Matricellular proteins in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is typically characterized by a prominent desmoplastic stroma that is often the most dominant feature of the tumor. This tumor reactive stroma is comprised of a dense fibro-collagenous-enriched extracellular matrix (ECM) surrounding the cancer cells, together with other ECM proteins/peptides, specifically secreted matricellular glycoproteins and proteolytic enzymes, growth factors, and cytokines. Moreover, as enjoined by cholangiocarcinoma cells, this enriched tumor microenvironment is populated by various stromal cell types, most prominently, cancer-associated myofibroblasts (CAFs), along with variable numbers of tumor-associated macrophages (TAMs), inflammatory and vascular cell types. While it is now well appreciated that the interplay between cholangiocarcinoma cells, CAFs, and TAMs in particular play a critical role in promoting cholangiocarcinoma progression, therapeutic resistance, and immune evasion, it is also becoming increasingly evident that over-expression and secretion into the tumor microenvironment of functionally overlapping matricellular glycoproteins, including periostin, osteopontin, tenascin-C, thrombospondin-1, mesothelin and others have an important role to play in regulating or modulating a variety of pro-oncogenic cellular functions, including cholangiocarcinoma cell proliferation, invasion, and metastasis, epithelial-mesenchymal transition, ECM remodeling, and immune evasion. Matricellular proteins have also shown promise as potential prognostic factors for iCCA and may provide unique therapeutic opportunities particularly in relation to targeting iCCA pre-metastatic and metastatic niches, tumor cell dormancy, and immune evasion. This review will highlight timely research and its translational implications for salient matricellular proteins in terms of their structure-function relationships, as modulators of intrahepatic cholangiocarcinoma microenvironment and progression, and potential clinical value for iCCA prognosis and therapy.

YAP1 activation and Hippo pathway signaling in the pathogenesis and treatment of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, is a highly lethal epithelial cell malignancy exhibiting features of cholangiocyte differentiation. iCCAs can potentially develop from multiple cell types of origin within liver, including immature or mature cholangiocytes, hepatic stem cells/progenitor cells, and from transdifferentiation of hepatocytes. Understanding the molecular mechanisms and genetic drivers that diversely drive specific cell lineage pathways leading to iCCA has important biological and clinical implications. In this context, activation of the YAP1-TEAD dependent transcription, driven by Hippo-dependent or -independent diverse mechanisms that lead to the stabilization of YAP1 is crucially important to biliary fate commitment in hepatobiliary cancer. In preclinical models, YAP1 activation in hepatocytes or cholangiocytes is sufficient to drive their malignant transformation into iCCA. Moreover, nuclear YAP1/TAZ is highly prevalent in human iCCA irrespective of the varied etiology, and significantly correlates with poor prognosis in iCCA patients. Based on the ubiquitous expression and diverse physiologic roles for YAP1/TAZ in the liver, recent studies have further revealed distinct functions of active YAP1/TAZ in regulating tumor metabolism, as well as the tumor immune microenvironment. In the current review, we discuss our current understanding of the various roles of the Hippo-YAP1 signaling in iCCA pathogenesis, with a specific focus on the roles played by the Hippo-YAP1 pathway in modulating biliary commitment and oncogenicity, iCCA metabolism, and immune microenvironment. We also discuss the therapeutic potential of targeting the YAP1/TAZ-TEAD transcriptional machinery in iCCA, its current limitations, and what future studies are needed to facilitate clinical translation.

Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly lethal adenocarcinoma of the hepatobiliary system, which can be classified as intrahepatic, perihilar and distal. Each anatomic subtype has distinct genetic aberrations, clinical presentations and therapeutic approaches. In endemic regions, liver fluke infection is associated with CCA, owing to the oncogenic effect of the associated chronic biliary tract inflammation. In other regions, CCA can be associated with chronic biliary tract inflammation owing to choledocholithiasis, cholelithiasis, or primary sclerosing cholangitis, but most CCAs have no identifiable cause. Administration of the anthelmintic drug praziquantel decreases the risk of CCA from liver flukes, but reinfection is common and future vaccination strategies may be more effective. Some patients with CCA are eligible for potentially curative surgical options, such as resection or liver transplantation. Genetic studies have provided new insights into the pathogenesis of CCA, and two aberrations that drive the pathogenesis of non-fluke-associated intrahepatic CCA, fibroblast growth factor receptor 2 fusions and isocitrate dehydrogenase gain-of-function mutations, can be therapeutically targeted. CCA is a highly desmoplastic cancer and targeting the tumour immune microenvironment might be a promising therapeutic approach. CCA remains a highly lethal disease and further scientific and clinical insights are needed to improve patient outcomes.

Intrahepatic cholangiocarcinoma: Morpho-molecular pathology, tumor reactive microenvironment, and malignant progression.

Intrahepatic cholangiocarcinoma (iCCA) is a relatively rare, but highly lethal and biologically complex primary biliary epithelial cancer arising within liver. After hepatocellular carcinoma, iCCA is the second most common primary liver cancer, accounting for approximately 10-20% of all primary hepatic malignancies. Over the last 10-20 years, iCCA has become the focus of increasing concern largely due to its rising incidence and high mortality rates in various parts of the world, including the United States. The challenges posed by iCCA are daunting and despite recent progress in the standard of care and management options for iCCA, the prognosis for this cancer continues to be dismal. In an effort to provide a framework for advancing our understanding of iCCA malignant aggressiveness and therapy resistance, this review will highlight key etiological, biological, molecular, and microenvironmental factors hindering more effective management of this hepatobiliary cancer. Particular focus will be on critically reviewing the cell origins and morpho-molecular heterogeneity of iCCAs, providing mechanistic insights into high risk fibroinflammatory cholangiopathies associated with iCCA development, and notably discussing the deleterious role played by the tumor reactive desmoplastic stroma in regulating iCCA malignant progression, lymphangiogenesis, and tumor immunobiology.

Intrahepatic Cholangiocarcinoma: Continuing Challenges and Translational Advances.

Intrahepatic cholangiocarcinoma (iCCA) has over the last 10-20 years become the focus of increasing concern, largely due to its rising incidence and high mortality rates worldwide. The significant increase in mortality rates from this primary hepatobiliary cancer, particularly over the past decade, has coincided with a rapidly growing interest among clinicians, investigators, and patient advocates to seek greater mechanistic insights and more effective biomarker-driven targeted approaches for managing and/or preventing this challenging liver cancer. In addition to discussing challenges posed by this aggressive cancer, this review will emphasize recent epidemiological, basic, and translational research findings for iCCA. In particular, we will highlight emerging demographic changes and evolving risk factors, the critical role of the tumor microenvironment, extracellular vesicle biomarkers and therapeutics, intertumoral and intratumoral heterogeneity, and current and emerging targeted therapies regarding iCCA. Specifically, recent evidence linking non-bile duct medical conditions, such as nonalcoholic fatty liver disease and nonspecific cirrhosis, to intrahepatic cholangiocarcinogenesis together with geographic and ethnic variation will be assessed. Recent developments concerning the roles played by transforming growth factor-ß and platelet-derived growth factor D in driving the recruitment and expansion of cancer-associated myofibroblasts within cholangiocarcinoma (CCA) stroma as well as their therapeutic implications will also be discussed. In addition, the potential significance of extracellular vesicles as bile and serum biomarkers and therapeutic delivery systems for iCCA will be described. An integrated systems approach to classifying heterogeneous iCCA subtypes will be further highlighted, and recent clinical trials and emerging targeted therapies will be reviewed, along with recommendations for future translational research opportunities. Established international CCA networks are now facilitating collaborations aimed at advancing iCCA translational and clinical research.

Overexpression of periostin and distinct mesothelin forms predict malignant progression in a rat cholangiocarcinoma model.

Periostin and mesothelin have each been suggested to be predictors of poor survival for patients with intrahepatic cholangiocarcinoma, although the clinical prognostic value of both of these biomarkers remains uncertain. The aim of the current study was to investigate these biomarkers for their potential to act as tumor progression factors when assessed in orthotopic tumor and three-dimensional culture models of rat cholangiocarcinoma progression. Using our orthotopic model, we demonstrated a strong positive correlation between tumor and serum periostin and mesothelin and increasing liver tumor mass and associated peritoneal metastases that also reflected differences in cholangiocarcinoma cell aggressiveness and malignant grade. Periostin immunostaining was most prominent in the desmoplastic stroma of larger sized more aggressive liver tumors and peritoneal metastases. In comparison, mesothelin was more highly expressed in the cholangiocarcinoma cells; the slower growing more highly differentiated liver tumors exhibited a luminal cancer cell surface immunostaining for this biomarker, and the rapidly growing less differentiated liver and metastatic tumor masses largely showed cytoplasmic mesothelin immunoreactivity. Two molecular weight forms of mesothelin were identified, one at ∼40 kDa and the other, a more heavily glycosylated form, at ∼50 kDa. Increased expression of the 40-kDa mesothelin over that of the 50 kDa form predicted increased malignant progression in both the orthotopic liver tumors and in cholangiocarcinoma cells of different malignant potential in three-dimensional culture. Moreover, coculturing of cancer-associated myofibroblasts with cholangiocarcinoma cells promoted overexpression of the 40-kDa mesothelin, which correlated with enhanced malignant progression in vitro. Conclusion: Periostin and mesothelin are useful predictors of tumor progression in our rat desmoplastic cholangiocarcinoma models. This supports their relevance to human intrahepatic cholangiocarcinoma. (Hepatology Communications 2018;2:155-172).

Transforming Growth Factors α and ß Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model.

To gain insight into the cellular and molecular interactions mediating the desmoplastic reaction and aggressive malignancy of mass-forming intrahepatic cholangiocarcinoma (ICC), we modeled ICC desmoplasia and progression in vitro. A unique three-dimensional (3D) organotypic culture model was established; within a dilute collagen-type I hydrogel, a novel clonal strain of rat cancer-associated myofibroblasts (TDFSM) was co-cultured with a pure rat cholangiocarcinoma cell strain (TDECC) derived from the same ICC type as TDFSM. This 3D organotypic culture model reproduced key features of desmoplastic reaction that closely mimicked those of the in situ tumor, as well as promoted cholangiocarcinoma cell growth and progression. Our results supported a resident liver mesenchymal cell origin of the TDFSM cells, which were not neoplastically transformed. Notably, 3D co-culturing of TDECC cells with TDFSM cells provoked the formation of a dense fibrocollagenous stroma in vitro that was associated with significant increases in both proliferative TDFSM myofibroblastic cells and TDECC cholangiocarcinoma cells accumulating within the gel matrix. This dramatic desmoplastic ICC-like phenotype, which was not observed in the TDECC or TDFSM controls, was highly dependent on transforming growth factor (TGF)-ß, but not promoted by TGF-α. However, TGF-α was determined to be a key factor for promoting cholangiocarcinoma cell anaplasia, hyperproliferation, and higher malignant grading in this 3D culture model of desmoplastic ICC.

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model.

The cancer microenvironment plays a central role in cancer development, growth, and homeostasis. This paradigm suggests that cancer fibroblasts support cancers, probably in response to stimuli received from the cancer cells. We aimed at investigating whether extracellular vesicles (EVs) can shuttle microRNA (miR) species between cancer-associated fibroblasts (CAFs) and cancer cells. To this end, we extracted EVs according to published protocols. EVs were studied for their miR content by quantitative reverse-transcription polymerase chain reaction. EVs were transfected with select miR species and utilized in vitro as well as in vivo in a rat model of cholangiocarcinoma (CCA). We found that miR-195 is down-regulated in CCA cells, as well as in adjoining fibroblasts. Furthermore, we report that EVs shuttle miR-195 from fibroblasts to cancer cells. Last, we show that fibroblast-derived EVs, loaded with miR-195, can be administered in a rat model of CCA, concentrate within the tumor, decrease the size of cancers, and improve survival of treated rats. CONCLUSION: EVs play a salient role in trafficking miR species between cancer cells and CAFs in human CCA. Understanding of these mechanisms may allow devising of novel therapeutics. (Hepatology 2017;65:501-514).

Periostin in intrahepatic cholangiocarcinoma: pathobiological insights and clinical implications.

Periostin is a modular glycoprotein frequently observed to be a major constituent of the extracellular milieu of mass-forming intrahepatic cholangiocarcinoma and other desmoplastic malignant tumors. In intrahepatic cholangiocarcinoma, as well as in desmoplastic pancreatic ductal adenocarcinoma, periostin is overexpressed and hypersecreted in large part, if not exclusively, by cancer-associated fibroblasts within the tumor stroma. Through its interaction with specific components of the extracellular tumor matrix, particularly collagen type I and tenascin-C, and with cell surface receptors, notably integrins leading to activation of the Akt and FAK signaling pathways, this TGF-ß family-inducible matricellular protein appears to be functioning as a key extracellular matrix molecule regulating such critically important and diverse malignant tumor behaviors as tumor fibrogenesis and desmoplasia, invasive malignant cell growth, chemoresistance, and metastatic colonization. This review will discuss current evidence and basic molecular mechanisms implicating periostin as a mediator of intrahepatic cholangiocarcinoma invasive growth. In addition, its significance as a potential prognostic biomarker for intrahepatic cholangiocarcinoma patients, as well as future possibilities and challenges as a molecular target for cholangiocarcinoma therapy and/or prevention, will be critically evaluated.

Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2.

UNLABELLED: Cholangiocarcinoma (CCA) is an often fatal primary malignancy of the intra- and extrahepatic biliary tract that is commonly associated with chronic cholestasis and significantly elevated levels of primary and conjugated bile acids (CBAs), which are correlated with bile duct obstruction (BDO). BDO has also recently been shown to promote CCA progression. However, whereas there is increasing evidence linking chronic cholestasis and abnormal bile acid profiles to CCA development and progression, the specific mechanisms by which bile acids may be acting to promote cholangiocarcinogenesis and invasive biliary tumor growth have not been fully established. Recent studies have shown that CBAs, but not free bile acids, stimulate CCA cell growth, and that an imbalance in the ratio of free to CBAs may play an important role in the tumorigenesis of CCA. Also, CBAs are able to activate extracellular signal-regulated kinase (ERK)1/2- and phosphatidylinositol-3-kinase/protein kinase B (AKT)-signaling pathways through sphingosine 1-phosphate receptor 2 (S1PR2) in rodent hepatocytes. In the current study, we demonstrate S1PR2 to be highly expressed in rat and human CCA cells, as well as in human CCA tissues. We further show that CBAs activate the ERK1/2- and AKT-signaling pathways and significantly stimulate CCA cell growth and invasion in vitro. Taurocholate (TCA)-mediated CCA cell proliferation, migration, and invasion were significantly inhibited by JTE-013, a chemical antagonist of S1PR2, or by lentiviral short hairpin RNA silencing of S1PR2. In a novel organotypic rat CCA coculture model, TCA was further found to significantly increase the growth of CCA cell spheroidal/"duct-like" structures, which was blocked by treatment with JTE-013. CONCLUSION: Our collective data support the hypothesis that CBAs promote CCA cell-invasive growth through S1PR2.

Non-canonical Hedgehog signaling contributes to chemotaxis in cholangiocarcinoma.

BACKGROUND & AIMS: The Hedgehog signaling pathway contributes to cholangiocarcinoma biology. However, canonical Hedgehog signaling requires cilia, and cholangiocarcinoma cells often do not express cilia. To resolve this paradox, we examined non-canonical (G-protein coupled, pertussis toxin sensitive) Hedgehog signaling in cholangiocarcinoma cells. METHODS: Human [non-malignant (H69), malignant (HuCC-T1 and Mz-ChA-1)] and rat [non-malignant (BDE1 and NRC), and malignant (BDEneu)] cell lines were employed for this study. A BDE(ΔLoop2) cell line with the dominant-negative receptor Patched-1 was generated with the Sleeping Beauty transposon transfection system. RESULTS: Cilia expression was readily identified in non-malignant, but not in malignant cholangiocarcinoma cell lines. Although the canonical Hh signaling pathway was markedly attenuated in cholangiocarcinoma cells, they were chemotactic to purmorphamine, a small-molecule direct Smoothened agonist. Purmorphamine also induced remodeling of the actin cytoskeleton with formation of filopodia and lamellipodia-like protrusions. All these biological features of cell migration were pertussis toxin sensitive, a feature of G-protein coupled (Gis) receptors. To further test the role of Hedgehog signaling in vivo, we employed a syngeneic orthotopic rat model of cholangiocarcinoma. In vivo, genetic inhibition of the Hedgehog signaling pathway employing BDE(ΔLoop2) cells or pharmacological inhibition with a small-molecule antagonist of Smoothened, vismodegib, was tumor and metastasis suppressive. CONCLUSIONS: Cholangiocarcinoma cells exhibit non-canonical Hedgehog signaling with chemotaxis despite impaired cilia expression. This non-canonical Hedgehog signaling pathway appears to contribute to cholangiocarcinoma progression, thereby, supporting a role for Hedgehog pathway inhibition in human cholangiocarcinoma.

Polo-like kinase 2 is a mediator of hedgehog survival signaling in cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and thus rely on potent survival signals to circumvent cell death by TRAIL. Hedgehog (Hh) signaling is an important survival pathway in CCA. Herein, we further examine the mechanisms whereby Hh signaling mediates apoptosis resistance in CCA, revealing a pivotal role for the cell division regulating serine/threonine kinase polo-like kinase 2 (PLK2). We employed 50 human CCA samples (25 intrahepatic and 25 extrahepatic CCA) as well as human KMCH-1, Mz-CHA-1, and HUCCT-1 CCA cells for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. In human samples, polo-like kinase (PLK)1/2/3-immunoreactive cancer cells were present in the preponderance of intra- and extrahepatic CCA specimens. Inhibition of Hh signaling by cyclopamine reduced PLK2, but not PLK1 or PLK3, messenger RNA and protein expression in vehicle-treated and sonic Hh-treated CCA cells, confirming our previous microarray study. PLK2 regulation by Hh signaling appears to be direct, because the Hh transcription factors, glioma-associated oncogene 1 and 2, bind to the PLK2 promotor. Moreover, inhibition of PLK2 by the PLK inhibitor, BI 6727 (volasertib), or PLK2 knockdown was proapoptotic in CCA cells. BI 6727 administration or PLK2 knockdown decreased cellular protein levels of antiapoptotic myeloid cell leukemia 1 (Mcl-1), an effect reversed by the proteasome inhibitor, MG-132. Finally, BI 6727 administration reduced Mcl-1 protein expression in CCA cells, resulting in CCA cell apoptosis and tumor suppression in vivo. CONCLUSION: PLK2 appears to be an important mediator of Hh survival signaling. These results suggest PLK inhibitors to be of therapeutic value for treatment of human CCA.

Therapeutic effects of deleting cancer-associated fibroblasts in cholangiocarcinoma.

Cancer-associated fibroblasts (CAF) are abundant in the stroma of desmoplastic cancers where they promote tumor progression. CAFs are "activated" and as such may be uniquely susceptible to apoptosis. Using cholangiocarcinoma as a desmoplastic tumor model, we investigated the sensitivity of liver CAFs to the cytotoxic drug navitoclax, a BH3 mimetic. Navitoclax induced apoptosis in CAF and in myofibroblastic human hepatic stellate cells but lacked similar effects in quiescent fibroblasts or cholangiocarcinoma cells. Unlike cholangiocarcinoma cells, neither CAF nor quiescent fibroblasts expressed Mcl-1, a known resistance factor for navitoclax cytotoxicity. Explaining this paradox, we found that mitochondria isolated from CAFs or cells treated with navitoclax both released the apoptogenic factors Smac and cytochrome c, suggesting that they are primed for cell death. Such death priming in CAFs appeared to be due, in part, to upregulation of the proapoptotic protein Bax. Short hairpin RNA-mediated attenuation of Bax repressed navitoclax-mediated mitochondrial dysfunction, release of apoptogenic factors, and apoptotic cell death. In a syngeneic rat model of cholangiocarcinoma, navitoclax treatment triggered CAF apoptosis, diminishing expression of the desmoplastic extracellular matrix protein tenascin C, suppressing tumor outgrowth, and improving host survival. Together, our findings argue that navitoclax may be useful for destroying CAFs in the tumor microenvironment as a general strategy to attack solid tumors.

Novel organotypic culture model of cholangiocarcinoma progression.

AIM: Recent studies have suggested that increased α-smooth muscle-actin positive myofibroblastic cells (α-SMA positive CAF) in the desmoplastic stroma may relate to a more aggressive cancer and worse survival outcomes for intrahepatic cholangiocarcinoma (ICC) patients. To facilitate investigating cellular and molecular interactions between α-SMA positive CAF and cholangiocarcinoma cells related to ICC progression, we developed a novel 3-D organotypic culture model of cholangiocarcinoma that more accurately mimics the stromal microenvironment, gene expression profile and select pathophysiological characteristics of desmoplastic ICC in vivo. METHODS: This unique model was established by co-culturing within a type I collagen gel matrix, a strain of cholangiocarcinoma cells (derived from an ICC formed in syngeneic rat liver following bile duct inoculation of spontaneously-transformed rat cholangiocytes) with varying numbers of clonal α-SMA positive CAF established from the same tumor type. RESULTS: Cholangiocarcinoma cells and α-SMA positive CAF in monoculture each exhibited cell-specific biomarker gene expression profiles characteristic of stromal myofibroblastic cell versus malignant cholangiocyte cell types. In comparison, the gene expression profile and histopathological characteristics exhibited by the organotypic co-culture closely resembled those of whole tissue samples of the parent orthotopic ICC. We further showed α-SMA positive CAF to significantly enhance cholangiocarcinoma cell "ductal-like" growth and cancer cell migration/invasiveness in vitro, as well as to promote upregulated expression of select genes known to be associated with ICC invasion. CONCLUSION: This novel organotypic model provides an important new resource for studying the effects of microenvironment on cholangiocarcinoma progression in vitro and may have potential as a preclinical model for identifying molecularly targeted therapies.

Immunization with aspartate-ß-hydroxylase-loaded dendritic cells produces antitumor effects in a rat model of intrahepatic cholangiocarcinoma.

UNLABELLED: Dendritic cells (DCs) capture and process proteins and present peptides on the cell surface in the context of major histocompatibility complex I and II molecules to induce antigen-specific T cell immune responses. The aims of this study were to (1) employ an expanded and purified DC population and load them with aspartate-ß-hydroxylase (ASPH), a highly expressed tumor-associated cell surface protein, and (2) to determine if immunization induced antitumor effects in an orthotopic rat model of intrahepatic cholangiocarcinoma. Splenocytes were incubated with ASPH-coated beads and passed through a magnetic field to yield an 80% pure DC OX62+ population. This DC subset was stimulated with granulocyte-macrophage colony-stimulating factor, interleukin-4, CD40L, and interferon-γ, resulting in a 40-fold increase in interleukin-12A messenger RNA expression to subsequently generate a T helper 1-type immune response. After incubation with the cytokine cocktail, DCs were found to have matured, as demonstrated by increased expression of CD40, CD80, and CD86 costimulatory molecules. Immunization with ASPH-loaded DCs induced antigen-specific immunity. A clone of the parental tumorigenic rat BDEneu cholangiocyte cell line, designated BDEneu-CL24, was found to have the highest number of cells expressing this surface protein (97%); it maintained the same phenotypic characteristics of the parental cell line and was used to produce intrahepatic tumors in immunocompetent syngeneic Fisher-344 rats. Immunization with ASPH-loaded DCs generated cytotoxicity against cholangiocarcinoma cells in vitro and significantly suppressed intrahepatic tumor growth and metastasis, and was associated with increased CD3+ lymphocyte infiltration into the tumors. CONCLUSION: These findings suggest that immunization with ASPH-loaded DCs may constitute a novel therapeutic approach for intrahepatic cholangiocarcinoma, because this protein also appears to be highly conserved and expressed on human hepatobiliary tumors.

Targeting PDGFR-ß in Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinomas (CCAs) are highly desmoplastic neoplasms with a tumour microenvironment plentiful in myofibroblasts (MFBs). MFB-derived PDGF-BB survival signalling is a mediator of CCA cell resistance to apoptotic stimuli. This raises the concept that targeting PDGFR-ß, a cognate receptor of PDGF-BB, represents a potential strategy for the treatment of human CCA. AIMS: Herein, we examine a role for inhibiting PDGFR-ß in restoring CCA cell sensitivity to apoptotic stimuli in vitro and in vivo. METHODS: We employed human CCA samples from 41 patients (19 intrahepatic and 22 extrahepatic CCA samples), the human CCA cell lines KMCH-1 and HUCCT-1 as well as shPDGFR-ß-KMCH-1 and human myofibroblastic LX-2 cells for these studies. In vivo-experiments were conducted using a syngeneic rat orthotopic CCA model. RESULTS: Of several MFB-derived growth factors profiled, PDGF-BB and CTGF were most abundantly expressed; however, only PDGF-BB attenuated tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Co-culturing CCA cells with PDGF-BB-secreting MFBs significantly decreased TRAIL-induced CCA cell apoptosis when compared with monoculture conditions; this cytoprotective effect was abrogated in the presence of the tyrosine kinase inhibitors imatinib mesylate or linifanib, which inhibit PDGFR-ß. Consistent with these findings, MFB-imparted cytoprotection also was abolished when PDGFR-ß was knocked down as demonstrated in shPDGFR-ß-KMCH-1 cells. Finally, administration of imatinib mesylate increased CCA cell apoptosis and reduced tumour growth in a rodent in vivo-CCA model that mimics the human disease. CONCLUSIONS: Targeting PDGFR-ß sensitizes CCA cells to apoptotic stimuli and appears to be therapeutic in vivo.