title: Use of multiple biomarkers for the localization and characterization             of colon cancer stem cells by indirect immunocytochemistry.
abstract: In this study, we used LGR5, γ-synuclein, p53, KRAS and epiregulin antibodies to localize stem cells by indirect immunocytochemistry in paraffin sections of normal and cancerous colon tissues. In the normal colon tissue, no staining of cells with LGR5, γ-synuclein, p53 and KRAS antibodies was observed, apart from a few scattered cells in between the colon villi that were faintly stained with antibodies to LGR5. Staining of highly differentiated cancer tissue with LGR5 antibodies revealed single cells or clusters of up to 4 cells in the interior space of the carcinoma cell layers. Staining of poorly differentiated cancer tissues (stage I-IV) revealed 9-81 clustered stem cells. The number of clustered stem cells increased significantly with the tumor stage, when comparing stage II to stage IV (p<00048). Occasionally, the clustered stem cells appeared in the interphase between the colon stroma and the tumor tissue. Surprisingly, antibodies to p53 clearly stained the clusters of stem cells both in the nuclei and the cytoplasm. The staining of the nuclei of other cells in the undifferentiated tumors was in general weaker, and no staining was found in the cytoplasm. Antibodies to γ-synuclein heavily stained the endothelial cells of the blood vessels and some other scattered cells in the highly differentiated tumors. Antibodies to γ-synuclein heavily stained the stem cells in both the cytoplasm and the nuclei of poorly differentiated tumors. Antibodies to KRAS stained the cytoplasm and the nuclei of stem cells in poorly differentiated tumors and also stained the cytoplasm of some scattered cells. Antibodies to epiregulin stained the cytoplasm of normal colon tissue cells in the crypt-villus axis. The antibodies weakly stained the highly differentiated tumor cells and moderately stained the moderately differentiated tumor cells. Of note, the antibodies intensively stained the clustered stem cells of the poorly differentiated tumor cells. These antibodies also clearly stained the clustered stem cells of poorly differentiated tumors but were not specific as they clearly stained cells in the crypt-villus axis of the normal colon wall. Our results show that LGR5 antibodies can serve as a reliable marker for colon cancer stem cells. Once the colon stem cells are identified, the targeting of specific drugs to kill these cells should be attempted in the future in order to cure this disease. Moreover, the fact that we did not find any stained cells with antibodies to LGR5 in normal tissues apart from a few scattered cells, suggests that the normal colon stem cells differ from the tumor stem cells at least as regards the expression of this protein. In addition, antibodies to γ-synuclein, p53 and KRAS only stained the tumor stem cells and not the normal tissue. Thus, they can serve as multiple biomarkers for the localization of colon cancer stem cells by indirect immunofluorescence.
authors.affiliation: Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel. abraham.amsterdam@weizmann.ac.il
authors.forename: Abraham
authors.initials: A
authors.lastname: Amsterdam
authors.forename: Calanit
authors.initials: C
authors.lastname: Raanan
authors.forename: Letizia
authors.initials: L
authors.lastname: Schreiber
authors.forename: Ora
authors.initials: O
authors.lastname: Freyhan
authors.forename: Yakov
authors.initials: Y
authors.lastname: Fabrikant
authors.forename: Ehud
authors.initials: E
authors.lastname: Melzer
mesh_terms: D003106:Colon / Q000378:metabolism / Q000473:pathology; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D004815:Epidermal Growth Factor / Q000378:metabolism; D066260:Epiregulin; D039621:Eukaryotic Initiation Factor-3 / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051845:gamma-Synuclein / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
chemical_list: C105988:EIF3A protein, human; C587880:EREG protein, human; D066260:Epiregulin; D039621:Eukaryotic Initiation Factor-3; C117307:KRAS protein, human; C116884:LGR5 protein, human; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051845:gamma-Synuclein; D004815:Epidermal Growth Factor; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins