title: Characterization of in vivo chemoresistant human hepatocellular carcinoma cells with transendothelial differentiation capacities.
abstract: BACKGROUND
  Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model.
  
  
  METHODS
  SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel.
  
  
  RESULTS
  HUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression.
  
  
  CONCLUSIONS
  Chemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.
authors.affiliation: Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität, Butenandtstrasse 5-13, D-81377, Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Marfels
authors.forename: Miriam
authors.initials: M
authors.lastname: Hoehn
authors.forename: Ernst
authors.initials: E
authors.lastname: Wagner
authors.forename: Michael
authors.initials: M
authors.lastname: Günther
mesh_terms: D059250:Administration, Metronomic; D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D054338:Cell Transdifferentiation; D004195:Disease Models, Animal; D019008:Drug Resistance, Neoplasm; D042783:Endothelial Cells / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D008297:Male; D051379:Mice; D000071317:Nanog Homeobox Protein; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D051881:Receptor, Notch1 / Q000235:genetics; D050697:Retinal Dehydrogenase / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics; D018874:Spheroids, Cellular; D018800:Thy-1 Antigens / Q000235:genetics; D000072056:Transcription Factor HES-1; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000080924:Aldehyde Dehydrogenase 1 Family; D000970:Antineoplastic Agents; D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D007527:Isoenzymes; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D051881:Receptor, Notch1; D050697:Retinal Dehydrogenase; D055748:SOXB1 Transcription Factors; D018800:Thy-1 Antigens; D000072056:Transcription Factor HES-1; C486780:NANOG protein, human; C497761:NOTCH1 protein, human; C078493:HES1 protein, human; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse
references.citation: N Engl J Med. 2008 Jul 24;359(4):378-90
references.pmid: 18650514
references.citation: Expert Rev Anticancer Ther. 2009 Apr;9(4):503-9
references.pmid: 19374603
references.citation: J Cell Mol Med. 2009 Feb;13(2):309-319
references.pmid: 18410528
references.citation: Mol Cancer Ther. 2007 Aug;6(8):2280-9
references.pmid: 17671082
references.citation: Cancer Gene Ther. 2006 Aug;13(8):771-9
references.pmid: 16543915
references.citation: Cancer Chemother Pharmacol. 2010 May;65(6):1029-37
references.pmid: 19701751
references.citation: Mol Cancer. 2009 Jun 09;8:35
references.pmid: 19508709
references.citation: Development. 2001 Oct;128(19):3675-83
references.pmid: 11585794
references.citation: Cell. 1989 Sep 8;58(5):933-43
references.pmid: 2528412
references.citation: Dev Cell. 2002 Jul;3(1):127-36
references.pmid: 12110173
references.citation: Acta Histochem. 2011 Dec;113(8):833-8
references.pmid: 21272924
references.citation: Am J Pathol. 2006 Dec;169(6):2223-35
references.pmid: 17148683
references.citation: Am J Pathol. 2001 Oct;159(4):1567-79
references.pmid: 11583982
references.citation: Cell. 2005 Sep 23;122(6):947-56
references.pmid: 16153702
references.citation: Mol Cells. 2000 Feb 29;10(1):1-7
references.pmid: 10774739
references.citation: Nat Rev Cancer. 2006 Sep;6(9):674-87
references.pmid: 16929323
references.citation: J Proteomics. 2010 May 7;73(7):1342-54
references.pmid: 20219715
references.citation: Cancer Res. 2008 Jun 1;68(11):4311-20
references.pmid: 18519691
references.citation: Drug Resist Updat. 2003 Jun;6(3):111-27
references.pmid: 12860459
references.citation: Int J Urol. 2006 Jan;13(1):47-57
references.pmid: 16448432
references.citation: Annu Rev Med. 2006;57:1-18
references.pmid: 16409133
references.citation: Brain. 2010 Apr;133(Pt 4):973-82
references.pmid: 20375132
references.citation: Cancer Lett. 2006 Oct 28;242(2):151-67
references.pmid: 16564617
references.citation: Cancer Res. 2007 May 15;67(10):4807-15
references.pmid: 17510410
references.citation: Cancer Res. 2000 Apr 1;60(7):1878-86
references.pmid: 10766175
references.citation: Breast Cancer Res. 2006;8(6):R69
references.pmid: 17156488
references.citation: Stem Cells. 2004;22(2):225-35
references.pmid: 14990861
references.citation: Oncogene. 2008 Jan 24;27(5):663-74
references.pmid: 17667938