title: A novel method for isolation of endothelial cells and macrophages from murine tumors based on Ac-LDL uptake and CD16 expression.
abstract: Analysis of specific properties of tumor endothelium should be useful for development of novel antiangiogenic strategies. However, the isolation of pure endothelial cells from tumor tissues is still a fundamental problem. In this study, we have attempted to develop a reliable method for the isolation of endothelial cells from murine tumors. We found that the labeling with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated-low density lipoprotein (Dil-Ac-LDL), commonly used for this purpose, can result in the contamination of isolated endothelium by macrophages due to the overlapping staining patterns of these two distinct cell types. Therefore, we chose the CD16, which is expressed on macrophages but not endothelial cells, to better distinguish them when labeled with Dil-Ac-LDL. By using this method, we obtained pure populations of endothelial cells and macrophages from murine colorectal cancer tissues, showing characteristic morphological and functional properties of the either cell type. The endothelial cells were long spindle-shaped, spread on gelatin, formed tube-like structures on Matrigel and expressed MECA-32 but not CD68. In contrast, the macrophages were round-shaped, partially spread on gelatin, formed unorganized aggregates on Matrigel and expressed CD68 but not MECA-32. The additional analysis of normal and tumor tissues revealed a positive correlation between the relative numbers of tumor endothelial cells and macrophages, calculated as % total cells, as well as the respective relative number and tumor weight. The present method is hoped to be useful for the evaluation of tumor angiogenesis and antitumor immunity.
authors.affiliation: Department of Surgical Oncology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yurai
authors.initials: Y
authors.lastname: Okaji
authors.forename: Nelson Hirokazu
authors.initials: NH
authors.lastname: Tsuno
authors.forename: Joji
authors.initials: J
authors.lastname: Kitayama
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Saito
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Takahashi
authors.forename: Kazushige
authors.initials: K
authors.lastname: Kawai
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yazawa
authors.forename: Masahiro
authors.initials: M
authors.lastname: Asakage
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sakurai
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Tsuchiya
authors.forename: Katsushi
authors.initials: K
authors.lastname: Tokunaga
authors.forename: Koki
authors.initials: K
authors.lastname: Takahashi
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Nagawa
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D003110:Colonic Neoplasms / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D017452:Receptors, IgG / Q000378:metabolism*
chemical_list: D008077:Lipoproteins, LDL; D017452:Receptors, IgG