Role of desmoplasia in cholangiocarcinoma and hepatocellular carcinoma
Authors: Jung Il Lee, Jean S. Campbell
Journal: Journal of Hepatology (2014)

COMMENTARY ON:Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor. Clapéron A, Mergey M, Aoudjehane L, Ho-Bouldoires TH, Wendum D, Prignon A, Merabtene F, Firrincieli D, Desbois-Mouthon C, Scatton O, Conti F, Housset C, Fouassier L. Hepatology. 2013 Dec;58(6):2001–2011. Copyright © 2013. Reprinted by permission from the American Association for the Study of Liver Diseases.http://www.ncbi.nlm.nih.gov/pubmed/23787814Abstract: Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs) in the stroma and with the sustained activation of the epidermal growth factor receptor (EGFR) in tumor cells. Among EGFR ligands, heparin-binding epidermal growth factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between MFs and tumor cells in several cancers. This study was designed to test whether hepatic MFs contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic MFs was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, cotransplantation of CCA cells with human liver myofibroblasts (HLMFs) increased tumor incidence, size, and metastatic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal MFs expressed HB-EGF, whereas EGFR was detected in cancer cells. In vitro, HLMFs produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF-neutralizing antibody. We also showed that CCA cells produced transforming growth factor beta 1, which, in turn, induced HB-EGF expression in HLMFs.CONCLUSION: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression.Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) are the two most common primary liver cancers worldwide [[1]Razumilava N. Gores G.J. Classification, diagnosis, and management of cholangiocarcinoma.Clin Gastroenterol Hepatol. 2013; 11 ([e11; quiz e13–14]): 13-21Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar] and are malignancies that arise from epithelial cells that share an early common developmental program. The risk of developing CCA has been linked to liver flukes and primary sclerosing cholangitis (PSC), although most cases are thought to be sporadic with chronic inflammation as a likely risk factor [[2]Palmer W.C. Patel T. Are common factors involved in the pathogenesis of primary liver cancers? A meta-analysis of risk factors for intrahepatic cholangiocarcinoma.J Hepatol. 2012; 57: 69-76Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar]. Cirrhosis is the primary risk factor for HCC, and results from a chronic inflammatory fibrotic milieu. This 'microenvironment' can be the consequence of diverse diseases including viral hepatitis, chronic alcoholism, metabolic syndrome, and inherited genetic mutations. Rather than occurring in a background of cirrhosis, CCA is often associated with desmoplasia, the deposition of fibrotic or connective tissue, which is frequently regarded as a response to malignant cholangiocytes. However, carcinogenesis research has illustrated the importance of the stroma or tumor microenvironment in tumor progression resulting in re-evaluation of impact of the "desmoplastic response" associated with CCA.In other solid tumors (e.g., pancreatic, prostrate or breast carcinomas), the term "desmoplastic response" refers to the abnormal activity of an epithelial neoplasia, wherein the tumor stimulates stromal fibroblasts to produce and deposit copious amounts of collagens and extracellular matrix (ECM), which surround the tumor. The desmoplastic stroma adjacent to these cancers commonly includes activated alpha smooth muscle actin (αSMA)-positive fibroblasts, which produce fibrillar collagens, fibronectins, proteoglycans, and tenascin C, in collaboration with other liver cell types. Thus formation of stroma in CCA appears as a secondary insult in response to malignant growth. Conversely, in hepatocellular carcinogenesis, fibrosis and cirrhosis are thought to generate a microenvironment that promotes tumorigenesis. Therapeutic implications for causal vs. consequential role(s) of fibrous tissue and collagenous stroma in liver tumors are obvious. Surgical resection offers the only curative therapy for either CCA or HCC, however few patients are cured of CCA or HCC because of late detection of the tumor or high surgical risk. Hence primary research goals for either liver malignancy are to identify risk factors that enable earlier detection, and define the molecular and cellular programs involved in their respective pathogenesis to develop effective treatments.The contribution of the stromal microenvironment to liver carcinogenesis has been more fully appreciated in the last decade (Fig. 1). In HCC molecular profiling studies from tumor and adjacent cirrhotic liver indicate that the gene signature of the adjacent non-tumor tissue contains critical molecular information on recurrence and prognosis [[3]Hoshida Y. Villanueva A. Kobayashi M. Peix J. Chiang D.Y. Camargo A. et al.Gene expression in fixed tissues and outcome in hepatocellular carcinoma.N Engl J Med. 2008; 359: 1995-2004Crossref PubMed Scopus (1018) Google Scholar], indicating that molecular prognostic determinants depend on both tumor and non-tumor tissue. Analogous studies with CCA and adjacent liver have not yet been performed, although CCA-genetic signatures have been identified [4Andersen J.B. Spee B. Blechacz B.R. Avital I. Komuta M. Barbour A. et al.Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.Gastroenterology. 2012; 142: e1015Abstract Full Text Full Text PDF Scopus (360) Google Scholar, 5Voss J.S. Holtegaard L.M. Kerr S.E. Fritcher E.G. Roberts L.R. Gores G.J. et al.Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions.Hum Pathol. 2013; 44: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar]. Recent studies in CCA development support the notion that cancer-associated fibroblasts (CAFs) promote tumor progression [[6]Sirica A.E. The role of cancer-associated myofibroblasts in intrahepatic cholangiocarcinoma.Nat Rev Gastroenterol Hepatol. 2012; 9: 44-54Crossref Scopus (110) Google Scholar]. While many different cell types influence the tumor microenvironment, CAFs are fundamentally important by virtue of their ability to overproduce ECM proteins and alter the cellular architecture [[7]Blaauboer M.E. Boeijen F.R. Emson C.L. Turner S.M. Zandieh-Doulabi B. Hanemaaijer R. et al.Extracellular matrix proteins: a positive feedback loop in lung fibrosis?.Matrix Biol. 2014; 34: 170-178Crossref PubMed Scopus (61) Google Scholar]. Yet the precise mechanisms by which CAFs facilitate tumor progression and its molecular evolution are not well understood. In particular, determining the cellular origin(s) of CAFs and whether CAFs have different functions when they participate in the desmoplastic response in CCA compared to cirrhosis and subsequent HCCs will facilitate novel therapeutic approaches. PSC may offer a unique vantage point for CAF-pathogenesis studies. The sequential pathogenesis in PSC, an autoimmune disease, begins with inflammation, bile duct collapse and subsequent concentric fibrosis. As CAFs are present in advanced PSC, they may be a consequence of inflammation and BEC cell death [[8]Eaton J.E. Talwalkar J.A. Lazaridis K.N. Gores G.J. Lindor K.D. Pathogenesis of primary sclerosing cholangitis and advances in diagnosis and management.Gastroenterology. 2013; 145: 521-536Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Recent cell fate tracing studies indicate that HSCs are the primary fibroblastic cell type in fibrosis that give rise to myofibroblasts regardless of mouse injury or surgical models used [[9]Mederacke I. Hsu C.C. Troeger J.S. Huebener P. Mu X. Dapito D.H. et al.Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology.Nat Commun. 2013; 4: 2823Crossref PubMed Scopus (792) Google Scholar]. Extension of cell fate mapping studies to animal models of CCA or HCC with desmoplastic or fibrotic pathogenesis will be a next logical step.A recent study furthers our mechanistic understanding of the contribution of CAFs through the use of an admixture xenographic mouse model of CCA [[10]Clapéron A. Mergey M. Aoudjehane L. Ho-Bouldoires T.H. Wendum D. Prignon A. et al.Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor.Hepatology. 2013; 58: 2001-2011Crossref PubMed Scopus (65) Google Scholar]. Clapéron and co-workers used a combination of in vivo and in vitro approaches to show that human hepatic myofibroblasts promote the growth of human CCAs. Human intrahepatic fibroblasts were isolated from livers from patients undergoing resection for metastases or benign tumors, allowed to expand in vitro (termed human liver myofibroblasts, HLMF) and then co-injected with human CCA cell lines into immune-compromised mice. HLMFs stimulated CCA growth in vivo and Gefitinib, a tyrosine kinase inhibitor (TKI) specific for EGFR1, blunted xenograph growth illustrating that EGFR signaling pathways contribute to CCA progression in this model. The authors further demonstrated that heparin-binding EGF (HB-EGF), one of the seven EGF ligands, was produced by HLMF and promoted CCA growth.The role of EGFR in carcinogenesis is well established [11Normanno N. De Luca A. Bianco C. Strizzi L. Mancino M. Maiello M.R. et al.Epidermal growth factor receptor (EGFR) signaling in cancer.Gene. 2006; 366: 2-16Crossref PubMed Scopus (1466) Google Scholar, 12Bianco R. Gelardi T. Damiano V. Ciardiello F. Tortora G. Rational bases for the development of EGFR inhibitors for cancer treatment.Int J Biochem Cell Biol. 2007; 39: 1416-1431Crossref PubMed Scopus (105) Google Scholar], yet challenges remain in developing selective therapies [[13]Yarden Y. Pines G. The ERBB network: at last, cancer therapy meets systems biology.Nat Rev Cancer. 2012; 12: 553-563Crossref PubMed Scopus (610) Google Scholar]. Genetic alterations in the EGFR ligand-receptor family have been identified in CCA, including amplifications, deletions, and point mutations [4Andersen J.B. Spee B. Blechacz B.R. Avital I. Komuta M. Barbour A. et al.Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.Gastroenterology. 2012; 142: e1015Abstract Full Text Full Text PDF Scopus (360) Google Scholar, 5Voss J.S. Holtegaard L.M. Kerr S.E. Fritcher E.G. Roberts L.R. Gores G.J. et al.Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions.Hum Pathol. 2013; 44: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar] with ErbB1 being the primary focus of prior investigations. The study by Clapéron and co-workers indicates yet another mechanism by which EGFR pathways appears to contribute to tumor progression, a mechanism whereby paracrine signaling is perturbed. As with any ligand-receptor family, subtle changes in cellular patterns of ligand or receptor expression manifest in exaggerated cell growth or migration. In the Clapéron study, HB-EGF expression was elevated in CCA specimens, in the xenographic tumors, and in cultures of HLMF implicating CAFs as a potential source of this EGF ligand. In vitro studies demonstrate that HB-EGF binds to ErbB4/ErbB2 receptor heterodimers in addition to ErbB1/ErbB2 dimers, albeit with a lower affinity [[14]Jones J.T. Akita R.W. Sliwkowski M.X. Binding specificities and affinities of egf domains for ErbB receptors.FEBS Lett. 1999; 447: 227-231Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar] expanding possible EGFR pathways that could contribute to CCA progression.The admixture xenographic CCA model described by Clapéron has a number of experimental advantages and therapeutic entrées. It would be interesting to create a variety of cell mixtures using different sources of human CAFs/HLMFs isolated from different etiologies or uninjured liver (e.g., quiescent HSCs). Will CCA cell lines evoke a more dramatic 'desmoplastic response' from freshly isolated HSCs in vivo? Evidence from gene profiling studies of isolated HSCs in human [[15]Sancho-Bru P. Bataller R. Gasull X. Colmenero J. Khurdayan V. Gual A. et al.Genomic and functional characterization of stellate cells isolated from human cirrhotic livers.J Hepatol. 2005; 43: 272-282Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar] and mice [[16]De Minicis S. Seki E. Uchinami H. Kluwe J. Zhang Y. Brenner D.A. et al.Gene expression profiles during hepatic stellate cell activation in culture and in vivo.Gastroenterology. 2007; 132: 1937-1946Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar] suggest that plastic-activation of HSCs does not fully recapitulate their in vivo activation profiles. Thus use of 'in vivo activated MFBs' will likely produce different results than plastic-activated MFBs. It would be also be interesting to determine whether CAFs execute their tumor-promoting effect when their numbers were less than CCA tumor cells, which may tease apart autocrine or juxtacrine signaling mechanisms from paracrine mechanisms. Therapeutically, CCA admixture xenographs could be used for testing combination therapies with cytostatic TKIs and targeted monoclonal antibodies to the various EFGR receptor isoforms.As with any study using preclinical models, there are limitations. In particular biliary architecture impacts the development of CCA, and intrahepatic vs. extrahepatic CCA origin may be important to delineate treatment [6Sirica A.E. The role of cancer-associated myofibroblasts in intrahepatic cholangiocarcinoma.Nat Rev Gastroenterol Hepatol. 2012; 9: 44-54Crossref Scopus (110) Google Scholar, 17Patel T. New insights into the molecular pathogenesis of intrahepatic cholangiocarcinoma.J Gastroenterol. 2014; 49: 165-172Crossref PubMed Scopus (40) Google Scholar]. A particular challenge in CCA models is mimicking the in vivo topographical variation observed in this malignancy. The microenvironmental mechanical forces resulting from excess collagen and ECM deposition continually and reciprocally alter MFB and epithelial cell phenotypes. How can the dynamically changing cellular architecture and mechanical forces be modeled in vivo [[7]Blaauboer M.E. Boeijen F.R. Emson C.L. Turner S.M. Zandieh-Doulabi B. Hanemaaijer R. et al.Extracellular matrix proteins: a positive feedback loop in lung fibrosis?.Matrix Biol. 2014; 34: 170-178Crossref PubMed Scopus (61) Google Scholar]? Nevertheless, the role of CAFs in CCA and HCC progression will offer great insight into the pathogenesis of these tumors.Conflict of interestThe authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. COMMENTARY ON: Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor. Clapéron A, Mergey M, Aoudjehane L, Ho-Bouldoires TH, Wendum D, Prignon A, Merabtene F, Firrincieli D, Desbois-Mouthon C, Scatton O, Conti F, Housset C, Fouassier L. Hepatology. 2013 Dec;58(6):2001–2011. Copyright © 2013. Reprinted by permission from the American Association for the Study of Liver Diseases.http://www.ncbi.nlm.nih.gov/pubmed/23787814 Abstract: Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs) in the stroma and with the sustained activation of the epidermal growth factor receptor (EGFR) in tumor cells. Among EGFR ligands, heparin-binding epidermal growth factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between MFs and tumor cells in several cancers. This study was designed to test whether hepatic MFs contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic MFs was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, cotransplantation of CCA cells with human liver myofibroblasts (HLMFs) increased tumor incidence, size, and metastatic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal MFs expressed HB-EGF, whereas EGFR was detected in cancer cells. In vitro, HLMFs produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF-neutralizing antibody. We also showed that CCA cells produced transforming growth factor beta 1, which, in turn, induced HB-EGF expression in HLMFs. CONCLUSION: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression. Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) are the two most common primary liver cancers worldwide [[1]Razumilava N. Gores G.J. Classification, diagnosis, and management of cholangiocarcinoma.Clin Gastroenterol Hepatol. 2013; 11 ([e11; quiz e13–14]): 13-21Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar] and are malignancies that arise from epithelial cells that share an early common developmental program. The risk of developing CCA has been linked to liver flukes and primary sclerosing cholangitis (PSC), although most cases are thought to be sporadic with chronic inflammation as a likely risk factor [[2]Palmer W.C. Patel T. Are common factors involved in the pathogenesis of primary liver cancers? A meta-analysis of risk factors for intrahepatic cholangiocarcinoma.J Hepatol. 2012; 57: 69-76Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar]. Cirrhosis is the primary risk factor for HCC, and results from a chronic inflammatory fibrotic milieu. This 'microenvironment' can be the consequence of diverse diseases including viral hepatitis, chronic alcoholism, metabolic syndrome, and inherited genetic mutations. Rather than occurring in a background of cirrhosis, CCA is often associated with desmoplasia, the deposition of fibrotic or connective tissue, which is frequently regarded as a response to malignant cholangiocytes. However, carcinogenesis research has illustrated the importance of the stroma or tumor microenvironment in tumor progression resulting in re-evaluation of impact of the "desmoplastic response" associated with CCA. In other solid tumors (e.g., pancreatic, prostrate or breast carcinomas), the term "desmoplastic response" refers to the abnormal activity of an epithelial neoplasia, wherein the tumor stimulates stromal fibroblasts to produce and deposit copious amounts of collagens and extracellular matrix (ECM), which surround the tumor. The desmoplastic stroma adjacent to these cancers commonly includes activated alpha smooth muscle actin (αSMA)-positive fibroblasts, which produce fibrillar collagens, fibronectins, proteoglycans, and tenascin C, in collaboration with other liver cell types. Thus formation of stroma in CCA appears as a secondary insult in response to malignant growth. Conversely, in hepatocellular carcinogenesis, fibrosis and cirrhosis are thought to generate a microenvironment that promotes tumorigenesis. Therapeutic implications for causal vs. consequential role(s) of fibrous tissue and collagenous stroma in liver tumors are obvious. Surgical resection offers the only curative therapy for either CCA or HCC, however few patients are cured of CCA or HCC because of late detection of the tumor or high surgical risk. Hence primary research goals for either liver malignancy are to identify risk factors that enable earlier detection, and define the molecular and cellular programs involved in their respective pathogenesis to develop effective treatments. The contribution of the stromal microenvironment to liver carcinogenesis has been more fully appreciated in the last decade (Fig. 1). In HCC molecular profiling studies from tumor and adjacent cirrhotic liver indicate that the gene signature of the adjacent non-tumor tissue contains critical molecular information on recurrence and prognosis [[3]Hoshida Y. Villanueva A. Kobayashi M. Peix J. Chiang D.Y. Camargo A. et al.Gene expression in fixed tissues and outcome in hepatocellular carcinoma.N Engl J Med. 2008; 359: 1995-2004Crossref PubMed Scopus (1018) Google Scholar], indicating that molecular prognostic determinants depend on both tumor and non-tumor tissue. Analogous studies with CCA and adjacent liver have not yet been performed, although CCA-genetic signatures have been identified [4Andersen J.B. Spee B. Blechacz B.R. Avital I. Komuta M. Barbour A. et al.Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.Gastroenterology. 2012; 142: e1015Abstract Full Text Full Text PDF Scopus (360) Google Scholar, 5Voss J.S. Holtegaard L.M. Kerr S.E. Fritcher E.G. Roberts L.R. Gores G.J. et al.Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions.Hum Pathol. 2013; 44: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar]. Recent studies in CCA development support the notion that cancer-associated fibroblasts (CAFs) promote tumor progression [[6]Sirica A.E. The role of cancer-associated myofibroblasts in intrahepatic cholangiocarcinoma.Nat Rev Gastroenterol Hepatol. 2012; 9: 44-54Crossref Scopus (110) Google Scholar]. While many different cell types influence the tumor microenvironment, CAFs are fundamentally important by virtue of their ability to overproduce ECM proteins and alter the cellular architecture [[7]Blaauboer M.E. Boeijen F.R. Emson C.L. Turner S.M. Zandieh-Doulabi B. Hanemaaijer R. et al.Extracellular matrix proteins: a positive feedback loop in lung fibrosis?.Matrix Biol. 2014; 34: 170-178Crossref PubMed Scopus (61) Google Scholar]. Yet the precise mechanisms by which CAFs facilitate tumor progression and its molecular evolution are not well understood. In particular, determining the cellular origin(s) of CAFs and whether CAFs have different functions when they participate in the desmoplastic response in CCA compared to cirrhosis and subsequent HCCs will facilitate novel therapeutic approaches. PSC may offer a unique vantage point for CAF-pathogenesis studies. The sequential pathogenesis in PSC, an autoimmune disease, begins with inflammation, bile duct collapse and subsequent concentric fibrosis. As CAFs are present in advanced PSC, they may be a consequence of inflammation and BEC cell death [[8]Eaton J.E. Talwalkar J.A. Lazaridis K.N. Gores G.J. Lindor K.D. Pathogenesis of primary sclerosing cholangitis and advances in diagnosis and management.Gastroenterology. 2013; 145: 521-536Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar]. Recent cell fate tracing studies indicate that HSCs are the primary fibroblastic cell type in fibrosis that give rise to myofibroblasts regardless of mouse injury or surgical models used [[9]Mederacke I. Hsu C.C. Troeger J.S. Huebener P. Mu X. Dapito D.H. et al.Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology.Nat Commun. 2013; 4: 2823Crossref PubMed Scopus (792) Google Scholar]. Extension of cell fate mapping studies to animal models of CCA or HCC with desmoplastic or fibrotic pathogenesis will be a next logical step. A recent study furthers our mechanistic understanding of the contribution of CAFs through the use of an admixture xenographic mouse model of CCA [[10]Clapéron A. Mergey M. Aoudjehane L. Ho-Bouldoires T.H. Wendum D. Prignon A. et al.Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor.Hepatology. 2013; 58: 2001-2011Crossref PubMed Scopus (65) Google Scholar]. Clapéron and co-workers used a combination of in vivo and in vitro approaches to show that human hepatic myofibroblasts promote the growth of human CCAs. Human intrahepatic fibroblasts were isolated from livers from patients undergoing resection for metastases or benign tumors, allowed to expand in vitro (termed human liver myofibroblasts, HLMF) and then co-injected with human CCA cell lines into immune-compromised mice. HLMFs stimulated CCA growth in vivo and Gefitinib, a tyrosine kinase inhibitor (TKI) specific for EGFR1, blunted xenograph growth illustrating that EGFR signaling pathways contribute to CCA progression in this model. The authors further demonstrated that heparin-binding EGF (HB-EGF), one of the seven EGF ligands, was produced by HLMF and promoted CCA growth. The role of EGFR in carcinogenesis is well established [11Normanno N. De Luca A. Bianco C. Strizzi L. Mancino M. Maiello M.R. et al.Epidermal growth factor receptor (EGFR) signaling in cancer.Gene. 2006; 366: 2-16Crossref PubMed Scopus (1466) Google Scholar, 12Bianco R. Gelardi T. Damiano V. Ciardiello F. Tortora G. Rational bases for the development of EGFR inhibitors for cancer treatment.Int J Biochem Cell Biol. 2007; 39: 1416-1431Crossref PubMed Scopus (105) Google Scholar], yet challenges remain in developing selective therapies [[13]Yarden Y. Pines G. The ERBB network: at last, cancer therapy meets systems biology.Nat Rev Cancer. 2012; 12: 553-563Crossref PubMed Scopus (610) Google Scholar]. Genetic alterations in the EGFR ligand-receptor family have been identified in CCA, including amplifications, deletions, and point mutations [4Andersen J.B. Spee B. Blechacz B.R. Avital I. Komuta M. Barbour A. et al.Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.Gastroenterology. 2012; 142: e1015Abstract Full Text Full Text PDF Scopus (360) Google Scholar, 5Voss J.S. Holtegaard L.M. Kerr S.E. Fritcher E.G. Roberts L.R. Gores G.J. et al.Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions.Hum Pathol. 2013; 44: 1216-1222Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar] with ErbB1 being the primary focus of prior investigations. The study by Clapéron and co-workers indicates yet another mechanism by which EGFR pathways appears to contribute to tumor progression, a mechanism whereby paracrine signaling is perturbed. As with any ligand-receptor family, subtle changes in cellular patterns of ligand or receptor expression manifest in exaggerated cell growth or migration. In the Clapéron study, HB-EGF expression was elevated in CCA specimens, in the xenographic tumors, and in cultures of HLMF implicating CAFs as a potential source of this EGF ligand. In vitro studies demonstrate that HB-EGF binds to ErbB4/ErbB2 receptor heterodimers in addition to ErbB1/ErbB2 dimers, albeit with a lower affinity [[14]Jones J.T. Akita R.W. Sliwkowski M.X. Binding specificities and affinities of egf domains for ErbB receptors.FEBS Lett. 1999; 447: 227-231Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar] expanding possible EGFR pathways that could contribute to CCA progression. The admixture xenographic CCA model described by Clapéron has a number of experimental advantages and therapeutic entrées. It would be interesting to create a variety of cell mixtures using different sources of human CAFs/HLMFs isolated from different etiologies or uninjured liver (e.g., quiescent HSCs). Will CCA cell lines evoke a more dramatic 'desmoplastic response' from freshly isolated HSCs in vivo? Evidence from gene profiling studies of isolated HSCs in human [[15]Sancho-Bru P. Bataller R. Gasull X. Colmenero J. Khurdayan V. Gual A. et al.Genomic and functional characterization of stellate cells isolated from human cirrhotic livers.J Hepatol. 2005; 43: 272-282Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar] and mice [[16]De Minicis S. Seki E. Uchinami H. Kluwe J. Zhang Y. Brenner D.A. et al.Gene expression profiles during hepatic stellate cell activation in culture and in vivo.Gastroenterology. 2007; 132: 1937-1946Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar] suggest that plastic-activation of HSCs does not fully recapitulate their in vivo activation profiles. Thus use of 'in vivo activated MFBs' will likely produce different results than plastic-activated MFBs. It would be also be interesting to determine whether CAFs execute their tumor-promoting effect when their numbers were less than CCA tumor cells, which may tease apart autocrine or juxtacrine signaling mechanisms from paracrine mechanisms. Therapeutically, CCA admixture xenographs could be used for testing combination therapies with cytostatic TKIs and targeted monoclonal antibodies to the various EFGR receptor isoforms. As with any study using preclinical models, there are limitations. In particular biliary architecture impacts the development of CCA, and intrahepatic vs. extrahepatic CCA origin may be important to delineate treatment [6Sirica A.E. The role of cancer-associated myofibroblasts in intrahepatic cholangiocarcinoma.Nat Rev Gastroenterol Hepatol. 2012; 9: 44-54Crossref Scopus (110) Google Scholar, 17Patel T. New insights into the molecular pathogenesis of intrahepatic cholangiocarcinoma.J Gastroenterol. 2014; 49: 165-172Crossref PubMed Scopus (40) Google Scholar]. A particular challenge in CCA models is mimicking the in vivo topographical variation observed in this malignancy. The microenvironmental mechanical forces resulting from excess collagen and ECM deposition continually and reciprocally alter MFB and epithelial cell phenotypes. How can the dynamically changing cellular architecture and mechanical forces be modeled in vivo [[7]Blaauboer M.E. Boeijen F.R. Emson C.L. Turner S.M. Zandieh-Doulabi B. Hanemaaijer R. et al.Extracellular matrix proteins: a positive feedback loop in lung fibrosis?.Matrix Biol. 2014; 34: 170-178Crossref PubMed Scopus (61) Google Scholar]? Nevertheless, the role of CAFs in CCA and HCC progression will offer great insight into the pathogenesis of these tumors. Conflict of interestThe authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.