Inhibition of Mast Cell-Derived Histamine Decreases Human Cholangiocarcinoma Growth and Differentiation via c-Kit/Stem Cell Factor–Dependent Signaling
2015; Elsevier BV; Volume: 186; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2015.09.016
ISSN1525-2191
AutoresChristopher M. Johnson, Victoria Huynh, Laura Hargrove, Lindsey Kennedy, Allyson Graf‐Eaton, Jennifer Owens, Jerome P. Trzeciakowski, Kyle Hodges, Sharon DeMorrow, Yuyan Han, Lucas Wong, Gianfranco Alpini, Heather Francis,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe tumor microenvironment of cholangiocarcinoma (CCA) is composed of numerous cells, including mast cells (MCs). MCs release histamine, which increases CCA progression and angiogenesis. Cholangiocytes secrete stem cell factor, which functions via the MC growth factor receptor c-Kit. Here, we show that cholangiocytes express histidine decarboxylase and its inhibition reduces CCA growth. MC recruitment in the tumor microenvironment increased CCA growth. MC infiltration and MC markers were detected by toluidine blue staining and real-time PCR in human biopsies and in tumors from athymic mice treated with saline, histamine, histidine decarboxylase inhibitor, or cromolyn sodium. Tumor growth, angiogenesis, and epithelial-mesenchymal transition (EMT)/extracellular matrix (ECM) markers were measured in mice treated with cromolyn sodium. In vitro, human CCA cells were treated with MC supernatant fluids before evaluating angiogenesis and EMT/ECM expression. Migration assays were performed with CCA cells treated with the stem cell factor inhibitor. MC supernatant fluids increased CCA histidine decarboxylase, vascular endothelial growth factor, and MC/EMT/ECM expression that decreased with pretreatment of cromolyn sodium. MCs were found in human biopsies. In mice treated with cromolyn sodium, MC infiltration and tumor growth decreased. Inhibition of CCA stem cell factor blocked MC migration and MC/EMT/ECM in CCA. MCs migrate into CCA tumor microenvironment via c-Kit/stem cell factor and increase tumor progression, angiogenesis, EMT switch, and ECM degradation. The tumor microenvironment of cholangiocarcinoma (CCA) is composed of numerous cells, including mast cells (MCs). MCs release histamine, which increases CCA progression and angiogenesis. Cholangiocytes secrete stem cell factor, which functions via the MC growth factor receptor c-Kit. Here, we show that cholangiocytes express histidine decarboxylase and its inhibition reduces CCA growth. MC recruitment in the tumor microenvironment increased CCA growth. MC infiltration and MC markers were detected by toluidine blue staining and real-time PCR in human biopsies and in tumors from athymic mice treated with saline, histamine, histidine decarboxylase inhibitor, or cromolyn sodium. Tumor growth, angiogenesis, and epithelial-mesenchymal transition (EMT)/extracellular matrix (ECM) markers were measured in mice treated with cromolyn sodium. In vitro, human CCA cells were treated with MC supernatant fluids before evaluating angiogenesis and EMT/ECM expression. Migration assays were performed with CCA cells treated with the stem cell factor inhibitor. MC supernatant fluids increased CCA histidine decarboxylase, vascular endothelial growth factor, and MC/EMT/ECM expression that decreased with pretreatment of cromolyn sodium. MCs were found in human biopsies. In mice treated with cromolyn sodium, MC infiltration and tumor growth decreased. Inhibition of CCA stem cell factor blocked MC migration and MC/EMT/ECM in CCA. MCs migrate into CCA tumor microenvironment via c-Kit/stem cell factor and increase tumor progression, angiogenesis, EMT switch, and ECM degradation. Cholangiocarcinoma (CCA) cancers are primary tumors that arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the intrahepatic and extrahepatic bile ducts of the liver.1Francis H. Alpini G. DeMorrow S. Recent advances in the regulation of cholangiocarcinoma growth.Am J Physiol Gastrointest Liver Physiol. 2010; 299: G1-G9Crossref PubMed Scopus (19) Google Scholar CCA is the second most prevalent liver tumor after hepatocellular carcinoma and accounts for 10% to 20% of deaths caused by hepatobiliary malignancies.2Gatto M. Bragazzi M.C. Semeraro R. Napoli C. Gentile R. Torrice A. Gaudio E. Alvaro D. Cholangiocarcinoma: update and future perspectives.Dig Liver Dis. 2010; 42: 253-260Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar Increased risk of developing CCA is associated with primary sclerosing cholangitis, liver fluke infestation, hepatitis C virus infection, and other diseases that lead to chronic biliary obstruction and inflammation.3Gatto M. Alvaro D. Cholangiocarcinoma: risk factors and clinical presentation.Eur Rev Med Pharmacol Sci. 2010; 14: 363-367PubMed Google Scholar CAA is a metastatic cancer, and studies have found its potential to migrate outside of the biliary tract.4Meng F. Han Y. Staloch D. Francis T. Stokes A. Francis H. The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis.Hepatology. 2011; 54: 1718-1728Crossref PubMed Scopus (56) Google Scholar We have found that histamine via the H4 histamine receptor (HR) promotes epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) breakdown and that globally blocking histamine synthesis decreases tumor growth.4Meng F. Han Y. Staloch D. Francis T. Stokes A. Francis H. The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis.Hepatology. 2011; 54: 1718-1728Crossref PubMed Scopus (56) Google Scholar, 5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar Because of its clinically silent nature, CCA is typically diagnosed in advanced stages, when chemotherapy is often ineffective and radical surgery may be contraindicated.2Gatto M. Bragazzi M.C. Semeraro R. Napoli C. Gentile R. Torrice A. Gaudio E. Alvaro D. Cholangiocarcinoma: update and future perspectives.Dig Liver Dis. 2010; 42: 253-260Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 3Gatto M. Alvaro D. Cholangiocarcinoma: risk factors and clinical presentation.Eur Rev Med Pharmacol Sci. 2010; 14: 363-367PubMed Google Scholar Thus, it is important to elucidate the mechanisms of CCA progression to provide alternative therapeutic targets and to allow for earlier detection. The tumor microenvironment of CCA consists of numerous cells, including mast cells.6Leyva-Illades D. McMillin M. Quinn M. DeMorrow S. Cholangiocarcinoma pathogenesis: role of the tumor microenvironment.Transl Gastrointest Cancer. 2012; 1: 71-80PubMed Google Scholar These cells contribute to CCA pathogenesis by releasing mediators that support tumor growth and suppress immune responses.6Leyva-Illades D. McMillin M. Quinn M. DeMorrow S. Cholangiocarcinoma pathogenesis: role of the tumor microenvironment.Transl Gastrointest Cancer. 2012; 1: 71-80PubMed Google Scholar, 7Dalton D.K. Noelle R.J. The roles of mast cells in anticancer immunity.Cancer Immunol Immunother. 2012; 61: 1511-1520Crossref PubMed Scopus (56) Google Scholar Although commonly associated with its role in mediating allergic response, mast cells play an important role in other disorders, although the exact mechanism by which they act is unknown.8Rao K.N. Brown M.A. Mast cells: multifaceted immune cells with diverse roles in health and disease.Ann N Y Acad Sci. 2008; 1143: 83-104Crossref PubMed Scopus (208) Google Scholar The role of mast cells in the tumor microenvironment is typically detrimental to the patient.9Hodges K. Kennedy L. Meng F. Alpini G. Francis H. Mast cells, disease and gastrointestinal cancer: a comprehensive review of recent findings.Transl Gastrointest Cancer. 2012; 1: 138-150PubMed Google Scholar On infiltration, mast cells release large amounts of inflammatory mediators into the tumor microenvironment, including histamine, ILs, and growth factors.9Hodges K. Kennedy L. Meng F. Alpini G. Francis H. Mast cells, disease and gastrointestinal cancer: a comprehensive review of recent findings.Transl Gastrointest Cancer. 2012; 1: 138-150PubMed Google Scholar, 10Chang D.Z. Ma Y. Ji B. Wang H. Deng D. Liu Y. Abbruzzese J.L. Liu Y.J. Logsdon C.D. Hwu P. Mast cells in tumor microenvironment promotes the in vivo growth of pancreatic ductal adenocarcinoma.Clin Cancer Res. 2011; 17: 7015-7023Crossref PubMed Scopus (103) Google Scholar These factors all contribute to the progression of tumor growth. Mast cells express numerous cellular ligands on their surface, including the stem cell factor (SCF) receptor, c-Kit that binds with SCF on other cell types.11Hermes B. Feldmann-Boddeker I. Welker P. Algermissen B. Steckelings M.U. Grabbe J. Henz B.M. Altered expression of mast cell chymase and tryptase and of c-Kit in human cutaneous scar tissue.J Invest Dermatol. 2000; 114: 51-55Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Lammie A. Drobnjak M. Gerald W. Saad A. Cote R. Cordon-Cardo C. Expression of c-kit and kit ligand proteins in normal human tissues.J Histochem Cytochem. 1994; 42: 1417-1425Crossref PubMed Scopus (230) Google Scholar CCA cells secrete SCF5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar; thus, there is the potential for mast cells to be drawn to the tumor microenvironment via this interaction. We have shown that histamine, via an autocrine pathway, increases CCA growth by increasing vascular endothelial growth factor (VEGF) expression and that blocking histamine with the use of an inhibitor for histidine decarboxylase (HDC) can substantially diminish tumor growth.5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar However, no studies have been performed to determine the role that mast cells or mast cell-derived histamine might play in CCA tumor growth, the EMT switch, or ECM breakdown. Thus, we aimed to determine whether mast cells were present in the tumor microenvironment of CCA and whether the recruitment of mast cells into the tumor microenvironment via SCF/c-Kit interaction increases CCA growth, EMT, and ECM breakdown. All reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise. Antibodies for immunoblots, immunofluorescence, and immunohistochemistry were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) unless otherwise stated. Cell culture reagents and media were obtained from Invitrogen Corporation (Carlsbad, CA). Human (for cell lines studies) and mouse (for tumor evaluation) sequenced primers and real-time PCR assay materials were obtained from SABiosciences (Frederick, MD). The following primers were used: Vimentin, Human-PPH00417F-200/Mouse-PPM04780B-200; Paxillin, Human-PPH02786A-200/Mouse-PPM25951B-200; s100A4, Human-PPH01313E-200/Mouse-PPM03811A-200; matrix metalloproteinase (MMP)2, Human-PPH00912B-200; MMP3, Human-PPH00235F-200; MMP9, Human-PPH00152E-200; proliferating cell nuclear antigen (PCNA), Human-PPH00216B-200/Mouse-PPM03456F-200; VEGF-A, Human-PPH00251C-200/Mouse-PPM03041F-200; VEGF-C, Human-PPH00673D-200/Mouse-PPM03061F-200; HDC, Human-PPH07080F-200/Mouse-PPM04804A-200; H1HR, Mouse-PPM04806B-200; H2HR, Mouse-PPM04805A-200; H3HR, Mouse-PPM04893A-200; H4HR, Mouse-PPM04894A-200; E-cadherin, Human-PPH00135F-200/Mouse-PPM03652F-200; C-kit, Human-PPH00432A-200/Mouse-PPM05195A-200; Chymase, Human-PPH05676A-200/Mouse-PPM24635B-200; and Tryptase, Human-PPH19168B-200 (α/β 1)/Mouse-PPM24954A-200 (α/β 1). Both the iScript cDNA Synthesis Kit (catalog no. 1708891) and the iTaq Universal SYBR-Green Supermix (catalog no. 1725124) were obtained from Bio-Rad/Life Science Research Division (Hercules, CA). We used tumors from xenograft mouse models to evaluate the presence of mast cells and expression of mast cell markers.5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar, 13Francis H. Onori P. Gaudio E. Franchitto A. DeMorrow S. Venter J. Kopriva S. Carpino G. Mancinelli R. White M. Meng F. Vetuschi A. Sferra R. Alpini G. H3 histamine receptor-mediated activation of protein kinase Calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo.Mol Cancer Res. 2009; 7: 1704-1713Crossref PubMed Scopus (51) Google Scholar Briefly, mice were injected with cells from an extrahepatic biliary cancer cell line (Mz-ChA-1) and, after tumor establishment, were treated by i.p. injections of 0.9% NaCl (vehicle), histamine (0.5 mg/kg in 100 μL of 0.9% NaCl), or α-methyl-dl-histidine (HDC inhibitor; 150 mg/kg in 100 μL of 0.9% NaCl).5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar Tumors were collected after 52 days.5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar In separate studies, we performed experiments to determine the effects of specifically blocking mast cell-derived histamine on tumor growth. Again, with the use of xenograft tumor models, we treated mice with 0.9% NaCl (saline) or cromolyn sodium (24 mg/kg body weight) for 38 days by i.p. injection three times per week. Tumor growth (length × width × height mm3) was measured every other day as described5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar; then, the presence of mast cells and mast cell markers was evaluated by immunohistochemistry and real-time PCR,5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar respectively. All experimental procedures were conducted with approval from the Baylor Scott & White Institutional Animal Care and Use Committee. Tumor samples were excised and fixed in 10% buffered formalin for 24 hours, embedded in low-temperature fusion paraffin, and sectioned (4 to 5 μm) for immunohistochemistry analysis.13Francis H. Onori P. Gaudio E. Franchitto A. DeMorrow S. Venter J. Kopriva S. Carpino G. Mancinelli R. White M. Meng F. Vetuschi A. Sferra R. Alpini G. H3 histamine receptor-mediated activation of protein kinase Calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo.Mol Cancer Res. 2009; 7: 1704-1713Crossref PubMed Scopus (51) Google Scholar, 14Onori P. DeMorrow S. Gaudio E. Franchitto A. Mancinelli R. Venter J. Kopriva S. Ueno Y. Alvaro D. Savage J. Alpini G. Francis H. Caffeic acid phenethyl ester decreases cholangiocarcinoma growth by inhibition of NF-kappaB and induction of apoptosis.Int J Cancer. 2009; 125: 565-576Crossref PubMed Scopus (83) Google Scholar Total mRNA was extracted with the Qiagen (Valencia, CA) RNeasy mini kit and, after amplification, a ΔΔCT (Δ threshold cycle) analysis was performed.15Onori P. Wise C. Gaudio E. Franchitto A. Francis H. Carpino G. Lee V. Lam I. Miller T. Dostal D.E. Glaser S.S. Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor.Int J Cancer. 2010; 127: 43-54Crossref PubMed Scopus (37) Google Scholar In commercially available human biopsy tissue arrays (AccuMax; BioCarta LLC, San Diego, CA) we evaluated mast cell presence by toluidine blue staining and performed immunohistochemistry for the following mast cell markers: c-Kit (dilution 1:200; anti-c-Kit polyclonal; MBL International Corporation, Woburn, MA), chymase, and tryptase (dilution 1:50; MC tryptase; Santa Cruz Biotechnology, Paso Robles, CA).16Boyce J.A. The biology of the mast cell.Allergy Asthma Proc. 2004; 25: 27-30PubMed Google Scholar, 17Castells M. Mast cell mediators in allergic inflammation and mastocytosis.Immunol Allergy Clin N Am. 2006; 26: 465-485Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Church M.K. Levi-Schaffer F. The human mast cell.J Allergy Clin Immunol. 1997; 99: 155-160Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar The staining index was calculated by multiplying the staining intensity by abundance.15Onori P. Wise C. Gaudio E. Franchitto A. Francis H. Carpino G. Lee V. Lam I. Miller T. Dostal D.E. Glaser S.S. Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor.Int J Cancer. 2010; 127: 43-54Crossref PubMed Scopus (37) Google Scholar Mast cell infiltration was measured by staining tumor sections from all animal groups for toluidine blue, which marks mature mast cells.16Boyce J.A. The biology of the mast cell.Allergy Asthma Proc. 2004; 25: 27-30PubMed Google Scholar Sections were visualized with a light microscope, and slides were scanned on Leica SCN400 (Wetzlar, Germany) and counted manually. The expression of mast cell markers, including c-Kit, chymase, and tryptase, was measured by real-time PCR in tumor mRNA of the above-mentioned animal groups. To quantitatively measure the expression of histamine enzyme and receptor mRNA in CCA, we used the RT2 real-time assay from SABiosciences.14Onori P. DeMorrow S. Gaudio E. Franchitto A. Mancinelli R. Venter J. Kopriva S. Ueno Y. Alvaro D. Savage J. Alpini G. Francis H. Caffeic acid phenethyl ester decreases cholangiocarcinoma growth by inhibition of NF-kappaB and induction of apoptosis.Int J Cancer. 2009; 125: 565-576Crossref PubMed Scopus (83) Google Scholar, 15Onori P. Wise C. Gaudio E. Franchitto A. Francis H. Carpino G. Lee V. Lam I. Miller T. Dostal D.E. Glaser S.S. Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor.Int J Cancer. 2010; 127: 43-54Crossref PubMed Scopus (37) Google Scholar Glyceraldehyde 3-phosphate dehydrogenase was used as the housekeeping gene. In sections from vehicle or cromolyn-treated mice, we measured c-Kit, chymase, and tryptase expression by immunohistochemistry as described.5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar We evaluated the effects of cromolyn sodium treatment in vivo by measuring tumor growth and the expression of PCNA and VEGF-C by immunoblots and real-time PCR in whole tumors from vehicle- and cromolyn sodium-treated mice as described.4Meng F. Han Y. Staloch D. Francis T. Stokes A. Francis H. The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis.Hepatology. 2011; 54: 1718-1728Crossref PubMed Scopus (56) Google Scholar, 5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar Tumor volume was measured as described5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar after establishment of tumors (day 7). Mice were treated with either saline or cromolyn sodium (24 mg/kg of body weight), and measurements were taken every other day with the use of a digital caliper. We measured the expression of EMT markers (paxillin, vimentin, E-cadherin, and s100A4) and ECM degradation markers (MMPs) by real-time PCR in whole tumor mRNA from vehicle- and cromolyn-treated mice as described.4Meng F. Han Y. Staloch D. Francis T. Stokes A. Francis H. The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis.Hepatology. 2011; 54: 1718-1728Crossref PubMed Scopus (56) Google Scholar, 5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar By immunohistochemistry we measured the expression of the epithelial and mesenchymal markers, CK-7, E-cadherin (dilution 1:50; Santa Cruz Biotechnology), and vimentin (dilution 1:200; Cell Signaling Technology, Danvers, MA) in sections from tumors treated with saline and cromolyn sodium as reported.4Meng F. Han Y. Staloch D. Francis T. Stokes A. Francis H. The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis.Hepatology. 2011; 54: 1718-1728Crossref PubMed Scopus (56) Google Scholar By real-time PCR we evaluated the expression of tumor HDC and the HRs (H1 to H4) from vehicle- and cromolyn-treated mice as described.5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar To evaluate the effects of mast cells in vitro, we used the extrahepatic biliary cancer cell line, Mz-ChA-1, derived from human gallbladder,19Knuth A. Gabbert H. Dippold W. Klein O. Sachsse W. Bitter-Suermann D. Prellwitz W. Meyer zum Buschenfelde K.H. Biliary adenocarcinoma. Characterisation of three new human tumor cell lines.J Hepatol. 1985; 1: 579-596Abstract Full Text PDF PubMed Scopus (168) Google Scholar obtained from Dr. G. Fitz (University of Texas Southwestern Medical Center, Dallas, TX) and cultured mast cells (derived from fetal mouse liver) that were obtained from ATCC (ATCC, Manassas, VA). All cultured lines were maintained as described19Knuth A. Gabbert H. Dippold W. Klein O. Sachsse W. Bitter-Suermann D. Prellwitz W. Meyer zum Buschenfelde K.H. Biliary adenocarcinoma. Characterisation of three new human tumor cell lines.J Hepatol. 1985; 1: 579-596Abstract Full Text PDF PubMed Scopus (168) Google Scholar or according to the supplier's protocol. Mast cells were treated with 0.1% bovine serum albumin (basal) or cromolyn sodium (10 μmol/L) for up to 30 minutes, and the conditioned medium was collected and frozen. Mz-ChA-1 cells were then stimulated for 72 hours with the following: 0.1% bovine serum albumin (basal), mast cell supernatant fluids, or supernatant fluids from mast cells treated with 0.1% bovine serum albumin (basal) or cromolyn sodium (10 μmol/L). Total mRNA was extracted as described above, and real-time PCR was performed for HDC, paxillin, and VEGF-C. The conditioned medium from Mz-ChA-1 cells treated as described was collected, and histamine release was measured by electroimmunoassay (Cayman Chemical, Ann Arbor, MI).5Francis H.L. DeMorrow S. Franchitto A. Venter J.K. Mancinelli R.A. White M.A. Meng F. Ueno Y. Carpino G. Renzi A. Baker K.K. Shine H.E. Francis T.C. Gaudio E. Alpini G. Onori P. Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms.Lab Invest. 2012; 92: 282-294Crossref PubMed Scopus (50) Google Scholar By immunofluorescence, we evaluated the expression of c-Kit in cultured mast cells and SCF in Mz-ChA-1 cells as described15Onori P. Wise C. Gaudio E. Franchitto A. Francis H. Carpino G. Lee V. Lam I. Miller T. Dostal D.E. Glaser S.S. Secretin inhibits cholangiocarcinoma growth via dysregulation of the cAMP-dependent signaling mechanisms of secretin receptor.Int J Cancer. 2010; 127: 43-54Crossref PubMed Scopus (37) Google Scholar with the use of specific c-Kit and SCF antibodies from Abcam (Cambridge, MA). Antibodies were diluted 1:100 in phosphate-buffered saline that contained 1% bovine serum albumin. Species-appropriate nonimmune serum was used for negative controls. Images were visualized with an Olympus IX-71 confocal microscope (Tokyo, Japan). To determine whether the effects of mast cells on CCA depend on c-Kit/SCF, we measured mast cell migration toward CCA cells with the use of Boyden Chambers and Transwell inserts.4Meng F. Han Y. Staloch D. Francis T. Stokes A. Francis H. The H4 histamine receptor agonist, clobenpropit, suppresses human cholangiocarcinoma progression by disruption of epithelial mesenchymal transition and tumor metastasis.Hepatology. 2011; 54: 1718-1728Crossref PubMed Scopus (56) Google Scholar Mz-ChA-1 cells were plated into the bottom of the chamber 24 hours before treatment and pretreated with either 0.1% ETOH (basal) or the SCF inhibitor, ISCK03, 1 μmol/L for 30 minutes before mast cells were added to the top of the Boyden Chamber insert. After 48 hours, mast cell presence was detected by toluidine blue staining, and the total percentage of migrating mast cells was manually calculated with a light microscope. We evaluated the effects of inhibition of the c-Kit/SCF interaction on CCA proliferation and angiogenesis, EMT switch, and ECM breakdown by real-time PCR in cells treated as described in Mast Cell Marker Analysis. All data are expressed as means ± SEM. Groups were analyzed by the Student's unpaired t-test when two groups are analyzed, followed by an appropriate post hoc test. P < 0.05 was considered significant. For tumor analysis, we estimated missing values by using missing at random and multiple imputation procedures.20White I.R. Royston P. Wood A.M. Multiple imputation using chained equations: issues and guidance for practice.Stat Med. 2011; 30: 377-399Crossref PubMed Scopus (4906) Google Scholar Predictive mean matching was used to impute volume. We obtained 24 multiple imputed data sets, based on the rule of thumb that the number of imputed sets should approximate the percentage of missing values.21Bodner T.E. What improves with increasing missing data imputations?.Struct Equ Modeling. 2008; 15: 651-675Crossref Scopus (407) Google Scholar We then estimated a multilevel mixed-effects regression model for volume with treatment type and days of treatment as fixed effects and the subject (mouse) as a random-effects variable. The model was fit by the restricted maximum likelihood method, and overall estimates of the regression coefficients, their standard errors, and significance were obtained with Rubin's rules.22Rubin D.B. Multiple Imputation for Nonresponse in Surveys. J Wiley & Sons, New York1987Crossref Google Scholar Both treatment (P = 0.003) and day (P < 0.001) were significant without evidence of a significant day by treatment interaction. Differences in volume between vehicle and cromolyn sodium were compared over days with the use of post-regression joint-hypothesis tests. Statistical analyses were performed with Stata 13.1 (StataCorp LP, College Station, TX). Figure 1A is a representative image of a CCA biopsy positive for toluidine blue staining. The expression of c-Kit, chymase, and tryptase was increased in human CCA biopsies compared with nonmalignant, uninvolved tissue (Figure 1, B and C). In tumors extracted from nude mice treated with histamine, mast cell infiltration increased as shown by toluidine blue staining, whereas in tumors from mice treated with the HDC inhibitor, α-methyl, or cromolyn sodium, no detectable mast cells were found (Figure 2A).
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