The radiosensitivity of endothelial cells isolated from human breast cancer and normal tissue in vitro Moon-Taek Park a, 1 , Eun-Taex Oh a, 1 , Min-Jeong Song a , Woo-Jean Kim b , Young Up Cho c , Sei Joong Kim c , Jee-Young Han d , Jun-Kyu Suh b , Eun Kyung Choi e , Byung Uk Lim a , Chang Won Song f , Heon Joo Park a, ⁎ a Department of Microbiology, Center for Advanced Medical Education by BK21 Project, College of Medicine, Inha University, Incheon 400–712, Republic of Korea b Department of Urology and National Research Laboratory of Regenerative Sexual Medicine, College of Medicine, Inha University, Incheon 400–712, Republic of Korea c Department of Surgery, College of Medicine, Inha University, Incheon 400–712, Republic of Korea d Department of Pathology, College of Medicine, Inha University, Incheon 400–712, Republic of Korea e Department of Radiation Oncology, Asan Medical Center, College of Medicine, University of Ulsan, Seoul 138–736, Republic of Korea f Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, Minnesota, USA abstract article info Article history: Accepted 4 June 2012 Available online 12 June 2012 We developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and compared the response of the cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) to ionizing radiation. In brief, when tissues were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth fac- tors, endothelial cells grew out of the tissues. The endothelial cells were harvested and cultured as monolayer cells in plates coated with gelatin, and the cells of 2nd–5th passages were used for experiments. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothe- lial cells. Furthermore, both CECs and NECs were able to migrate into experimental wound in the monolayer culture, and also to form capillary-like tubes on Matrigel-coated plates. However, the radiation-induced sup- pressions of migration and capillary-like tube formations were greater for CECs than NECs from the same pa- tients. In addition, in vitro clonogenic survival assays demonstrated that CECs were far more radiosensitive than NECs. In summary, we have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue, and demonstrated for the first time that endothelial cells of human breast cancer are significantly more radiosensitive than their normal counterparts from the same patients. © 2012 Elsevier Inc. All rights reserved. Introduction In human and animal tumors, nutrients including oxygen must be supplied through vascular networks in order for tumor cells to survive and proliferate (Folkman, 1995; Hida et al., 2004). Tumor blood vessels are formed by three processes: angiogenesis, growth of new blood vessels from pre-existing vessels; vasculogenesis, formation of blood vessels from endothelial progenitors and other stem-like cells found in the blood and bone marrow, and intussusception, the splitting of new vessels off exis- ting vessels (Ahn and Brown, 2009; Folkman, 1995; Hida et al., 2004; Kim et al., 2010). In contrast to the vascular beds of normal tissues which are hierarchically organized microarchitectures formed of mature vessels, tumor vasculature is structurally tortuous, and the vessels are often formed from only a single layer of endothelial cells frequently sepa- rated by gaps and lack underlying basement membrane and smooth mus- cle layers. These immature tumor vessels are hyper-permeable leading to an increase in extravasation of plasma protein (Konerding et al., 2001; Song and Levitt, 1971). Compressive stresses generated by cancer cells and other stromal components can cause collapse of microvasculature in the tumor (Boucher and Jain, 1992; Padera et al., 2004). Consequently, the intratumor microenvironment is hypoxic, nutritionally deprived and acidic (Choi et al., 2004; Lunt et al., 2009; Park et al., 2000; Vaupel et al., 1987), which greatly affects the response of tumor cells to various treat- ments such as radiotherapy or certain chemotherapies. Because of the importance of the functional integrity of blood vessels for the survival, proliferation and radioresponse of tumor cells, the changes in vascular functions in human tumors after irradiation have been studied by a Microvascular Research 84 (2012) 140–148 Abbreviations: EC, endothelial cell; HUVEC, human umbilical-vein endothelial cell; CECs, cancer-derived endothelial cells; NECs, normal tissue-derived endothelial cells; HDMEC, human dermal microvascular endothelial cell; TEM, tumor endothelial mark- er; SMA, α-smooth muscle actin; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; FACS, fluorescence-activated cell sorting; ECM, endothelial cell medium; PBS, phosphate buffered saline. ⁎ Corresponding author at: Department of Microbiology, Inha University College of Medicine, Jungsuck B/D B-Dong 3F, 7–241, 3rd street, Shinheung-Dong, Jung-Gu, Incheon, 400–712, Republic of Korea. Fax: +82 32 881 8559. E-mail address: park001@inha.ac.kr (H.J. Park). 1 Note: Moon-Taek Park and Eun-Taex Oh contributed equally to this work. 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2012.06.002 Contents lists available at SciVerse ScienceDirect Microvascular Research journal homepage: www.elsevier.com/locate/ymvre