title: Establishment and characterization of cell lines from chromosomal instable colorectal cancer.
abstract: OBJECTIVE
  To generate novel tumor models for preclinical validation of biomarkers that allow drug response prediction and individual therapeutic decisions.
  
  
  METHODS
  Cell line establishment was conducted by both direct in vitro culturing and in vivo xenografting followed by in vitro culturing procedure. A comprehensive characterization was subsequently performed. This included quality control, consisting of the confirmation of human and colorectal cancer (CRC) origin by DNA fingerprint and epithelial cell adhesion molecule (EpCAM) staining, as well as mycoplasma and human virus testing. Phenotypic analysis was done by light microscopy and multicolor flow cytometry. Histopathological examination (β-catenin and cytokeratin staining) was conducted in direct comparison to parental tumor tissues. Extensive molecular-pathological profiling included mutation analysis for CRC-associated driver mutations, assessment of chromosomal and microsatellite instability, and the grade of CpG island methylation. Additionally, an array-based comparative genomic hybridization analysis was performed. Drug responsiveness was assessed for a panel of classical and novel substances in clinical use for the treatment of solid cancers. Finally, tumorigenicity of the cell lines was tested by xenografting into immunocompromised nude mice.
  
  
  RESULTS
  Herein we describe the establishment of three ultra-low passage cell lines from two individual patients suffering from sporadic CRC. One cell line was derived directly from an early stage case (HROC18), whereas two cell lines could be established both direct from patient material and after xenografting from a late stage tumor (HROC32). All cell lines were free of contaminating mycoplasma and viruses. Molecular-pathological analysis allowed all cell lines to be classified as chromosomal instable (CIN(+)). They were aneuploid, with CpG island promoter methylation and microsatellite instability being absent. The following mutational profile was observed both in the cell lines and the parental tumor tissue: HROC18: APC(mut), p53(mut), K-ras(wt); HROC32: APC(wt), p53(mut), K-ras(mut). All cell lines were characterized as epithelial (EpCAM(+)) cells, showing distinct morphology and migration speed, but comparable growth kinetics. The cell lines showed different patterns of response towards clinically approved and novel drugs, with HROC18 being more resistant than HROC32 cells. Finally, in vivo tumorigenicity was demonstrated.
  
  
  CONCLUSIONS
  We successfully established and characterized novel ultra-low passage patient-derived CRC models as useful instruments for analyzing biological characteristics associated with the CIN(+) phenotype.
authors.affiliation: Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Maletzki
authors.affiliation: Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Gock
authors.affiliation: Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Randow
authors.affiliation: Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
authors.forename: Ernst
authors.initials: E
authors.lastname: Klar
authors.affiliation: Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
authors.forename: Maja
authors.initials: M
authors.lastname: Huehns
authors.affiliation: Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Prall
authors.affiliation: Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Linnebacher
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D048430:Cell Shape / Q000187:drug effects; D043171:Chromosomal Instability; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D055028:Comparative Genomic Hybridization; D018899:CpG Islands; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm; D044127:Epigenesis, Genetic; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D020022:Genetic Predisposition to Disease; D006801:Humans; D020128:Inhibitory Concentration 50; D008819:Mice, Nude; D053842:Microsatellite Instability; D009154:Mutation; D009361:Neoplasm Invasiveness; D009367:Neoplasm Staging; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D013997:Time Factors; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor
keywords: Colorectal cancer; Drug response; Individualized medicine; Molecular characterization; Patient-derived tumor model; Translational research