangiocarcinoma. This involves a multipronged strategy incorporating advanced imaging, endoscopic methods, multiple approaches to tissue sampling, and molecular markers. We also provide an algorithm for the sequential use of these tools.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Colangitis Esclerosante , Humanos , Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/patología , Endoscopía Gastrointestinal , Biomarcadores , Conductos Biliares Intrahepáticos/patología , Colangitis Esclerosante/diagnósticoRESUMEN
Congenital hepatic fibrosis (CHF), a genetic cholangiopathy characterized by fibropolycystic changes in the biliary tree, is caused by mutations in the PKHD1 gene, leading to defective fibrocystin (FPC), changes in planar cell polarity (PCP) and increased ß-catenin-dependent chemokine secretion. In this study, we aimed at understanding the role of Scribble (a protein involved in PCP), Yes-associated protein (YAP), and ß-catenin in the regulation of the fibroinflammatory phenotype of FPC-defective cholangiocytes. Immunohistochemistry showed that compared with wild type (WT) mice, in FPC-defective (Pkhd1del4/del4 ) mice nuclear expression of YAP/TAZ in cystic cholangiocytes, significantly increased and correlated with connective tissue growth factor (CTGF) expression and pericystic fibrosis, while Scribble expression on biliary cyst cells was markedly decreased. Cholangiocytes isolated from WT mice showed intense Scribble immunoreactivity at the membrane, but minimal nuclear expression of YAP, which conversely increased, together with CTGF, after small interfering RNA (siRNA) silencing of Scribble. In FPC-defective cholangiocytes, inhibition of YAP nuclear import reduced ß-catenin nuclear expression, and CTGF, integrin ß6, CXCL1, and CXCL10 mRNA levels, whereas inhibition of ß-catenin signaling did not affect nuclear translocation of YAP. Notably, siRNA silencing of Scribble and YAP in WT cholangiocytes mimics the fibroinflammatory changes of FPC-defective cholangiocytes. Conditional deletion of ß-catenin in Pkhd1del4/del4 mice reduced cyst growth, inflammation and fibrosis, without affecting YAP nuclear expression. In conclusion, the defective anchor of Scribble to the membrane facilitates the nuclear translocation of YAP and ß-catenin with gain of a fibroinflammatory phenotype. The Scribble/YAP/ß-catenin axis is a critical factor in the sequence of events linking the genetic defect to fibrocystic trait of cholangiocytes in CHF.
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Quistes , beta Catenina , Animales , Modelos Animales de Enfermedad , Enfermedades Genéticas Congénitas , Péptidos y Proteínas de Señalización Intracelular , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Ratones , ARN Interferente Pequeño , Receptores de Superficie Celular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Oncolytic viruses (OVs) are promising therapeutics for tumors with a poor prognosis. An OV based on herpes simplex virus type 1 (oHSV-1), talimogene laherparepvec (T-VEC), has been recently approved by the Food and Drug Administration (FDA) and by the European Medicines Agency (EMA) for the treatment of unresectable melanoma. T-VEC, like most OVs, is administered via intratumoral injection, underlining the unresolved problem of the systemic delivery of the oncolytic agent for the treatment of metastases and deep-seated tumors. To address this drawback, cells with a tropism for tumors can be loaded ex vivo with OVs and used as carriers for systemic oncolytic virotherapy. Here, we evaluated human monocytes as carrier cells for a prototype oHSV-1 with a similar genetic backbone as T-VEC. Many tumors specifically recruit monocytes from the bloodstream, and autologous monocytes can be obtained from peripheral blood. We demonstrate here that oHSV-1-loaded primary human monocytes migrated in vitro towards epithelial cancer cells of different origin. Moreover, human monocytic leukemia cells selectively delivered oHSV-1 to human head-and-neck xenograft tumors grown on the chorioallantoic membrane (CAM) of fertilized chicken eggs after intravascular injection. Thus, our work shows that monocytes are promising carriers for the delivery of oHSV-1s in vivo, deserving further investigation in animal models.
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Herpesvirus Humano 1 , Melanoma , Viroterapia Oncolítica , Virus Oncolíticos , Embrión de Pollo , Animales , Humanos , Herpesvirus Humano 1/genética , Melanoma/terapia , Pollos , Monocitos , Membrana Corioalantoides , Virus Oncolíticos/genéticaRESUMEN
Breast cancer (BC) is the most frequent cancer and the second leading cause of death in women. A typical feature of BC cells is the metabolic shift toward increased glycolysis, which has become an interesting therapeutic target for metabolic drugs such as metformin (MET). Recently, the administration of the antihypertensive syrosingopine (SYRO) in combination with MET has shown a synergistic effect toward a variety of cancers. However, a fundamental need remains, which is the development of in vivo biomarkers that are able to detect early clinical response. In this study, we exploited a triple-negative murine BC cell line (4T1) and a metastatic ER+ murine BC cell line (TS/A) in order to investigate, in vivo, the early response to treatment, based on MET and/or SYRO administration, evaluating [18F]FDG and [18F]FLT as potential biomarkers via PET/CT. The study provides evidence that SYRO plus MET has a synergistic effect on tumor growth inhibition in both 4T1 and TS/A experimental models and has showed the highest efficacy on the TNBC xenograft mice (4T1) via the expression reduction in the lactate transporter MCT4 and in the epithelial-mesenchymal transition biomarker Snail, promoting its potential application in therapy settings. In addition, the selective reduction in the [18F]FLT tumor uptake (at 7 dd), observed in the SYRO plus MET treated mice in comparison with the vehicle group, suggests that this radiotracer could be potentially used as a biomarker for the early detection of therapy response, in both evaluated xenografts models.
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Metformina , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Animales , Ratones , Fluorodesoxiglucosa F18 , Tomografía Computarizada por Tomografía de Emisión de Positrones , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Biomarcadores , EstrógenosRESUMEN
Metabolic syndrome (MetS) is a common condition closely associated with non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH). Recent meta-analyses show that MetS can be prodromal to intrahepatic cholangiocarcinoma (iCCA) development, a liver tumor with features of biliary differentiation characterized by dense extracellular matrix (ECM) deposition. Since ECM remodeling is a key event in the vascular complications of MetS, we aimed at evaluating whether MetS patients with iCCA present qualitative and quantitative changes in the ECM able to incite biliary tumorigenesis. In 22 iCCAs with MetS undergoing surgical resection, we found a significantly increased deposition of osteopontin (OPN), tenascin C (TnC), and periostin (POSTN) compared to the matched peritumoral areas. Moreover, OPN deposition in MetS iCCAs was also significantly increased when compared to iCCA samples without MetS (non-MetS iCCAs, n = 44). OPN, TnC, and POSTN significantly stimulated cell motility and the cancer-stem-cell-like phenotype in HuCCT-1 (human iCCA cell line). In MetS iCCAs, fibrosis distribution and components differed quantitatively and qualitatively from non-MetS iCCAs. We therefore propose overexpression of OPN as a distinctive trait of MetS iCCA. Since OPN stimulates malignant properties of iCCA cells, it may provide an interesting predictive biomarker and a putative therapeutic target in MetS patients with iCCA.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Síndrome Metabólico , Humanos , Neoplasias de los Conductos Biliares/complicaciones , Conductos Biliares Intrahepáticos/metabolismo , Colangiocarcinoma/complicaciones , Síndrome Metabólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Osteopontina/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide, and affects 25% of the population in Western countries. NAFLD is the hepatic manifestation of the metabolic syndrome, linked to insulin resistance, which is the common pathogenetic mechanism. In approximately 40% of NAFLD patients, steatosis is associated with necro-inflammation and fibrosis, resulting in nonalcoholic steatohepatitis (NASH), a severe condition that may progress to cirrhosis and liver cancer. Although the hepatocyte represents the main target of the disease, involvement of the bile ducts occurs in a subset of patients with NASH, and is characterized by ductular reaction and activation of the progenitor cell compartment, which incites portal fibrosis and disease progression. We aim to dissect the multiple biological effects that adipokines and metabolic alterations exert on cholangiocytes to derive novel information on the mechanisms driven by insulin resistance, which promote fibro-inflammation and carcinogenesis in NASH.
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Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico , Conductos Biliares/metabolismo , Conductos Biliares/patología , Células Epiteliales/metabolismo , Humanos , Inflamación/metabolismo , Hígado/metabolismo , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
BACKGROUND & AIMS: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. METHODS: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. RESULTS: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. CONCLUSION: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. LAY SUMMARY: Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Animales , Neoplasias de los Conductos Biliares/etiología , Conductos Biliares Intrahepáticos , Línea Celular Tumoral , Colangiocarcinoma/etiología , Humanos , Ratones , Modelos Teóricos , Proteómica , Transducción de SeñalRESUMEN
Benign biliary tumor are common lesions that are often an incidental finding in subjects who undergo medical imaging tests for other conditions. Most are true neoplasms while few result from reactive or malformative proliferation. Benign tumors have no clinical consequences, although the premalignant nature or potential for malignant transformation is of concern in some cases. The main practical problem for pathologists is the need to differentiate them from malignant biliary tumours, which is not always straightforward.Premalignant lesions of the bile duct have been described, although their incidence has been poorly characterized. These lesions include biliary mucinous cystic neoplasms, intraductal papillary neoplasms of the bile duct, and biliary intraepithelial neoplasia. In this article, histopathology of benign biliary tumors and biliary tumor precursors is discussed, with a focus on the main diagnostic criteria.
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Neoplasias de los Conductos Biliares , Carcinoma in Situ , Lesiones Precancerosas , Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/epidemiología , Diagnóstico por Imagen , Humanos , Patólogos , Lesiones Precancerosas/epidemiologíaRESUMEN
Liver cancer represents the third leading cause of cancer-related death worldwide. Cholangiocarcinoma (CCA) is the second most common type of liver cancer after hepatocellular carcinoma, accounting for 10-15% of all primary liver malignancies. Both the incidence and mortality of CCA have been steadily increasing during the last decade. Moreover, most CCAs are diagnosed at an advanced stage, when therapeutic options are very limited.CCA may arise from any tract of the biliary system and it is classified into intrahepatic, perihilar, and distal CCA, according to the anatomical site of origin. This topographical classification also reflects distinct genetic and histological features, risk factors, and clinical outcomes. This review focuses on histopathology of CCA, its differential diagnoses, and its diagnostic pitfalls.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/epidemiología , Conductos Biliares Intrahepáticos , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/epidemiología , HumanosRESUMEN
Autoimmune cholestatic liver diseases are rare hepato-biliary disorders characterized by a progressive, inflammatory destruction of bile ducts. Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are the main autoimmune cholestatic liver diseases. Both may evolve into secondary biliary cirrhosis and its complications. Therapeutic options are limited and liver transplantation remains the only definitive treatment for PBC and PSC.Most PBC and PSC patients have a typical presentation, which does not require liver biopsy. However, in routine clinical practice, important variants or specific subgroups that benefit from liver biopsy for proper management may be observed. Herein, we provide a general overview of clinical and pathological characteristic of PBC and PSC, highlighting the most important features for routine diagnostic practice.
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Enfermedades Autoinmunes , Colangitis Esclerosante , Cirrosis Hepática Biliar , Enfermedades Autoinmunes/complicaciones , Colangitis Esclerosante/complicaciones , Colangitis Esclerosante/diagnóstico , Colangitis Esclerosante/epidemiología , Humanos , Cirrosis Hepática Biliar/complicaciones , Cirrosis Hepática Biliar/diagnósticoRESUMEN
The extracellular matrix is a highly reactive scaffold formed by a wide array of multifunctional molecules, encompassing collagens and noncollagenous glycoproteins, proteoglycans, glycosaminoglycans, and polysaccharides. Besides outlining the tissue borders, the extracellular matrix profoundly regulates the behavior of resident cells by transducing mechanical signals, and by integrating multiple cues derived from the microenvironment. Evidence is mounting that changes in the biostructure of the extracellular matrix are instrumental for biliary repair. Following biliary damage and eventually, malignant transformation, the extracellular matrix undergoes several quantitative and qualitative modifications, which direct interactions among hepatic progenitor cells, reactive ductular cells, activated myofibroblasts and macrophages, to generate the ductular reaction. Herein, we will give an overview of the main molecular factors contributing to extracellular matrix remodeling in cholangiopathies. Then, we will discuss the structural alterations in terms of biochemical composition and physical stiffness featuring the "desmoplastic matrix" of cholangiocarcinoma along with their pro-oncogenic effects.
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Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/patología , Matriz Extracelular/metabolismo , Hígado/patología , Sistema Biliar/patología , HumanosRESUMEN
BACKGROUND & AIMS: In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. METHODS: Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo. RESULTS: In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRß inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases. CONCLUSION: PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis. LAY SUMMARY: Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.
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Neoplasias de los Conductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Hígado/patología , Linfangiogénesis/efectos de los fármacos , Linfocinas/metabolismo , Linfocinas/farmacología , Miofibroblastos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Neoplasias de los Conductos Biliares/patología , Fibroblastos Asociados al Cáncer/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/patología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Xenoinjertos , Humanos , Mesilato de Imatinib/farmacología , Masculino , Ratones , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Endogámicas F344 , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor C de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
In end-stage chronic kidney disease, the option of organ transplantation is limited because of the scarce availability of kidneys. The combination of stem cell research, regenerative medicine, and tissue engineering seems a promising approach to produce new transplantable kidneys. Currently, the possibility to repopulate naturally obtained scaffolds with cells of different sources is advancing. Our aim was to test, for the first time, whether the nephrosphere (NS) cells, composed by renal stem/progenitor-like cells, were able to repopulate different nephron portions of renal extracellular matrix scaffolds obtained after decellularization of human renal tissue slices. Our decellularization protocol enabled us to obtain a completely acellular renal scaffold while maintaining the extracellular matrix structure and composition in terms of collagen IV, laminin, and fibronectin. NS cells, cultured on decellularized renal scaffolds with basal medium, differentiated into proximal and distal tubules as well as endothelium, as highlighted by histology and by the specific expression of epithelial cytokeratin 8.18, proximal tubular CD10, distal tubular cytokeratin 7, and endothelial von Willebrand factor markers. Endothelial medium promoted the differentiation toward the endothelium, whereas epithelial medium promoted the differentiation toward the epithelium. NS cells seem to be a good tool for scaffold repopulation, paving the way for experimental investigations focused on whole-kidney reconstruction.
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Diferenciación Celular/fisiología , Riñón/citología , Andamios del Tejido , Anciano , Anciano de 80 o más Años , Células Cultivadas , Colágeno Tipo IV/metabolismo , Matriz Extracelular/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Congenital hepatic fibrosis (CHF), a genetic disease caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, encoding for the protein fibrocystin/polyductin complex, is characterized by biliary dysgenesis, progressive portal fibrosis, and a protein kinase A-mediated activating phosphorylation of ß-catenin at Ser675. Biliary structures of Pkhd1del4/del4 mice, a mouse model of CHF, secrete chemokine (C-X-C motif) ligand 10 (CXCL10), a chemokine able to recruit macrophages. The aim of this study was to clarify whether CXCL10 plays a pathogenetic role in disease progression in CHF/Caroli disease and to understand the mechanisms leading to increased CXCL10 secretion. We demonstrate that treatment of Pkhd1del4/del4 mice for 3 months with AMG-487, an inhibitor of CXC chemokine receptor family 3, the cognate receptor of CXCL10, reduces the peribiliary recruitment of alternative activated macrophages (cluster of differentiation 45+ F4/80+ cells), spleen size, liver fibrosis (sirius red), and cyst growth (cytokeratin 19-positive area), consistent with a pathogenetic role of CXCL10. Furthermore, we show that in fibrocystin/polyductin complex-defective cholangiocytes, isolated from Pkhd1del4/del4 mice, CXCL10 production is mediated by Janus kinase/signal transducer and activator of transcription 3 in response to interleukin 1beta (IL-1ß) and ß-catenin. Specifically, IL-1ß promotes signal transducer and activator of transcription 3 phosphorylation, whereas ß-catenin promotes its nuclear translocation. Increased pro-IL-1ß was regulated by nuclear factor kappa-light-chain-enhancer of activated B cells, and increased secretion of active IL-1ß was mediated by the activation of Nod-like receptors, pyrin domain containing 3 inflammasome (increased expression of caspase 1 and Nod-like receptors, pyrin domain containing 3). CONCLUSION: In fibrocystin/polyductin complex-defective cholangiocytes, ß-catenin and IL-1ß are responsible for signal transducer and activator of transcription 3-dependent secretion of CXCL10; in vivo experiments show that the CXCL10/CXC chemokine receptor family 3 axis prevents the recruitment of macrophages, reduces inflammation, and halts the progression of the disease; the increased production of IL-1ß highlights the autoinflammatory nature of CHF and may open novel therapeutic avenues. (Hepatology 2018;67:1903-1919).
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Quimiocina CXCL10/metabolismo , Enfermedades Genéticas Congénitas/metabolismo , Interleucina-1beta/metabolismo , Cirrosis Hepática/metabolismo , beta Catenina/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Citometría de Flujo , Inmunohistoquímica , Hígado/metabolismo , Hígado/patología , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR3/metabolismo , Transducción de SeñalRESUMEN
The high mortality rate of cholangiocarcinoma (CCA) is due, in part, to the lack of non-invasive approaches able to accurately detect this silent tumour at early stages, when therapeutic options can be potentially curative or may at least increase the overall survival of patients. The fact that the majority of CCA tumours are not linked to any known aetiological factor highly compromises the monitoring of patients at risk for tumour development and also their early diagnosis. Combination of clinical/biochemical features, imaging techniques and analysis of non-specific tumour biomarkers in serum are commonly used to help in the diagnosis of CCA, but tumour biopsy is usually required to confirm the diagnosis. Moreover, no prognostic biomarkers are currently used in the clinical setting, deserving more innovative research, and international validation and consensus. Important efforts have been made in the last few years to identify accurate non-invasive biomarkers, by using innovative techniques and high-throughput omics technologies. This review summarizes and discusses the advances in the investigation of novel diagnostic and prognostic biomarkers in CCA and envisions the future directions in this field of research.
Asunto(s)
Neoplasias de los Conductos Biliares/diagnóstico , Biomarcadores de Tumor/análisis , Colangiocarcinoma/diagnóstico , Neoplasias de los Conductos Biliares/mortalidad , Neoplasias de los Conductos Biliares/patología , Biopsia , Colangiocarcinoma/mortalidad , Colangiocarcinoma/patología , Diagnóstico Precoz , Humanos , Pronóstico , ProteómicaRESUMEN
Prognosis of cholangiocarcinoma, a devastating liver epithelial malignancy characterized by early invasiveness, remains very dismal, though its incidence has been steadily increasing. Evidence is mounting that in cholangiocarcinoma, tumor epithelial cells establish an intricate web of mutual interactions with multiple stromal components, largely determining the pervasive behavior of the tumor. The main cellular components of the tumor microenvironment (i.e. myofibroblasts, macrophages, lymphatic endothelial cells), which has been recently termed as 'tumor reactive stroma', are recruited and activated by neoplastic cells, and in turn, deleteriously mold tumor behavior by releasing a huge variety of paracrine signals, including cyto/chemokines, growth factors, morphogens and proteinases. An abnormally remodeled and stiff extracellular matrix favors and supports these cell interactions. Although the mechanisms responsible for the generation of tumor reactive stroma are largely uncertain, hypoxia presumably plays a central role. In this review, we will dissect the intimate relationship among the different cell elements cooperating within this complex 'ecosystem', with the ultimate goal to pave the way for a deeper understanding of the mechanisms underlying cholangiocarcinoma aggressiveness, and possibly, to foster the development of innovative, combinatorial therapies aimed at halting tumor progression. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.
Asunto(s)
Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/patología , Células Epiteliales/patología , Comunicación Paracrina , Células del Estroma/patología , Animales , Conductos Biliares/citología , Conductos Biliares/patología , Hipoxia de la Célula , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Invasividad Neoplásica/patología , Transducción de Señal , Células del Estroma/metabolismo , Microambiente TumoralRESUMEN
The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangitis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.