Liver Matrix in Benign and Malignant Biliary Tract Disease

Luca Fabris, MD, PhD1,2 Massimiliano Cadamuro, PhD1 Silvia Cagnin, MD1 Mario Strazzabosco, MD, PhD2 Gregory J. Gores, MD, PhD3

1 Department of Molecular Medicine, University of Padua, Padua, Italy Address for correspondence Luca Fabris, MD, PhD, Department of 2 Liver Center, Department of Medicine, Yale University, New Haven, Molecular Medicine, University of Padua, Via A. Gabelli 63, Connecticut Padua 35121, Italy (e-mail: [email protected]). 3 Division of Gastroenterology and Hepatology and the Mayo Clinic Center for Cell Signaling in Gastroenterology, Mayo Clinic, Rochester, Michigan

Semin Liver Dis

Abstract The extracellular matrix is a highly reactive scaffold formed by a wide array of multifunctional molecules, encompassing collagens and noncollagenous glycopro- teins, 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 Keywords quantitative and qualitative modifications, which direct interactions among hepatic ► basement membrane progenitor cells, reactive ductular cells, activated myofibroblasts and macrophages, to ► cholangiocytes generate the ductular reaction. Herein, we will give an overview of the main molecular ► ductular reaction factors contributing to extracellular matrix remodeling in cholangiopathies. Then, we ► tumor reactive will discuss the structural alterations in terms of biochemical composition and physical stroma stiffness featuring the “desmoplastic matrix” of cholangiocarcinoma along with their ► biliary fibrosis pro-oncogenic effects.

Architectural organization of organs is based on a three- The ECM can be divided into two main components, the dimensional scaffold holding together different cell types basement membrane (BM) and the interstitial matrix, which and enabling them to communicate. This extracellular matrix variably support cell polarization and migration. The BM is a (ECM) is not a simple inert substrate, but rather, a highly finely assembled membrane, underlining tubular structures, dynamic biostructure composed by a complex meshwork of such as blood and lymphatic vessels, and ductal epithelia. multifunctional molecules, formed by cross-linked proteins The BM mediates cell attachment and adhesion, and pro- (collagens and noncollagenous glycoproteins), proteoglycans, motes polarization and transport activities.6,7 The interstitial glycosaminoglycans, and polysaccharides. The ECM defines matrix is the main component of the stroma, which fills the tissue boundaries, but also regulates cell behavior.1,2 Indeed, space among the resident mesenchymal cell types, and besides providing structural anchorage for the attachment of instructs their contribution to several fundamental tissue – parenchymal cells, and transducing mechanical signals,3 5 functions, such as development, regeneration and repair, and ECM can play a range of functions, including presentation of angiogenesis.8 Although changes in ECM composition are growth factors to their cognate receptors, storage of soluble considered relevant to the progression of several chronic factors, cytokines and chemokines, thereby integrating multi- diseases including those of the biliary epithelium (also called ple cues released in the microenvironment.2 “cholangiopathies”), in recent years this topic has received

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– less attention.9 12 Thus, the aim of the present review is to associated large heparan sulfate proteoglycan (HSPG) express- reawaken interest in the role of the biliary ECM, first by ing several binding domains for laminins and collagens, acts as discussing ECM structure and composition in the normal a bridge between these structural molecules. In the normal liver, and then addressing ECM remodeling as it occurs in liver, small leucine-rich proteoglycans such as decorin and benign and malignant diseases of the biliary tract. biglycan are contained in the space of Disse, likely acting as antifibrotic agents due to their ability to bind and neutralize β 22 ECM in the Normal Liver biologically active transforming growth factor (TGF)- 1. On the other hand, proteoglycans can serve as co-receptors for In the normal liver, the ECM is localized in the portal area, cytokines and growth factors, as betaglycan does for TGF-β1, around the central veins and in the thin space surrounding and syndecans for basic fibroblast growth factor (FGF). The role liver sinusoids (space of Disse). The liver sinusoids lack a BM, of ECM as reservoir for proinflammatory cytokines andgrowth but some ECM components are contained in the space of factors able to regulate profibrotic responses will be discussed Disse. The normal constituents of the hepatic ECM in both in the following study. rodents and humans include collagens, laminins, fibronectin, Interactions of ECM components with cells are mediated by nidogens (or entactin), and perlecan, which distribute vari- specific cell membrane receptors, among which integrins are ably in the BM and in the interstitial matrix.2,13 best studied. Integrins are transmembrane heterodimers Collagens are the major constituents in the ECM; 28 differ- formed by α and β subunits, variably combined in more than ent types of collagens have been identified so far, and are 20 members, with binding domains located in their globular categorized into “fibrillar-forming” and “network-forming” portion constituted by an Arg-Gly-Asp (RGD) sequence with collagens.14 In the normal liver, the most represented member affinity for both ECM components (collagens, laminins, fibro- of the collagen family is collagen-IV, a network-forming colla- nectin) and cell adhesion molecules.23 Besides providing gen.15 It is expressed in the BM lining of the bile ducts, as well adhesive functions, integrins modulate several signaling path- as in the interstitial matrix of the portal tracts, where it ways by recruiting adaptor molecules (caveolin, paxillin)24,25 assembles with the fibrillary-forming collagens -III, -V, and and tyrosinekinases (platelet-derivedgrowth factors [PDGF]-β -I (though less abundantly expressed), to generate anchoring receptors)26 to form focal adhesion complexes. In the normal sites for cells. Notably, collagen-IV is also expressed in the liver, integrin receptors are expressed, though at low levels, by space of Disse.16 hepatocytes (α1β1, α5β1, α9β1) and hepatic stellate cells Laminins are a large family of noncollagenousglycoproteins (α1β1, α2β1, α5β1, α6β4). Studies in mouse models of liver highly expressed in the BM, composed of α (1–5), β (1–4), and γ fibrosis have shown that laminin-binding (α6β1, α2β1) and (1–3) disulfide-linked chains, with structural and functional fibronectin-binding (α5β1, αvβ6) integrins are upregulated.27 roles.17 Laminins affect cell differentiation, adhesion, and In particular, upregulation of integrin αvβ6 in the biliary migration. In the mouse liver, the most abundant is the β2 epithelium is a hallmark of biliary fibrosis, as discussed later.28 laminin, which is a constitutive BM component in the bile ►Table 1 outlines the structural components characteriz- ducts, and in portal vessels.18 By combining with collagen type ing the “healthy” ECM phenotype in the liver. Noteworthy, IV, laminin generates a BM around the hepatic sinusoid, an these macromolecules are normally secreted at low levels by element of the so-called “capillarization” of sinusoids, typical stromal cells usually present in the hepatic microenvironment of cirrhosis.13 (hepatic stellate cells and portal fibroblasts). Following biliary Another major component of the hepatic ECM is fibronec- damage and eventually malignant transformation, other cell tin, a high molecular weight (around 440 kDa) glycoprotein elements, including hepatic progenitor cells (HPCs), reactive largely expressed in the interstitial matrix surrounding the ductular cells, activated myofibroblasts and macrophages, gain portal vein and in the BM of the bile ducts in rodent the ability to secrete pre-existing or new ECM components, models.19,20 Thanks to its pronounced binding ability to leading to quantitative and qualitative changes which are several ligands (collagens, heparin, fibrin) and cell surface instrumental for ECM remodeling, as discussed in the follow- receptors, in particular with integrins, fibronectin undergoes ing sections.29 polymerization and stabilizes fibrils, serving as a hook for the attachment of several cell types to the ECM, to regulate cell fl 21 ECM Remodeling in In ammatory adhesion and cytoskeletal organization. Cholangiopathies Nidogens (also called “entactins”) are a class of non- collagenous glycoproteins expressed by the BM. Two forms, ECM remodeling is a functional hallmark of liver repair and nidogen-1 and nidogen-2, have been described, and are fibrogenesis. Some of the major changes of ECM during fibro- critically involved in the development of BM by providing genesis are an increased expression of collagen-I and fibronec- a link for laminins and collagen type IV, to generate a sheath tin.2,4 Surprisingly, only few studies have been conducted in where tubular structures may align in the portal tract.20 human conditions to investigate ECM remodeling in portal Proteoglycans are a distinct family of glycoproteins con- (biliary) fibrosis, although this is the main mechanism of taining glycosaminoglycan side chains, which in the interstitial progression of cholangiopathies such as primary biliary chol- matrix mediate interactions with multiple ECM components angitis (PBC)30 and primary sclerosing cholangitis (PSC).31 It is and regulate their spatial arrangement (aggrecan, fibromodu- knownthatinPBC,theBMbeneaththeinflamed bile ducts lin, decorin, biglycan). Similar to nidogens, perlecan, a BM- is focally destroyed and this lesion is accompanied by an early

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

Table 1 Structural components characterizing the “healthy” ECM phenotype in the liver

ECM component Biochemical structure Localization in the portal tract Collagens Proteins composed of a triple helix Collagen-IV, in the basement membrane of the • Type IV (two α1chainsandoneα2chain). bile ducts, in the interstitial matrix and in the (most expressed) Fibrillar-forming (I, III, and V) and space of Disse. • Type III network-forming (IV). Collagen-III, -V, and -I, in the interstitial matrix. • Type V • Type I Laminins Noncollagenous high molecular weight Basement membrane surrounding bile ducts • β2 laminin glycoproteins, with a heterotrimeric structure and portal vessels. (most expressed) composed of α (1–5), β (1–4), and γ (1–3) disulfide-linked chains. Fibronectin High molecular weight dimeric glycoprotein, Basement membrane surrounding bile ducts. consisting of two nearly identical monomers linked by a pair of disulfide bonds. Nidogens (entactin) Noncollagenous sulfated monomeric Basement membrane of bile ducts and portal • Nidogen-1 glycoproteins. vessels in the portal tract. • Nidogen-2 Perlecan Large heparan sulfate multidomain Basement membrane surrounding the portal proteoglycan (glycoprotein with vessels. glycosaminoglycan [GAG] side chains). Decorin and biglycan Small leucine-rich proteoglycans, with a protein Not in the portal tract (space of Disse). core containing leucine repeats and GAG chains. Integrins Transmembrane heterodimers formed by Cell membrane receptors expressed at low different combinations of α and β subunits, with levels by hepatocytes and hepatic stellate cells, an Arg-Gly-Asp (RGD) binding sequence in their but not by bile ducts. globular portion.

Abbreviation: ECM, extracellular matrix. loss of the periductal capillaries, likely responsible for an prominent role in biliary epithelium diseases, as discussed ischemic damage that may contribute to ductopenia. This is below. ►Table 2 summarizes soluble factors coupled with their accompanied by increased expression of fibronectin, which ECM storing constituents, which are mainly involved in biliary interacts with integrin α4 to facilitate adhesion and infiltration repair. Besides serving as storage sites, ECM proteins themselves of α4-expressing lymphocytes across thebiliary epithelial layer, may behave as potent activators of the cell elements engaged in a process described as “epitheliotropism,” which is a typical the tissue repair (matricellular proteins). In the liver, activation feature of PBC.30 Conversely, in PSC, the BM is reported to be not of the HPCs is a typical response of biliary damage eventually only intact but also thickened, and this aspect contributes to the evolving into the ductular reaction.35 Ductular reactive cells typical “onion-like” periductal fibrosis. In this context, the (DRC) areepithelialcellsclosely interplaying withinflammatory periductal capillaries are preserved, but physically kept distant cells, immune cells, endothelial cells, and fibroblasts, which are from the BM by concentric collagen deposition.31 not present in the healthy liver. Ductular reactions can be Alongside these morphological alterations, ECM compo- categorized in type 1, 2A/B, and 3, depending upon their cell nents establish a close functional relationship with multiple origin, anatomical location, and type of liver insult, involving cellular elements involved in the hepatic reparative response.32 both acute and chronic liver diseases. DRC type 1 originate from Storage regulation of several cytokines and growth factors is a the elongation and proliferation of pre-existing bile ducts, as crucial function of ECM in this context. As aforementioned for observed in response to α-naphthyl isothiocyanate toxicity or to proteoglycans, ECM components can bind soluble factors via biliary obstruction. In contrast with DRC type 1, DRC type 2 low-affinity noncovalent interactions, to create gradients that show a less differentiated biliary phenotype, arranged in irreg- direct trafficking of inflammatory cells and myofibroblasts to ularly shaped, and highly anastomosed structures, lacking an the site of injury to sustain not only ECM remodeling, but also identifiable lumen that can be further distinguished by their tissue regeneration and neoangiogenesis. Growth factors stim- hepatic localization and the liver insult eliciting their genera- ulating angiogenesis and epithelial cell proliferation, such as tion. Whereas DRC type 2A are usually localized at the periphery vascular endothelial growth factor, epidermal growth factor of the portal space and are induced by chronic cholestasis (EGF), hepatocyte growth factor (HGF), and FGF, possess affinity (►Fig. 1A), type 2B develop along cirrhotic nodules and are – for heparan sulfate.33 Others, such as PDGFs, IL-2, and yet HGF, evoked by a hypoxic stimulation.36 38 DRC type 2A and 2B can interact with collagen fibers to regulate myofibroblast recruit- derive either from HPC activation or ductular metaplasia of ment and activation.34 Fibronectin and laminins also show periportal hepatocytes. DRC type 3 originate from the potent binding abilities for connective tissue growth factor (CTGF) activation of the HPC compartment following massive hepato- and tumor-necrosis factor (TNF)-α, two mediators with a cyte loss as it occurs in acute hepatitis or fulminant hepatic

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

Table 2 Soluble factors (growth factors, cytokines, chemokines) stored by ECM components relevant in biliary repair

Soluble factors ECM binding Effect on biliary repair (growth factors, component cytokines, chemokines) VEGF Heparan sulfate • Increases peribiliary vascularization. • Stimulates cholangiocyte proliferation. EGF Heparan sulfate • Stimulates cholangiocyte proliferation. HGF Heparan sulfate • Stimulates cholangiocyte proliferation and promotes ductular reaction. and collagen fibers • Stimulates myofibroblast recruitment and activation. FGF Heparan sulfate • Induces bile duct proliferation. PDGFs Collagen fibers • Most potent mitogen for myofibroblasts. • Stimulates myofibroblast recruitment and activation. • Induces myofibroblasts to acquire an angiogenic phenotype. IL-2 Collagen fibers • Stimulates myofibroblast recruitment and activation. CTGF Fibronectin • Stimulates myofibroblast recruitment and activation. and laminins • Promotes proliferation and collagen production in myofibroblasts. TNF-α Fibronectin • Stimulates proliferation of HPCs. and laminins • Activates ductular reaction. • Upregulates αvβ6 integrin in cholangiocytes. TWEAK FGF-inducible • Stimulates proliferation of HPCs. 14 (Fn14) • Activates ductular reaction.

Abbreviations: CTGF, connective tissue growth factor; ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HPC, hepatic progenitor cell; IL-2, interleukin-2; PDGF, platelet-derived growth factor; TNF, tumor-necrosis factor; TWEAK, TNF-like weak inducer of apoptosis; VEGF, vascular endothelial growth factor. failure.38 Since this review deals with chronic cholangiopathies, isoforms created by alternative splicing and post-transla- when discussing ductular reaction, we will refer to as DRC type tional modifications.39 Similar to CTGF, OPN belongs to a 2A. In cooperationwith portal myofibroblasts and macrophages subclass of ECM proteins that instead of providing a struc- (►Fig. 1B,C), DRC promote ECM deposition and remodeling to tural function, molds the cell phenotype. OPN is secreted by create an environment conducive to liver cell differentiation several cell types and can bind to ECM as phosphoglycopro- and repair.37 Herein, we will first review the main factors tein, or act as a cytokine and mediate autocrine and paracrine regulating HPC activation and ductular reaction (summarized communications relevant to ECM remodeling, cell survival in ►Table 3), and then the ECM components that favor differ- and proliferation and inflammatory cell recruitment. Thanks entiation of progenitor cells toward the biliary lineage. to its ability to bind multiple receptors, including integrin receptors, OPN regulates cell–cell (homotypic) and cell–ECM ECM Remodeling, HPC Activation, and Ductular (heterotypic) interactions, resulting in cell adhesion, migra- Reaction tion, and ECM invasion. OPN is expressed at low levels by Osteopontin (OPN) is a complex matricellular soluble protein normal cholangiocytes, whereby it is upregulated following initially identified in bone remodeling, that exists in different liver damage in both human and rodents, as observed in

Fig. 1 Morphology of hepatic progenitor cells and ductular reactive cells in primary sclerosing cholangitis, identified by their immunoreactivity for K19 (A). Ductular reactive cells are assisted by portal myofibroblasts (immunohistochemistry for α-SMA, B), and by macrophages (immunohistochemistry for CD68, C), in the generation of the ductular reaction, as shown by serial sections. Original magnification: 100X.

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

Table 3 ECM components regulating ductular reaction

ECM Biochemical structure Effect on biliary repair protein Osteopontin Glycosylated phosphoprotein, • Stimulates HPC proliferation and migration, leading to ductular reaction. intracellular, or secreted. • Stimulates myofibroblast activation and collagen production. CTGF Cysteine-rich heparin-binding • Stimulates HPC activation. protein, secreted. • Induces ductular reaction and peribiliary collagen deposition. Integrin Transmembrane heterodimer • Local activator of TGFβ1, stimulates HPC activation. αvβ6 formed by an α and a β subunit. • Induces ductular reaction and peribiliary collagen deposition. LOXL2 Enzyme belonging to the • Stimulates HPC proliferation and migration, leading to ductular reaction. lysyl-oxidase gene family. • Reduces bile duct barrier integrity. • Stimulates myofibroblast activation and collagen production. Polysialic • Negatively charged highly polar • Involved in DRC migration from the HPC niche. acid carbohydrate polymer with affinity for NCAM.

Abbreviations: CTGF, connective tissue growth factor; DRC, ductular reactive cells; ECM, extracellular matrix; HPC, hepatic progenitor cells; LOXL2, lysyl oxidase-like protein 2; NCAM, neural cell adhesion molecule.

40,41 biliary atresia and in CCl4-treated mice, while, in mice CTGF is a member of the cysteine-rich, heparin-binding CCN fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (cysteine-rich 61, connective tissue growth factor, nephroblas- to induce chronic cholangitis, OPN was upregulated also in toma-overexpressed) protein family (CCN2), with strong affin- periportal hepatocytes.42 Recent in vivo studies by Wang43 ity for HSPGs. CTGF does not function as a traditional growth and Coombes44 pinpoint OPN as an important regulator of the factor or cytokine since it does not bind to a single receptor, HPC-driven repair response. Following liver damage, OPN is rather it acts as a matricellular protein able to interact at produced by HPC and stimulates via an autocrine loop, their multiple sites with ECM components (like fibronectin), cell proliferation and migration ultimately leading to ductular surface glycoprotein receptors (like integrins), and growth reaction, while reducing hepatocyte proliferation.44 In addi- factors (including TGFβ1) to modulate cellular functions and tion, OPN regulates the interactions of HPC with stromal cells, signaling.50 In cholangiocytes, CTGF works in concert with thereby stimulating myofibroblast activation and collagen integrin αvβ6, the local activator of TGFβ1, which is secreted in production, through TGF-β45 (see below for the role of latent a latent form, to regulate biliary repair. Cooperation between TGF-β1 activation by integrins). OPN manipulation can lead to CTGF and integrin αvβ6 is paradigmatic of the interactions therapeutic earnings. In fact, OPN deficiency or OPN inactiva- between ECM components and cell receptors in sensing and tion using specific aptamers or neutralizing antibodies, transducing mechanical signals to cells.51 Similar to other attenuates HPC activation, ductular reaction, and fibrogenesis peptides and transcription factors involved in liver reparative in different mouse models of chronic liver disease, including mechanisms, integrin αvβ6 is usually not expressed by the cholangiopathies.44 Conversely, in DDC-fed OPN-knockout (KO) normal biliary epithelium, but it is expressed in the embryonic mice, Fickert et al46 failed to observe significant differences ductal plate during liver development. In the postnatal life, in fibrosis deposition and inflammatory cell recruitment, in αvβ6 is upregulated in response to injury and inflammation of particular in CD11bþ/F4–80þ macrophages, compared the ductal epithelia, and it is rapidly downregulated once with DDC-fed WT littermates. Noteworthy, a recent study inflammation has resolved. Thus, its persistent expression by performed in OPN-KO mice undergoing bile duct ligation, ductal epithelial cells is a distinctive trait of a “woundthatdoes has shown that OPN released by cholangiocytes is the not heal.” mediator initiating the early neutrophil response during A recent study by Pi et al51 demonstrates that integrin αvβ6 obstructive cholestasis.47 Recent studies have also unraveled and CTGF are co-expressed by HPC and DRC after experimental the mechanisms by which OPN stimulates collagen-I biliary damage in mice; they cooperate to regulate HPC activa- synthesis by liver myofibroblasts. In liver fibroblasts, OPN tion and therefore fibrogenesis, interacting with fibronectin induced upregulation of collagen-I through acetylation of and TGFβ1. Activation of latent TGFβ1 empowered by integrin the intracellular high-mobility group box-1 (HMGB1), an endo- αvβ6 ispivotal for HPC function. In fact, the invitro inhibition of genous molecular effector of damage (damage-associated either αvβ6orTGFβ1 halts HPC differentiation into cholangio- molecular pattern) that acts as a downstream alarmin to cyte or hepatocyte, which instead, is rescued after supplemen- amplify inflammatory responses. Moreover, HMGB1 can be tation of bioactive TGFβ1.52 Moreover, in experimental models also secreted by the liver myofibroblasts to behave as autocrine of cholangiopathy, the genetic ablation or pharmacological ligand for the receptor for advanced glycation end-products targeting of αvβ6 decreases the extent of ductular reaction they express to further potentiate collagen-I deposition.48,49 and prevents progression of biliary fibrosis.28 Altogether, these data indicate OPN as a key matricellular The crucial profibrogenic role of integrin αvβ6 in biliary protein promoting HPC expansion, ductular reaction, and tract diseases has been confirmed in a murine model of myofibroblast activation, to create a profibrogenic loop in congenital hepatic fibrosis (CHF), a genetic cholangiopathy cooperation with TGF-β and HMBG1.45 due to a defect in the ciliary protein fibrocystin (Pkhd1del4/del4

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al. mouse) and characterized by biliary dysgenesis and peribiliary by CCR2 genetic deletion results in marked improvement of fibrosis.53 Similar to the DRC phenotype, integrin αvβ6is liver injury and fibrosis in both acute and chronic mouse upregulated in dysgenetic bile ducts of Pkhd1del4/del4 mouse. models.55 Overall, these observations have lent support to the Expression of integrin αvβ6 increases over maturation, as it is notion that monocyte-derived macrophages are important exacerbated by proinflammatory cytokines (TNF-α,TGFβ1) upstream regulators of biliary fibrogenesis, and unveil their released by infiltrating macrophages. Interestingly, macro- potential as a therapeutic target in “orphan” conditions, such as phage recruitment is driven by chemokines (CXCL1, CXCL10, CHF and PSC, with lack of effective treatments.53,55 CXCL12) secreted by fibrocystin-defective cholangiocytes due The lysyl oxidase-like protein 2 (LOXL2) belongs to a family to an overactivation of the β-catenin signaling.53,54 The impor- of enzymes encompassing five members that mediate collagen tance of macrophages as effectors of biliary fibrogenesis at covalent crosslinking and ECM stabilization.56 LOXL2 is upre- least in the first month, is confirmed by the finding that gulated in several scarring conditions, also affecting the liver. In macrophage depletion induced by clodronate leads to a signif- mouse models of biliary fibrosis, induced LOXL2 expression is icant amelioration of biliary fibrosis. Portal myofibroblasts, the typically seen in the portal region in close association with main ECM-producing cells, accumulate only in a later phase, ductular reaction, mostly by portal myofibroblasts, but also by with further increase in peribiliary fibrosis and development DRC and macrophages. Noteworthy, in the Mdr2/ mouse of portal hypertension. (a well-characterized model of fibrosing cholangiopathy), The significant contribution of macrophages to biliary fibro- selective targeting of LOXL2 using neutralizing antibodies genesis has been further addressed in PSC, where alike CHF, suppresses the progression of biliary fibrosis, and promotes biliary lesions are characterized by an abundant fibrotic tissue reversal of pre-established fibrosis.56,57 Beyond collagen cross- closely abutting the bile ducts (►Fig. 2A,B). In both human PSC linking, LOXL2 plays other biological functions with profibrotic and acute and chronic murine sclerosing cholangitis models, a effect, and involves liver fibroblast activation, HPC expansion dense peribiliary accumulation of macrophages is observed.55 and ductular reaction. In fact, in vitro blockade of LOXL2 In these settings, cultured cholangiocytes release in vitro spe- switches the differentiation of primary HPC toward the hepa- cific monocyte chemoattractants (CCL2, IL-8) and macrophage- tocytelineage, while inhibitingexpression of biliary phenotypic activating factors. Attenuation of macrophage accumulation by markers, indicating that autocrine/paracrine LOXL2 suppresses pharmacological inhibition of monocyte recruitment by cen- differentiation of HPC into functional hepatocytes, and favors in icriviroc, antagonist of the CCL2 cognate receptors (CCR2/5) or turn their commitment to ductular reaction. Moreover, LOXL2

Fig. 2 In primary sclerosing cholangitis (PSC) (A, C) and congenital hepatic fibrosis (CHF) (B, D), fibrotic biliary lesions are characterized by a nearby accumulation of portal myofibroblasts (immunolabeled by α-SMA, A), in conjunction with a prominent recruitment of macrophages (immunolabeled by CD68, B). Original magnification: 100X.

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al. may affect the barrier integrity of the biliary epithelium, thus During liver development, whereas laminin containing α1- contributing to the cholestatic injury in both PSC patients and disulfide-linked chains are important for cholangiocyte speci- Mdr2/ mice.58 In PSC, LOXL2 overexpression is located to the fication, α5-containing laminin is essential for bile duct for- characteristic periductal onion skin-type fibrosis, and it is mation.64 However, laminin overexpression in the BM in the paralleled by E-cadherin downregulation in medium-sized postnatal life is associated to biliary dysgenesis and cyst bile ducts. Interestingly, in vitro pharmacological LOX-inhibi- formation, hallmark of genetic cholangiopathies, as observed tion restores the tight junction integrity in cholangiocyte in CHF and polycystic liver.65 monolayers. However, in a phase 2 clinical trial, administration Biomechanical signals related to ECM stiffness are also for 96 weeks of simtuzumab, an inhibitor of LOXL2, did not major determinants of HPC differentiation toward the biliary improve biliary fibrosis nor hamper disease progression in phenotype. Recent studies have shown that there is gradient of patients with PSC, evaluated as total amount of hepatic colla- ECM stiffness across the liver lobule, being stiffness greater at gen, Ishak fibrosis stage, and frequency of PSC-related clinical the periportal region, where biliary morphogenesis typically events.59 occurs during embryonic development.66 Furthermore, ECM Polysialic acid (PolySia) is a highly polar carbohydrate stiffness modulates cholangiocyte differentiation according to polymer structurally associated with the ECM, whose devel- its biochemical composition.66 In particular, stiffness induced opmental role was originally reported in the neural tissue, cholangiocyte differentiation when cells were cultured on where it regulates neuronal guidance and synapse forma- fibronectin, while collagen-IV promoted cholangiocyte differ- tion.60 PolySia is often conjugated with the neural cell adhesion entiation independently from the stiffness of the ECM. Stiffness molecule (NCAM), which is its major carrier. NCAM is a surface promoting effects on cholangiocyte differentiation dependent glycoprotein, member of the Ig superfamily of cell adhesion on fibronectin were related to enhanced myosin II contractility, molecules that mediate cell–cell and cell–ECM interactions. and these effects were associated with ERK activation; in fact, NCAM is transiently expressed in the embryo by ductal plate inhibition of ERK significantly halted cholangiocyte differenti- cells before the stage of lumen formation, but it is absent in the ation, particularly on stiff substrates.66 normal bile ducts, becoming upregulated again in HPC and Furthermore, as aforementioned, ECM storage abilities are DRC, in the course of chronic cholangiopathies.61 Consistent important to integrate paracrine effects of secreted factors, with this observation, expression of PolySia-conjugated NCAM including morphogens. The TNF-like weak inducer of apoptosis increased significantly in both HPC and DRC in experimental (TWEAK) isparadigmaticof thisfunction, since TWEAK induces chronic cholestatic injury.62 NCAM conjugation with PolySia is proliferation of HPC by interacting with its receptor FGF- a fundamental process in biliary repair which is controlled by inducible 14 and leads to ductular reaction via activation of several polysialyltransferases expressed by DRC. Since PolySia NFκB signaling.67 Since the main cell sources of TWEAK are residues have a high hydrophilic content, thanks to this macrophages and natural killer (NK) cells, TWEAK provides a conjugation the adhesive properties of NCAM are turned molecular link between bile duct injury and ductular reaction into antiadhesive, enabling DRC to lose their homotypic and in cholangiopathies. However, whether other morphogens like heterotypic cell interactions, and to gain the migratory func- Notch, Wnt, and Hedgehog that are relevant for HPC activa- tions necessary to detach from the HPC niche nearby the canals tion68 and differentiation might be modulated by ECM com- of Hering, and to extend into the portal region. At this level, ponents has not been uncovered yet, and this topic will deserve DRC may cross talk intensely with different cell types, includ- strong attention by future studies. ing portal myofibroblasts, to build the “hepatic reparative ”63 complex. In vitro and in vivo cleavage of NCAM from PolySia ECM Remodeling in Malignant Diseases of by endosialidase reduces HPC migration, and hampers duct- þ the Bile Ducts: the Desmoplastic ECM of ular reaction, since NCAM DRC unbound to PolySia are not Cholangiocarcinoma (CCA) suffice to form branching structures. Of note, dysregulation of NCAM-PolySia conjugation leads to an increased biliary dam- An exuberant deposition of ECM, histologically recognized by age, in line with the concept that ductular reaction basically pathologists for many decades as “desmoplasia,” is a distinc- develops as an adaptive/compensatory response, and defective tive feature of many adenocarcinomas with a particularly branching mechanisms due to thelackof ECM support impinge invasive behavior, such as biliary tract, pancreatic, breast, on biliary repair.61 prostate cancers, and some forms of gastric and colorectal cancer. In these ductal epithelial cancers, an abnormally ECM Remodeling and Biliary Differentiation remodeled and mechanically stiffer ECM closely adjoins the Following HPC activation, generation of immature DRC neoplastic ducts (►Fig. 3), providing a “dynamic niche,” which requires biliary lineage commitment, which is a process strongly influences the ability of tumor cells to grow, invade, particularly susceptible to ECM organization, in terms of and metastasize.69 Driven by the tumor epithelial counterpart, both biochemical composition and biophysical stiffness. the surrounding stroma is gradually converted from the Among ECM components, collagen-I and laminins have been normal thin framework underlying the basal aspect of the shown to influence expression of biliary markers and ductal ductal epithelial cell layers, into a thick and dense scaffolding morphogenesis by interacting with β1-integrins displayed by wherebya crowd of multiple stromal cell types is recruited and ductal plate cells.63,64 There are different isoforms of laminin activated (tumor reactive stroma).70 They comprise activated and they have different effects on biliary differentiation. fibroblasts (also termed cancer-associated fibroblasts [CAF])

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

Fig. 3 In intrahepatic cholangiocarcinoma, a prominent stromal reaction extensively populated by cancer-associated fibroblasts aligns tumoral bile ducts (immunohistochemistry for α-SMA, A). Periostin (B), tenascin-C (C), and osteopontin (D) decorate the external profile of the neoplastic bile ducts, while these markers are negative in the matched nontumoral counterpart. Histological sections were obtained from a surgical sample of a patient undergoing hepatic resection. Original magnification: 100X. and inflammatory cells (e.g., tumor-associated macrophages CCA induced by Opisthorchis viverrini infestation in hamsters, [TAM], and tumor-associated neutrophils [TAN]), generating a MMP-9 expression correlated with the accumulation of vast array of cues that variably shape the tumor cell traits.71 myofibroblasts, extent of fibrosis, and malignant transforma- While it was first believed that the stromal reaction harbored tion of cholangiocytes; furthermore, the upregulation of tumor-promoting properties, recent data derived from both MMP-9 was related to increased expression of Rac1 and of experimental models and clinical trials have called into ques- inducible nitric oxide synthase, which promoted DNA dam- tions this assumption. In pancreatic ductal adenocarcinoma age.76 Break of the BM is a prerequisite of tumor progression (PDAC), ablation of CAF induced a more aggressive tumor from the stage of carcinoma in situ to dissemination to phenotype with reduced survival in mouse models,72 whereas adjacent tissues. Cancer cell invasion through the BM can be stroma-targeting drugs were proven to be ineffective in clini- induced by CAF via an MMP-independent mechanism. In an ex cal trials.73 Conversely, in experimental models of PDAC, vivo model of colorectal carcinoma, CAF modify the organiza- reprogramming fibroblasts into their quiescent state has tion and the physical properties of the BM by exerting con- been successful to tackle tumor progression.74 Altogether, tractile forces which pull, stretch, and weaken the BM. This these studies indicate that tumor stromal reaction is a highly mechanical perturbation leads to the formation of gaps heterogeneous “ecosystem,” where multiple cell elements are exploited by cancer cells to invade the below-sided tunica hosted and hold both tumor-promoting and tumor-restraining propria.77 properties. Within the complex tumor microenvironment, where opposing effects coexist in, EMT changes induced by Altered Composition of ECM in CCA stromal and tumor cells are important determinants of tumor In CCA, inadditiontothe degradation of the native constituents, invasiveness. During carcinogenesis, CAF, the largest cell pop- ECM is extensively modified by the deposition of newly syn- ulation in the tumor mass (►Fig. 3A), secrete several types of thesized components, such as periostin, tenascin-C, and osteo- proteolytic enzymes, including matrix metalloproteases pontin, proteins that normally are not expressed in the ECM (MMPs), the best characterized ECM degrading enzymes. surrounding bile ducts (►Fig. 3B, D). Of note, overexpression of MMP1, MMP2, MMP3, MMP7, and MMP9 are intensely these proteins has clinical significance, since it correlates with expressed in samples from CCA patients and are associated an increase in tumor size and lymph node metastatization, – with an invasive phenotype.75 In an experimental model of and with shorter overall survival of patients.78 80 Structural

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

Table 4 Structural alterations featuring the “desmoplastic” ECM

ECM proteins Role in “desmoplastic” ECM Periostin • Not expressed in the normal ECM. • Promotes cell migration and metastatization. • Promotes angiogenesis. • Promotes fibrosis by inducing collagen-I fibrillogenesis and stimulating lysyl-oxidase activity for collagen cross-linkage. Tenascin C • Not expressed in the normal ECM. • Stimulates tumor cell proliferation. • Promotes cell migration and metastatization. • Promotes angiogenesis. • Induction of EMT. Osteopontin • Not expressed in the normal ECM. • Stimulates tumor cell proliferation. • Promotes cell migration and metastatization. • Promotes angiogenesis. • Stimulates myofibroblast recruitment and activation, leading to stromal reaction. Collagens • Increases ECM stiffness. – " collagen-I, -III, and -IV – ↓ collagen-V Laminins • Reduces the integrity of the basement membrane. – ↓,especiallyβ2 laminin Fibronectin • Increases ECM stiffness. Proteoglycans (Decorin,Biglycan) • Binding ability for different cytokines and growth factors acting as a reservoir. – "/↓ • Recruitment of inflammatory cells and myofibroblasts to sites of damage to promote tissue regeneration and angiogenesis. Nidogen • Increases ECM stiffness. Perlecan • Increases ECM stiffness.

Abbreviation: ECM, extracellular matrix. alterations discriminating the “desmoplastic” ECM from its promote tumor growth.84 In iCCA, the malignant potential “normal” counterpart are highlighted in ►Table 4. stimulated by periostin was mediated by induction of EMT Among the “ex novo” constituents expressed by tumoral changes instigating cell proliferation, migration, and invasion, ECM, periostin has drawn a considerable interest not only as a as well as enhancing chemoresistance to gemcitabine; inter- potential prognostic biomarker, but also as putative molecular estingly, these proinvasive functions were all inhibited by target in the intrahepatic variant of CCA (iCCA).79,80 Periostin is knockdown of periostin in iCCA cells.85 a glycoprotein belonging to the TGF-β family-inducible matri- Likewise periostin, tenascin-C is a hexameric, multimodular cellular proteins, extensively represented also in the tumoral ECM protein, which contains adhesive and antiadhesive ECM of other malignancies with abundant stroma, as in the sequences enabling competitive binding to multiple ECM pancreatic ductal adenocarcinoma, where it is mainly pro- components, soluble factors, and cell surface receptors. Tenas- duced by CAF. Periostin structure is formed by several domains cin-C is also highly expressed in embryonic tissues, where it that enable its interaction with other ECM proteins, such as regulates cell migration (as shown in the neural crest develop- collagen-I and -V, fibronectin, tenascin-C, and with cellular ment), and can be upregulated in tissue repair, chronic inflam- receptors, integrins in particular, such as α5β1, α5β3, α5β5, mation, and in some cancer types. In adult liver, tenascin-C is and α6β4. Through these interactions, periostin contributes to normally expressed only by sinusoidal walls, but in patients cell motility, angiogenesis, tumor invasiveness, and metasta- with PSC it has been found to be upregulated also in areas of tization. In human mammary ductal epithelial cells and breast brisk ductular reaction and ongoing inflammation, consistent cancer cells, periostin expression mediates EMT changes and with a role in increased ECM remodeling.86 Biological effects promotes a stem cell-like phenotype.81 In CCA, periostin are multiple, yet poorly understood and probably related to cell activates phosphoinositide 3-kinase (PI3K)/AKT signaling, type-specific contexts. For instance, in cancer cells, tenascin-C through interaction with integrin α5, its specific receptor, can activate Wnt and MAPK signaling leading to enhanced cell expressed by CCA cells; in turn, this signaling stimulates tumor proliferation, whereas in fibroblasts it inhibits proliferation, cell proliferation and invasion. Knockdown of integrin α5 while favoring secretory program that may promote angiogen- reduced CCA proliferation and invasion.82 Recent data indicate esis.87 Interestingly, in desmoplastic tumors, such as breast and that iCCA cells are also able to secrete periostin and to recruit colorectal cancers, tenascin-C produced by CAF accumulates TAM,83 similar to what is described for glioblastoma, where within the ECM to generate specialized tracks that direct cancer periostin generated by the cancer stem cells recruited TAM to cell invasion and dissemination through mechanisms mediated

