title: Detection of ALDH1 activity in rabbit hepatic VX2 tumors and isolation of ALDH1 positive cancer stem cells.
abstract: BACKGROUND
  Aldehyde dehydrogenase 1 (ALDH1) activity has been implicated in the therapeutic drug resistance of many malignancies and has been widely used as a marker to identify stem-like cells, including in primary liver cancer. Cancer stem cells (CSCs) are thought to play a crucial role in cancer progression and metastasis. In order to clarify the validity of the rabbit VX2 liver cancer model, we questioned if it expresses ALDH1 as a potential marker of CSCs. Hepatocellular carcinoma is a common malignancy worldwide and has poor prognosis. Most of the animal models used to study hepatocellular carcinoma are rodent models which lack clinical relevance. The rabbit VX2 model is a large animal model useful for preclinical and for developing drugs targeting cancer stem cells.
  
  
  METHODS
  We used flow cytometry to identify rabbit VX2 liver tumor cells that express ALDH1A1 activity at a high level and confirmed the results with RT-PCR, immunohistochemical and western blot analyses. Further, mRNA and protein expression analysis of tumor samples also express the markers for stemness like klf4, oct3/4, CD44 and nanog as well as the differentiation marker α-fetoprotein.
  
  
  RESULTS
  We used Aldefluor flow cytometry-based assay to identify cells with high ALDH1 activity in the rabbit VX2 liver cancer model. We used the brightest 4.39 % of the total cancer cell population in our study. We performed semi-quantitative as well as real time PCR to characterize the stemness derived from VX2 tumors and tissues from normal rabbit liver. We demonstrated that VX2 tumors have higher expression of cancer stem cell markers such as AlDH1A1 and CD44 in comparison to normal rabbit liver cells. Additionally, real time PCR analysis of the same samples using syber-green demonstrated the significant change (p > 0.05) in the expression of genes. We validated the gene expression of the stemness markers by performing western blot and immunofluorescence. We showed that cancer stem cell markers (AlDH1A1, CD44) and the differentiation marker α-fetoprotein were upregulated in VX2 tumor cells. The same extent of upregulation was observed in stemness markers (klf4, oct3/4 and nanog) in VX2 tumors in comparison to normal rabbit liver.
  
  
  CONCLUSIONS
  The overall results of this study indicate that ALDH1 is a valid CSC marker for VX2 cancer. This finding suggests that the rabbit VX2 liver cancer model is useful in studying drug resistance in hepatocellular carcinoma and may be useful for basic and preclinical studies of other types of human cancer.
authors.affiliation: Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. pgehlot@med.umich.edu.
authors.forename: Prashasnika
authors.initials: P
authors.lastname: Gehlot
authors.affiliation: TGIB Branch, NCI, Betehsda, MD, USA. yashvita@gmail.com.
authors.forename: Vivek
authors.initials: V
authors.lastname: Shukla
authors.affiliation: Department of Diagnostic Radiology-Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. sgupta@mdanderson.org.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Gupta
authors.affiliation: The Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, MI, 48109, USA. Pmakidon@umich.edu.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Makidon
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D015153:Blotting, Western; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D007527:Isoenzymes / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D008113:Liver Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D050697:Retinal Dehydrogenase / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D012333:RNA, Messenger; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase