title: Identification of colorectal cancer metastasis markers by an angiogenesis-related cytokine-antibody array.
abstract: OBJECTIVE
  To investigate the angiogenesis-related protein expression profile characterizing metastatic colorectal cancer (mCRC) with the aim of identifying prognostic markers.
  
  
  METHODS
  The expression of 44 angiogenesis-secreted factors was measured by a novel cytokine antibody array methodology. The study evaluated vascular endothelial growth factor (VEGF) and its soluble vascular endothelial growth factor receptor (sVEGFR)-1 protein levels by enzyme immunoassay (EIA) in a panel of 16 CRC cell lines. mRNA VEGF and VEGF-A isoforms were quantified by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) and vascular endothelial growth factor receptor (VEGFR)-2 expression was analyzed by flow cytometry.
  
  
  RESULTS
  Metastasis-derived CRC cell lines expressed a distinctive molecular profile as compared with those isolated from a primary tumor site. Metastatic CRC cell lines were characterized by higher expression of angiogenin-2 (Ang-2), macrophage chemoattractant proteins-3/4 (MCP-3/4), matrix metalloproteinase-1 (MMP-1), and the chemokines interferon γ inducible T cell α chemoattractant protein (I-TAC), monocyte chemoattractant protein I-309, and interleukins interleukin (IL)-2 and IL-1α, as compared to primary tumor cell lines. In contrast, primary CRC cell lines expressed higher levels of interferon γ (IFN-γ), insulin-like growth factor-1 (IGF-1), IL-6, leptin, epidermal growth factor (EGF), placental growth factor (PlGF), thrombopoietin, transforming growth factor β1 (TGF-β1) and VEGF-D, as compared with the metastatic cell lines. VEGF expression does not significantly differ according to the CRC cellular origin in normoxia. Severe hypoxia induced VEGF expression up-regulation but contrary to expectations, metastatic CRC cell lines did not respond as much as primary cell lines to the hypoxic stimulus. In CRC primary-derived cell lines, we observed a two-fold increase in VEGF expression between normoxia and hypoxia as compared to metastatic cell lines. CRC cell lines express a similar pattern of VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅ and VEGF₁₈₉) despite variability in VEGF expression, where the major transcript was VEGF₁₂₁. No relevant expression of VEGFR-2 was found in CRC cell lines, as compared to that of human umbilical vein endothelial cells and sVEGFR-1 expression did not depend on the CRC cellular origin.
  
  
  CONCLUSIONS
  A distinct angiogenesis-related expression pattern characterizes metastatic CRC cell lines. Factors other than VEGF appear as prognostic markers and intervention targets in the metastatic CRC setting.
authors.affiliation: Laboratory of Pharmacogenomics, Division of Oncology, Center for Applied Medical Research, University of Navarra, Avda Pio XII 55, 31008 Pamplona, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Abajo
authors.forename: Nerea
authors.initials: N
authors.lastname: Bitarte
authors.forename: Ruth
authors.initials: R
authors.lastname: Zarate
authors.forename: Valentina
authors.initials: V
authors.lastname: Boni
authors.forename: Ines
authors.initials: I
authors.lastname: Lopez
authors.forename: Marisol
authors.initials: M
authors.lastname: Gonzalez-Huarriz
authors.forename: Javier
authors.initials: J
authors.lastname: Rodriguez
authors.forename: Eva
authors.initials: E
authors.lastname: Bandres
authors.forename: Jesus
authors.initials: J
authors.lastname: Garcia-Foncillas
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000378:metabolism* / Q000473:pathology*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D046228:Microarray Analysis / Q000379:methods; D009362:Neoplasm Metastasis / Q000473:pathology / Q000503:physiopathology*; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D014408:Biomarkers, Tumor; D016207:Cytokines; D020033:Protein Isoforms; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2