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

– by c-MET and EGFR activation.88 90 A similar process coined by collagen-I, and its expression is increased in many epithelial Hendrix et al as “vasculogenic mimicry,” has been associated to cancers with intense desmoplasia, as breast, colorectal, and increased metastatic spread.91 In iCCA, Aishima et al found that pancreatic cancer, where it cooperated with collagen-I to tenascin-C decorates the desmoplastic stroma around the increase proliferation and migration of tumor cells.99 Within malignant bile ducts and at the invasive front of the tumor, the tumoral ECM, the collagen fiber pattern is profoundly being produced by both CAF and tumoral cholangiocytes. altered by cross-linking and tight packaging. This structural Expression of tenascin-C correlated with tumor size, lymphatic rearrangement is operated by remodeling enzymes belonging spread, and proliferative activity; interestingly, patients to MMP and LOX families, variably released either by stromal expressing tenascin-C at the invasive front had the worst (CAF above all) or tumor cells, and results in an increased prognosis.78 stiffening of the ECM.100,101 Moreover, the secreted protein As previously discussed, OPN can behave as either an ECM acidic and rich in cysteine (SPARC) family members influence protein or a cytokine. Since OPN may exist in different isoforms, deposition, aggregation, and folding of collagen fibers, even at both extracellular (secreted) and intracellular, it can play the level of the BM. SPARC are highly conserved matricellular distinct roles in tumorigenesis and cancer progression.39 As proteins (also called osteonectin), which possess a strong is the case for periostin and tenascin-C, both cancer cells and affinity for collagens, in particular for collagen-IV, that bind stromal cells, including CAF and TAM, can produce OPN. in a Ca2þ-dependent manner.102 Within the SPARC protein Interestingly, OPN originating from CAF and TAM can inhibit family, recent findings identified Sparc/Osteonectin, Cwcv and tumor growth by promoting macrophage recruitment, and Kazal‐like domains proteoglycan (SPOCK1), as an important furthermore, support the antitumor activity of NK and NKT mediator of the ECM remodeling to become permissive to cells. Conversely, tumor-derived OPN can decrease macrophage tumor growth and dissemination in PDAC. Stimulated by cytotoxicity against the neoplastic lesions. Moreover, when TGFβ1 produced by tumor epithelial cells, SPOCK1 is expressed expressed at the ECM level, OPN may interact with many predominantly in the stromal fraction, and acts by modifying different integrins to activate pathways of cell proliferation, the collagen fiber arrangement, to favor the invasive growth of adhesion, invasion, and migration, thus supporting its involve- tumor cells, as shown in organotypic cocultures.101 ment in carcinogenesis. Overexpression of OPN has been These structural changes involving multiple components of observed in several carcinomas associated with an intense the ECM result in a solid framework, which exerts several stroma, such as breast, prostate, lung, stomach, pancreatic promalignant effects that have been mostly elucidated by and colorectal cancer, with prognostic significance especially studies performed in epithelial cancers with abundant stroma in breast and lung cancer.39,92 Thanks to its secretory proper- akin to CCA, and will be discussed below. In this context, it is ties, OPN can be also detected in biological fluids, including worth mentioning that while the profound changes in ECM plasma, and thus used as tumor biomarker to monitor disease composition between normal and diseased livers have been progression. In a recent translational study, increased serum well described and characterized,103 very few studies investi- levels of OPN discriminated patients with CCA from patients gating the differences in ECM in premalignant biliary condi- with PSC, and high OPN in pre- and postoperative settings was tions, such as PSC and CHF/Caroli’s disease, compared with strongly associated with poor survival after resection.93 Of diseases without predisposition to develop CCA, as PBC, cystic note, OPN promotes iCCA growth and metastasis by recruiting fibrosis-related cholangiopathy or biliary atresia, are available MEK/MAPK1 and activating Wnt/β-Catenin signaling.94 Clini- so far. In particular, seminal papers from thelate 1990s showed cal relevance of OPN as putative biomarker was confirmed by no differences in quality and patterns of collagen deposition in molecular profiling of laser-capture microdissected stroma biliary diseases with or without premalignant potential.104,105 derived from human iCCA, showing marked increase in OPN Similar results were then observed on the deregulation of gene expression levels compared with nontumor tissue, and different isoforms of integrins in human samples.30,52,106,107 also, a significant correlation with poor prognosis.95 Notewor- With respect to the expression in human samples of the thy, stromal overexpression of OPN and TGF-β2 had the specific ECM-related proteins endowed with protumorigenic strongest independent predictive power on overall and dis- properties, such as periostin, tenascin-C, and OPN, while a ease-free survival.95 These data, combined with the previously substantial similarity in OPN expression has been reported discussed ability of OPN to promote expansion of HPC,43,44 between PSC and PBC/biliary atresia,40,93,108 comparative conceivably suggest that OPN might playa role in thoseforms of studies dealing with tenascin-C109,110 and periostin111 are iCCA, such as the bile ductular (mixed)-type and the cholan- still lacking (►Table 5). This is indeed a remarkable gap in giolocellular carcinoma, thought to originate from HPCs.96 knowledge of the ECM pathobiology and of its pro-oncogenic The tumoral ECM is also skewed by a change in the expres- attitudes toward biliary transformation, which will deserve sion of collagens. In CCA, ECM is characterized by an excessive consideration by indepth studies in the near future. deposition ofcollagen-I, the most represented collagen protein in the interstitial matrix and of collagen-III. Given the high Altered Stiffness of ECM in CCA affinity for many growth factors (EGF, HGF, keratinocyte The “desmoplastic” ECM possesses an increased rigidity, which growth factor), collagen-I may stimulate tumor cell prolifera- exerts a range of proinvasive mechanical effects. A stiff and tion, reduce cell apoptosis (as shown in PDAC cells),97 and dense ECM may impinge on the vascular assembly, with promote the metastatic potential (as shown in breast cancer generation of leaky vessels,112 and on the transport of immune cells).98 Collagen-III is distributed in close alignment with cells to thetumoral area, reducing the migration ofantitumor T

Seminars in Liver Disease Table 5 Differences in ECM components between cholangiopathies with emphasis on strengths and weaknesses of relative studies

ECM proteins PSC PBC Biliary atresia CHF/Caroli Strengths Weaknesses disease Periostin N/A N/A " vs. healthy N/A Large cohorts of patients.111 ELISA analysis in serum only.111 controls.111 Tenascin-C þ in ECM.109 N/A þ in ECM.110 N/A Thorough histological Small cohorts and examination.109 nonisoform-specificAb.109 Clear histology, appropriate IHC only.110 cohort.110 Osteopontin " vs. healthy þ in mononuclear " vs. healthy N/A Approach based on Small PSC cohorts.93 controls.93 cells and DRC.108 controls.40 different techniques ECM expression not evaluated.108 (ELISA, PCR, IHC), and appropriate controls.93 Large cohorts and use of different techniques.108

Thorough histological Disease Tract Biliary Malignant and Benign in Matrix Liver examination.39 Collagens Col IVþ in Col IVþ in Col I and IVþ in ↓ Col IVþ in IHC of good quality.30 Small series of patients; IHC only.104 periportal periportal periportal area.105 pericystic area.65 Combined use of IHC and ISH.105 Small series of patients; IHC only.30 area.104 area.30 Multimodal approach.65 Small series of biliary atresia patients.105 Small number of CHF patients.65 Integrins αVβ6þ in DRC52 "α4 vs. healthy "β8 vs. healthy αVβ6þ in IHC of good quality.30 Small series of patients; IHC only.30 controls.30 controls.107 biliary cysts.53 Multimodal approach; Small number of human subjects.52 αVβ6þ in DRC.52 "α3and↓β6vs. use of both human Small number of CHF patients.53 healthy controls.106 subjects and animal models.52 IHC only.106 Use of both human subjects IHC only.107 and animal models; multimodal approach.53 Appropriate cohorts of patients.106 Large cohorts of patients.107

Abbreviations: þ, positive expression; " increased; ↓ decreased; Ab, antibody; CHF, congenital hepatic fibrosis; DRC, ductular reactive cells; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; ISH, in situ hybridization; N/A, not assessed; PBC, primary biliary cholangitis; PCR, polymerase chain reaction; PSC, primary sclerosing cholangitis. eiasi ie Disease Liver in Seminars arse al. et Fabris Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al. cells.113 More importantly, in tumor cells, ECM stiffness stim- Rho-associated coiled-coil forming kinase, a downstream ulates the activity of intracellular mechanosensors, such as mediator of the small GTPase RhoA that directs the organiza- Yes-associated protein (YAP) and transcriptional coactivator tion of the actin cytoskeleton, the formation of stress fibers, with postsynaptic density (PSD95)/disc large (Dlg)/zonula and overall, the cell motility, blocked this feed-forward loop, occludens (ZO1) (PDZ)-binding motif (TAZ). YAP/TAZ super- finally reverting the CAF phenotype.122 vise a range of cell functions, relevant for tumor initiation and progression, such as proliferation, survival, plasticity, and 114 Conclusions and Rationale for Drug Design invasion. These observations stem from the well-estab- and Intervention lished pathobiological concept that in normal epithelia, me- chanical inputs generated from the tissue architecture exert a Dynamic changes in the ECM phenotype are instrumental in strong oncosuppressor stimulus. Among the pathways regu- biliary repair, as observed in inflammatory cholangiopathies, lated by YAP/TAZ, several of them, including hyperprolifera- where they regulate activation of HPC and ductular reaction. tion mediated by the mTOR/cyclin D1, and escape from However, the web of interactions that ECM establishes with apoptosis mediated by AKT, promote a malignant pheno- the cell elements engaged in the reparative system are bidi- type.115 Further important tumor-promoting functions regu- rectional, since once activated, these cells intensely contribute lated by YAP/TAZ are progressively emerging. In prostate to the ECM remodeling. Although portal myofibroblasts cancer, YAP/TAZ enable tumor cells to recruit myeloid-derived remain the predominant cell source of the fibrotic ECM suppressor cells which facilitate tumor growth by upregulat- ultimately leading to portal hypertension, other cell types, ing proinflammatory chemokines, such as CXCL5 and TNF- like macrophages, are actively involved, in particular in the α∙116 In malignant hepatocytes, YAP/TAZ induce expression of early phase of the “pathological” ECM remodeling. CCA rep- MCP-1 thereby promoting infiltration of M2 macrophages, resents a further example of how ECM well beyond behaving as which results in immunological tolerance.117 A recent study an inert scaffold may profoundly affect cell phenotype and has shown how the increased rigidity of ECM can activate biological activities. However, in CCA, the interplay of the ECM YAP/TAZ in CCA. The proinvasive functions played by YAP/TAZ with both the stromal and the tumor components is even more are mediated by their association with downstream transcrip- complex. Signals derived from ECM components cooperate to tion factors, including the transcriptional enhanced associate stimulate several intracellular pathways that are integrated domain (TEAD1–4), and this mechanism is inhibited by the into the neoplastic cell to enhance the malignant behavior. switching defective/sucrose nonfermenting (SWI/SNF) chro- Combinatorial interactions between redundant signal path- matin-remodeling complex, which alternatively to TEAD, ways activated by integrins, periostin, tenascin-C, and OPN binds YAP/TAZ in the nucleus through AT-rich interacting regulate cell proliferation, invasiveness, and inflammation. As domain-containing protein 1A (ARID1A).118 Of note, genetic illustrated in ►Fig. 4, both periostin and OPN can bind differ- inactivation of ARID1A has been reported in nearly 7% of ent integrins, including α4β1, α4β7, αvβ1, αvβ3, αvβ5, and iCCA.119 The ability of the complex ARID1A–SWI/SNF to α9β1, to activate the PI3K/AKT and NF-kB leading to cell assemble with YAP/TAZ is regulated by mechanical inputs of proliferation and survival. These effects are further supported the ECM. On a soft ECM, YAP/TAZ are sequestrated within the and amplified by OPN acting on tyrosine kinase receptors such ARID1A-containing SWI/SNF, whereby they are kept at bay, as EGFR, c-Met, and TGFβ receptors, resulting in the activation while on a stiff ECM, YAP/TAZ detach from SWI/SNF, thus of the MAPK signaling. On the other hand, invasive properties becoming free to interact with TEAD to unfold the transcrip- are unfolded by periostin that activate both Notch1 and WNT/ tional program.120 In the liver, a putative mechanoactivator of β-catenin signaling, the latter behaving also as a downstream YAP in ECM is agrin, a proteoglycan that interacts with the effector of tenascin-C. Besides the usefulness as biomarker of integrin-focal adhesion-low-density lipoprotein receptor- fibrosis, serological assessment of tenascin-C and OPN could related protein 4/muscle-specific kinase (Lrp4/MuSK) receptor provide a tool to detect early CCA development, and eventually pathway, to suppress the Hippo pathway, inhibitor of YAP/TAZ a target for therapeutic intervention since the initial via a phosphorylation mediated by large tumor suppressor 1 stages.59,123 Harnessing redundancies of multiple pro-onco- and 2 (LATS1/2) kinases. Cooperation between agrin and YAP genic pathways originating from different ECM components led to liver cancer development, HCC in particular.121 might have great translational value and indeed, points out Notably, in addition to its effects on cancer cells, a stiff ECM ECM as an interesting target for the generation of novel may enhance the activity of YAP/TAZ also in cells of the tumor compounds aimed at both fibrosing cholangiopathies and microenvironment, and CAFs are indeed very sensitive to CCA. With this respect, several molecules targeting different mechanical changes in the ECM. In breast cancer, gene sig- ECM components have been designed and are now in the natures of YAP/TAZ signaling were enriched in CAFs compared pipeline. Overall, despite the promising results obtained in in with the normal fibroblast counterpart. CAF expressed active vitro and in rodent models, so far phase 1 and 2 clinical trials in nuclear YAP, and YAP depletion reduced their tumor-promot- humans have not confirmed the preclinical data, and have not ing functions. Since YAP activation is further stimulated by lived up to the expectations of the scientific community.121 For ECM rigidity, this mechanism involving actomyosin contrac- instance, simtuzumab, a LOXL2 blocking antibody, failed to tility and Src function, establishes a feed-forward self-reinforc- provide clinical benefit in patients with PSC, in a phase 2 study ing loop that on one side, maintains the CAF phenotype and on involving PSC patients.59 Unsatisfactory results were also the other, enhances ECM stiffening. Notably, inhibition of the obtained with pegylated recombinant human hyaluronidase

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

Fig. 4 Intracellular pathways activated by periostin, tenascin-C, and osteopontin modulating protumorigenic functions. De novo expression of matrix components may stimulate a range of pro-oncogenic functions in cholangiocytes, including cell proliferation, invasion, metastatization, and secretion of proinflammatory mediators. These effects are governed by the activation of multiple downstream effectors, such as PI3K/AKT, MAPK, β-catenin, and NF-kB, with a variable degree of overlap among the three matricellular proteins, depending on the binding to the specific receptor. Of note, direct Notch activation by periostin and tenascin-C may also induce a prometastatic cell phenotype.

(PEGPH20) in patients with PDAC treated with/without mFol- by DK034989 Silvio O. Conte Digestive Diseases Research firinox in a clinical trial phase 1b/2 123. However, such disap- Core Center, and by PSC Partners Seeking a Cure; G.J.G. was pointment is justified by the extreme complexity of the ECM- supported by Chris M. Carlos and Catharine Nicole Jockisch driven pathways promoting the fibrosis deposition and shap- Carlos Endowment Fund in Primary Sclerosing Cholangitis. ing a malignant behavior of ductal epithelial cells, which Conflicts of Interest cooperate in the absence of a single causative master regulator M.S. is member of the advisory board of Esiai/Merk, Bayer, of ECM functions. Thus, the engagement of multiple signals and Engitix. hereby originating, including increased organ stiffness and storage of de novo secreted proteins among others, might overcome the effect of a compound aimed at a single molecule, References indicating that a multitargeted approach is likely necessary. 1 Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracel- Moreover, it must be recognized that once transformed in a lular matrix structure. Adv Drug Deliv Rev 2016;97:4–27 stiff and biochemically perturbed biostructure, ECM finely 2 Klaas M, Kangur T, Viil J, et al. The alterations in the extracellular matrix composition guide the repair of damaged liver tissue. Sci modulates an intricate balance involving tumor-promoting Rep 2016;6:27398 as well as tumor-restricting activities. Discriminating between 3 Lu P, Takai K, Weaver VM, Werb Z. Extracellular matrix degrada- the two activities, is one of the major challenges that future tion and remodeling in development and disease. Cold Spring research will have to face to grab the therapeutic potential of Harb Perspect Biol 2011;3(12):a005058 targeting ECM in malignant conditions. 4 Daley WP, Peters SB, Larsen M. Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci 2008;121 (Pt 3):255–264 Financial Support 5 Miller RT. Mechanical properties of basement membrane in L.F. was supported by Progetti di Ricerca di Dipartimento health and disease. Matrix Biol 2017;57-58:366–373 (PRID-DMM) 2017, University of Padua; M.S. was sup- 6 Jayadev R, Sherwood DR. Basement membranes. Curr Biol 2017; ported by the National Institutes of Health RO1DK096096I, 27(06):R207–R211

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

7 Kalluri R. Basement membranes: structure, assembly and role in 31 Washington K, Clavien PA, Killenberg P. Peribiliary vascular tumour angiogenesis. Nat Rev Cancer 2003;3(06):422–433 plexus in primary sclerosing cholangitis and primary biliary 8 Marchand M, Monnot C, Muller L, Germain S. Extracellular cirrhosis. Hum Pathol 1997;28(07):791–795 matrix scaffolding in angiogenesis and capillary homeostasis. 32 Arriazu E, Ruiz de Galarreta M, Cubero FJ, et al. Extracellular Semin Cell Dev Biol 2019;89:147–156 matrix and liver disease. Antioxid Redox Signal 2014;21(07): 9 Insua-Rodríguez J, Oskarsson T. The extracellular matrix in 1078–1097 breast cancer. Adv Drug Deliv Rev 2016;97:41–55 33 Forsten-Williams K, Chu CL, Fannon M, Buczek-Thomas JA, 10 Burgstaller G, Oehrle B, Gerckens M, White ES, Schiller HB, Nugent MA. Control of growth factor networks by heparan Eickelberg O. The instructive extracellular matrix of the lung: sulfate proteoglycans. Ann Biomed Eng 2008;36(12):2134–2148 basic composition and alterations in chronic lung disease. Eur 34 Somasundaram R, Ruehl M, Tiling N, et al. Collagens serve as an Respir J 2017;50(01):1601805 extracellular store of bioactive interleukin 2. J Biol Chem 2000; 11 Sonbol HS. Extracellular matrix remodeling in human disease. 275(49):38170–38175 J Microsc Ultrastruct 2018;6(03):123–128 35 Van Hul NK, Abarca-Quinones J, Sempoux C, Horsmans Y, Leclercq 12 Parola M, Pinzani M. Liver fibrosis: pathophysiology, pathogenetic IA. Relation between liver progenitor cell expansion and extracel- targets and clinical issues. Mol Aspects Med 2019;65:37–55 lular matrix deposition in a CDE-induced murine model of chronic 13 Mak KM, Mei R. Basement membrane type IV collagen and liver injury. Hepatology 2009;49(05):1625–1635 laminin: an overview of their biology and value as fibrosis 36 Roskams TA, Theise ND, Balabaud C, et al. Nomenclature of the biomarkers of liver disease. Anat Rec (Hoboken) 2017;300(08): finer branches of the biliary tree: canals, ductules, and ductular 1371–1390 reactions in human livers. Hepatology 2004;39(06):1739–1745 14 Ricard-Blum S. The collagen family. Cold Spring Harb Perspect 37 Fabris L, Spirli C, Cadamuro M, Fiorotto R, Strazzabosco M. Biol 2011;3(01):a004978 Emerging concepts in biliary repair and fibrosis. Am J Physiol 15 Aycock RS, Seyer JM. Collagens of normal and cirrhotic human Gastrointest Liver Physiol 2017;313(02):G102–G116 liver. Connect Tissue Res 1989;23(01):19–31 38 Desmet VJ. Ductal plates in hepatic ductular reactions. Hypothesis 16 Griffiths MR, Keir S, Burt AD. Basement membrane proteins in the and implications. III. Implications for liver pathology. Virchows space of Disse: a reappraisal. J Clin Pathol 1991;44(08):646–648 Arch 2011;458(03):271–279 17 Tunggal P, Smyth N, Paulsson M, Ott MC. Laminins: structure and 39 Hao C, Cui Y, Owen S, Li W, Cheng S, Jiang WG. Human genetic regulation. Microsc Res Tech 2000;51(03):214–227 osteopontin: potential clinical applications in cancer (Review). 18 Kikkawa Y, Mochizuki Y, Miner JH, Mitaka T. Transient expres- Int J Mol Med 2017;39(06):1327–1337 sion of laminin alpha1 chain in regenerating murine liver: 40 Whitington PF, Malladi P, Melin-Aldana H, Azzam R, Mack CL, restricted localization of laminin chains and nidogen-1. Exp Sahai A. Expression of osteopontin correlates with portal biliary Cell Res 2005;305(01):99–109 proliferation and fibrosis in biliary atresia. Pediatr Res 2005;57 19 Amenta PS, Harrison D. Expression and potential role of the (06):837–844 extracellular matrix in hepatic ontogenesis: a review. Microsc 41 Lorena D, Darby IA, Gadeau AP, et al. Osteopontin expression in Res Tech 1997;39(04):372–386 normal and fibrotic liver. altered liver healing in osteopontin- 20 Geerts A, Geuze HJ, Slot JW, et al. Immunogold localization of deficient mice. J Hepatol 2006;44(02):383–390 procollagen III, fibronectin and heparan sulfate proteoglycan on 42 Fickert P, Stöger U, Fuchsbichler A, et al. A new xenobiotic- ultrathin frozen sections of the normal rat liver. Histochemistry induced mouse model of sclerosing cholangitis and biliary 1986;84(4-6):355–362 fibrosis. Am J Pathol 2007;171(02):525–536 21 Bachmann M, Kukkurainen S, Hytönen VP, Wehrle-Haller B. Cell 43 Wang X, Lopategi A, Ge X, et al. Osteopontin induces ductular adhesion by integrins. Physiol Rev 2019;99(04):1655–1699 reaction contributing to liver fibrosis. Gut 2014;63(11): 22 Schaefer L. Small leucine-rich proteoglycans in kidney disease. 1805–1818 J Am Soc Nephrol 2011;22(07):1200–1207 44 Coombes JD, Swiderska-Syn M, Dollé L, et al. Osteopontin 23 Anderson LR, Owens TW, Naylor MJ. Structural and mechanical neutralisation abrogates the liver progenitor cell response and functions of integrins. Biophys Rev 2014;6(02):203–213 fibrogenesis in mice. Gut 2015;64(07):1120–1131 24 Salanueva IJ, Cerezo A, Guadamillas MC, del Pozo MA. Integrin 45 Xiao X, Gang Y, Gu Y, et al. Osteopontin contributes to TGF-β1 regulation of caveolin function. J Cell Mol Med 2007;11(05): mediated hepatic stellate cell activation. Dig Dis Sci 2012;57 969–980 (11):2883–2891 25 Harburger DS, Calderwood DA. Integrin signalling at a glance. 46 Fickert P, Thueringer A, Moustafa T, et al. The role of osteopontin J Cell Sci 2009;122(Pt 2):159–163 and tumor necrosis factor alpha receptor-1 in xenobiotic-in- 26 Zemskov EA, Loukinova E, Mikhailenko I, Coleman RA, Strickland duced cholangitis and biliary fibrosis in mice. Lab Invest 2010;90 DK, Belkin AM. Regulation of platelet-derived growth factor (06):844–852 receptor function by integrin-associated cell surface transglu- 47 Yang M, Ramachandran A, Yan HM, et al. Osteopontin is an initial taminase. J Biol Chem 2009;284(24):16693–16703 mediator of inflammation and liver injury during obstructive 27 Schnittert J, Bansal R, Storm G, Prakash J. Integrins in wound cholestasis after bile duct ligation in mice. Toxicol Lett 2014;224 healing, fibrosis and tumor stroma: high potential targets for (02):186–195 therapeutics and drug delivery. Adv Drug Deliv Rev 2018; 48 Lenga Y, Koh A, Perera AS, McCulloch CA, Sodek J, Zohar R. 129:37–53 Osteopontin expression is required for myofibroblast differenti- 28 Popov Y, Patsenker E, Stickel F, et al. Integrin alphavbeta6 is a ation. Circ Res 2008;102(03):319–327 marker of the progression of biliary and portal liver fibrosis and a 49 Arriazu E, Ge X, Leung TM, et al. Signalling via the osteopontin and novel target for antifibrotic therapies. J Hepatol 2008;48(03): high mobility group box-1 axis drives the fibrogenic response to 453–464 liver injury. Gut 2017;66(06):1123–1137 29 Cannito S, Milani C, Cappon A, Parola M, Strazzabosco M, 50 Ramazani Y, Knops N, Elmonem MA, et al. Connective tissue Cadamuro M. Fibroinflammatory liver injuries as preneoplastic growth factor (CTGF) from basics to clinics. Matrix Biol 2018; condition in cholangiopathies. Int J Mol Sci 2018;19(12):E3875 68-69:44–66 30 Yasoshima M, Tsuneyama K, Harada K, Sasaki M, Gershwin ME, 51 Pi L, Robinson PM, Jorgensen M, et al. Connective tissue growth Nakanuma Y. Immunohistochemical analysis of cell-matrix adhe- factor and integrin αvβ6: a new pair of regulators critical for sion molecules and their ligands in the portal tracts of primary ductular reaction and biliary fibrosis in mice. Hepatology 2015; biliary cirrhosis. J Pathol 2000;190(01):93–99 61(02):678–691

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

52 Peng ZW, Ikenaga N, Liu SB, et al. Integrin αvβ6 critically 72 Özdemir BC, Pentcheva-Hoang T, Carstens JL, et al. Depletion of regulates hepatic progenitor cell function and promotes ductular carcinoma-associated fibroblasts and fibrosis induces immuno- reaction, fibrosis, and tumorigenesis. Hepatology 2016;63(01): suppression and accelerates pancreas cancer with reduced sur- 217–232 vival. Cancer Cell 2014;25(06):719–734 53 Locatelli L, Cadamuro M, Spirlì C, et al. Macrophage recruitment 73 Kim EJ, Sahai V, Abel EV, et al. Pilot clinical trial of hedgehog by fibrocystin-defective biliary epithelial cells promotes portal pathway inhibitor GDC-0449 (vismodegib) in combination with fibrosis in congenital hepatic fibrosis. Hepatology 2016;63(03): gemcitabine in patients with metastatic pancreatic adenocarci- 965–982 noma. Clin Cancer Res 2014;20(23):5937–5945 54 Spirli C, Locatelli L, Morell CM, et al. Protein kinase A-dependent 74 Khoshchehreh R, Totonchi M, Carlos Ramirez J, et al. Epigenetic pSer(675)-β-catenin, a novel signaling defect in a mouse model of reprogramming of primary pancreatic cancer cells counteracts congenital hepatic fibrosis. Hepatology 2013;58(05):1713–1723 their in vivo tumourigenicity. Oncogene 2019;38(34): 55 Guicciardi ME, Trussoni CE, Krishnan A, et al. Macrophages 6226–6239 contribute to the pathogenesis of sclerosing cholangitis in 75 Terada T, Okada Y, Nakanuma Y. Expression of immunoreactive mice. J Hepatol 2018;69(03):676–686 matrix metalloproteinases and tissue inhibitors of matrix metal- 56 Liu SB, Ikenaga N, Peng ZW, et al. Lysyl oxidase activity contrib- loproteinases in human normal livers and primary liver tumors. utes to collagen stabilization during liver fibrosis progression Hepatology 1996;23(06):1341–1344 and limits spontaneous fibrosis reversal in mice. FASEB J 2016;30 76 Prakobwong S, Yongvanit P, Hiraku Y, et al. Involvement of MMP- (04):1599–1609 9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1- 57 Ikenaga N, Peng ZW, Vaid KA, et al. Selective targeting of lysyl dependent DNA damage in a hamster model. Int J Cancer 2010; oxidase-like 2 (LOXL2) suppresses hepatic fibrosis progression 127(11):2576–2587 and accelerates its reversal. Gut 2017;66(09):1697–1708 77 Glentis A, Oertle P, Mariani P, et al. Cancer-associated fibroblasts 58 Pollheimer MJ, Racedo S, Mikels-Vigdal A, et al. Lysyl oxidase-like induce metalloprotease-independent cancer cell invasion of the protein 2 (LOXL2) modulates barrier function in cholangiocytes basement membrane. Nat Commun 2017;8(01):924 in cholestasis. J Hepatol 2018;69(02):368–377 78 Aishima S, Taguchi K, Terashi T, Matsuura S, Shimada M, Tsu- 59 Muir AJ, Levy C, Janssen HLA, et al; GS-US-321-0102 Investiga- neyoshi M. Tenascin expression at the invasive front is associated tors. Simtuzumab for primary sclerosing cholangitis: phase 2 with poor prognosis in intrahepatic cholangiocarcinoma. Mod study results with insights on the natural history of the disease. Pathol 2003;16(10):1019–1027 Hepatology 2019;69(02):684–698 79 Utispan K, Thuwajit P, Abiko Y, et al. Gene expression profiling of 60 Tsuchiya A, Lu WY, Weinhold B, et al. Polysialic acid/neural cell cholangiocarcinoma-derived fibroblast reveals alterations relat- adhesion molecule modulates the formation of ductular reac- ed to tumor progression and indicates periostin as a poor tions in liver injury. Hepatology 2014;60(05):1727–1740 prognostic marker. Mol Cancer 2010;9:13 61 Fabris L, Strazzabosco M, Crosby HA, et al. Characterization and 80 Sirica AE, Almenara JA, Li C. Periostin in intrahepatic cholangio- isolation of ductular cells coexpressing neural cell adhesion carcinoma: pathobiological insights and clinical implications. molecule and Bcl-2 from primary cholangiopathies and ductal Exp Mol Pathol 2014;97(03):515–524 plate malformations. Am J Pathol 2000;156(05):1599–1612 81 Huang Y, Liu W, Xiao H, et al. Matricellular protein periostin 62 Strazzabosco M, Fabris L. Neural cell adhesion molecule and contributes to hepatic inflammation and fibrosis. Am J Pathol polysialic acid in ductular reaction: the puzzle is far from 2015;185(03):786–797 completed, but the picture is becoming more clear. Hepatology 82 Utispan K, Sonongbua J, Thuwajit P, et al. Periostin activates 2014;60(05):1469–1472 integrin α5β1 through a PI3K/AKT–dependent pathway in inva- 63 Tanimizu N, Miyajima A, Mostov KE. Liver progenitor cells sion of cholangiocarcinoma. Int J Oncol 2012;41(03):1110–1118 develop cholangiocyte-type epithelial polarity in three-dimen- 83 Zeng J, Liu Z, Sun S, et al. Tumor-associated macrophages sional culture. Mol Biol Cell 2007;18(04):1472–1479 recruited by periostin in intrahepatic cholangiocarcinoma 64 Tanimizu N, Kikkawa Y, Mitaka T, Miyajima A. α1- and α5- stem cells. Oncol Lett 2018;15(06):8681–8686 containing laminins regulate the development of bile ducts via 84 Zhou W, Ke SQ, Huang Z, et al. Periostin secreted by glioblasto- β1 integrin signals. J Biol Chem 2012;287(34):28586–28597 ma stem cells recruits M2 tumour-associated macrophages and 65 Yasoshima M, Sato Y, Furubo S, et al. Matrix proteins of basement promotes malignant growth. Nat Cell Biol 2015;17(02): membrane of intrahepatic bile ducts are degraded in congenital 170–182 hepatic fibrosis and Caroli’s disease. J Pathol 2009;217(03): 85 Mino M, Kanno K, Okimoto K, et al. Periostin promotes malignant 442–451 potential by induction of epithelial-mesenchymal transition in 66 Kourouklis AP, Kaylan KB, Underhill GH. Substrate stiffness and intrahepatic cholangiocarcinoma. Hepatol Commun 2017;1(10): matrix composition coordinately control the differentiation of 1099–1109 liver progenitor cells. Biomaterials 2016;99:82–94 86 Midwood KS, Chiquet M, Tucker RP, Orend G. Tenascin-C at a 67 Yanai M, Tatsumi N, Hasunuma N, Katsu K, Endo F, Yokouchi Y. glance. J Cell Sci 2016;129(23):4321–4327 FGF signaling segregates biliary cell-lineage from chick hepato- 87 Lowy CM, Oskarsson T. Tenascin C in metastasis: a view from the blasts cooperatively with BMP4 and ECM components in vitro. invasive front. Cell Adhes Migr 2015;9(1-2):112–124 Dev Dyn 2008;237(05):1268–1283 88 De Wever O, Nguyen QD, Van Hoorde L, et al. Tenascin-C and 68 Kaylan KB, Ermilova V, Yada RC, Underhill GH. Combinatorial SF/HGF produced by myofibroblasts in vitro provide convergent microenvironmental regulation of liver progenitor differentia- pro-invasive signals to human colon cancer cells through RhoA tion by Notch ligands, TGFβ, and extracellular matrix. Sci Rep and Rac. FASEB J 2004;18(09):1016–1018 2016;6:23490 89 Degen M, Brellier F, Kain R, et al. Tenascin-W is a novel marker for 69 Lu P, Weaver VM, Werb Z. The extracellular matrix: a dynamic activated tumor stroma in low-grade human breast cancer and niche in cancer progression. J Cell Biol 2012;196(04):395–406 influences cell behavior. Cancer Res 2007;67(19):9169–9179 70 Brivio S, Cadamuro M, Strazzabosco M, Fabris L. Tumor reactive 90 Degen M, Brellier F, Schenk S, et al. Tenascin-W, a new marker of stroma in cholangiocarcinoma: the fuel behind cancer aggres- cancer stroma, is elevated in sera of colon and breast cancer siveness. World J Hepatol 2017;9(09):455–468 patients. Int J Cancer 2008;122(11):2454–2461 71 Fabris L, Perugorria MJ, Mertens J, et al. The tumour microenvi- 91 Hendrix MJ, Seftor EA, Hess AR, Seftor RE. Vasculogenic mimicry ronment and immune milieu of cholangiocarcinoma. Liver Int and tumour-cell plasticity: lessons from melanoma. Nat Rev 2019;39(Suppl 1):63–78 Cancer 2003;3(06):411–421

Seminars in Liver Disease Liver Matrix in Benign and Malignant Biliary Tract Disease Fabris et al.

92 Zhao H, Chen Q, Alam A, et al. The role of osteopontin in the biliary atresia. J Pediatr Gastroenterol Nutr 2014;59(06): progression of solid organ tumour. Cell Death Dis 2018;9(03): 679–683 356 108 Harada K, Ozaki S, Sudo Y, Tsuneyama K, Ohta H, Nakanuma Y. 93 Loosen SH, Roderburg C, Kauertz KL, et al. Elevated levels of Osteopontin is involved in the formation of epithelioid granulo- circulating osteopontin are associated with a poor survival after ma and bile duct injury in primary biliary cirrhosis. Pathol Int resection of cholangiocarcinoma. J Hepatol 2017;67(04): 2003;53(01):8–17 749–757 109 Koukoulis GK, Koso-Thomas AK, Zardi L, Gabbiani G, Gould VE. 94 Zheng Y, Zhou C, Yu XX, et al. Osteopontin promotes metastasis Enhanced tenascin expression correlates with inflammation in of intrahepatic cholangiocarcinoma through recruiting MAPK1 primary sclerosing cholangitis. Pathol Res Pract 1999;195(11): and mediating Ser675 phosphorylation of β-Catenin. Cell Death 727–731 Dis 2018;9(02):179 110 Noguchi H, Yoshida H, Takamatsu H, Akiyama H. Immunohisto- 95 Sulpice L, Rayar M, Desille M, et al. Molecular profiling of stroma chemical studies on tenascin in extrahepatic bile duct remnants identifies osteopontin as an independent predictor of poor of biliary atresia. In Vivo 2000;14(06):715–720 prognosis in intrahepatic cholangiocarcinoma. Hepatology 111 Honsawek S, Udomsinprasert W, Vejchapipat P, Chongsrisawat V, 2013;58(06):1992–2000 Phavichitr N, Poovorawan Y. Elevated serum periostin is associ- 96 Banales JM, Cardinale V, Carpino G, et al. Expert consensus ated with liver stiffness and clinical outcome in biliary atresia. document: cholangiocarcinoma: current knowledge and future Biomarkers 2015;20(02):157–161 perspectives consensus statement from the European Network 112 Bordeleau F, Mason BN, Lollis EM, et al. Matrix stiffening for the Study of Cholangiocarcinoma (ENS-CCA). Nat Rev Gastro- promotes a tumor vasculature phenotype. Proc Natl Acad Sci U enterol Hepatol 2016;13(05):261–280 S A 2017;114(03):492–497 97 Armstrong T, Packham G, Murphy LB, et al. Type I collagen 113 Salmon H, Franciszkiewicz K, Damotte D, et al. Matrix architec- promotes the malignant phenotype of pancreatic ductal adeno- ture defines the preferential localization and migration of T cells carcinoma. Clin Cancer Res 2004;10(21):7427–7437 into the stroma of human lung tumors. J Clin Invest 2012;122 98 Barcus CE, O’Leary KA, Brockman JL, et al. Elevated collagen-I (03):899–910 augments tumor progressive signals, intravasation and metasta- 114 Zanconato F, Cordenonsi M, Piccolo S. YAP/TAZ at the roots of sis of prolactin-induced estrogen receptor alpha positive mam- cancer. Cancer Cell 2016;29(06):783–803 mary tumor cells. Breast Cancer Res 2017;19(01):9 115 Domínguez-Calderón A, Ávila-Flores A, Ponce A, et al. ZO-2 99 Menke A, Philippi C, Vogelmann R, et al. Down-regulation of E- silencing induces renal hypertrophy through a cell cycle mecha- cadherin gene expression by collagen type I and type III in nism and the activation of YAP and the mTOR pathway. Mol Biol pancreatic cancer cell lines. Cancer Res 2001;61(08):3508–3517 Cell 2016;27(10):1581–1595 100 Nissen NI, Karsdal M, Willumsen N. Collagens and cancer asso- 116 Wang G, Lu X, Dey P, et al. Targeting YAP-dependent mdsc ciated fibroblasts in the reactive stroma and its relation to cancer infiltration impairs tumor progression. Cancer Discov 2016;6 biology. J Exp Clin Cancer Res 2019;38(01):115 (01):80–95 101 Veenstra VL, Damhofer H, Waasdorp C, et al. Stromal SPOCK1 117 Kim W, Khan SK, Liu Y, et al. Hepatic hippo signaling inhibits supports invasive pancreatic cancer growth. Mol Oncol 2017;11 protumoural microenvironment to suppress hepatocellular car- (08):1050–1064 cinoma. Gut 2018;67(09):1692–1703 102 Bradshaw AD. The role of SPARC in extracellular matrix assem- 118 Chang L, Azzolin L, Di Biagio D, et al. The SWI/SNF complex is a bly. J Cell Commun Signal 2009;3(3-4)239–246 mechanoregulated inhibitor of YAP and TAZ. Nature 2018;563 103 Karsdal MA, Manon-Jensen T, Genovese F, et al. Novel insights (7730):265–269 into the function and dynamics of extracellular matrix in liver 119 Zou S, Li J, Zhou H, et al. Mutational landscape of intrahepatic fibrosis. Am J Physiol Gastrointest Liver Physiol 2015;308(10): cholangiocarcinoma. Nat Commun 2014;5:5696 G807–G830 120 Chakraborty S, Njah K, Pobbati AV, et al. Agrin as a mechano- 104 Matsumoto S, Yamamoto K, Nagano T, et al. Immunohistochemi- transduction signal regulating YAP through the hippo pathway. cal study on phenotypical changes of hepatocytes in liver disease Cell Rep 2017;18(10):2464–2479 with reference to extracellular matrix composition. Liver 1999; 121 Schuppan D, Ashfaq-Khan M, Yang AT, Kim YO. Liver fibrosis: 19(01):32–38 direct antifibrotic agents and targeted therapies. Matrix Biol 105 Lamireau T, Le Bail B, Boussarie L, et al. Expression of collagens 2018;68-69:435–451 type I and IV, osteonectin and transforming growth factor beta-1 122 Macias RIR, Kornek M, Rodrigues PM, et al. Diagnostic and (TGFbeta1) in biliary atresia and paucity of intrahepatic bile prognostic biomarkers in cholangiocarcinoma. Liver Int 2019; ducts during infancy. J Hepatol 1999;31(02):248–255 39(Suppl 1):108–122 106 Whitby T, Schroeder D, Kim HS, et al. Modifications in integrin 123 Ramanathan RK, McDonough SL, Philip PA, et al. Phase IB/II expression and extracellular matrix composition in children randomized study of FOLFIRINOX plus pegylated recombinant with biliary atresia. Klin Padiatr 2015;227(01):15–22 human hyaluronidase versus FOLFIRINOX alone in patients with 107 Iordanskaia T, Koeck E, Rossi C, et al. Integrin β-8, but not β-5 or metastatic pancreatic adenocarcinoma: SWOG S1313. J Clin -6, protein expression is increased in livers of children with Oncol 2019;37(13):1062–1069

Seminars in Liver Disease