Hong-Bo Wei1*. Abstract Coaction of Spheroid-Derived Stem-Like Cells and Endothelial Progenitor Cells Promotes Development of Colon Cancer Bo Wei1., Xiao-Yan Han2, Cui-Ling Qi3., Shi Zhang4, Zong-Heng Zheng1, Yong Huang1, Tu-Feng Chen1, 1 Department of Gastrointestinal Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, 2 Central Laboratory, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, 3 Institute of Vascular Biology, Guangdong Pharmaceutical University, Guangzhou, China, 4 Department of Surgery, the University of Hong Kong, Hong Kong Although some studies described the characteristics of colon cancer stem cells (CSCs) and the role of endothelial progenitor cells (EPCs) in neovascularization, it is still controversial whether an interaction exists or not between CSCs and EPCs. In the present study, HCT116 and HT29 sphere models, which are known to be the cells enriching CSCs, were established to investigate the roles of this interaction in development and metastasis of colon cancer. Compared with their parental counterparts, spheroid cells demonstrated higher capacity of invasion, higher tumorigenic and metastatic potential. Then the in vitro and in vivo relationship between CSCs and EPCs were studied by using capillary tube formation assay and xenograft models. Our results showed that spheroid cells could promote the proliferation, migration and tube formation of EPCs through secretion of vascular endothelial growth factor (VEGF). Meanwhile, the EPCs could increase tumorigenic capacity of spheroid cells through angiogenesis. Furthermore, higher microvessel density was detected in the area enriching cancer stem cells in human colon cancer tissue. Our findings indicate that spheroid cells possess the characteristics of cancer stem cells, and the coaction of CSCs and EPCs may play an important role in the development of colon cancer. Citation: Wei B, Han X-Y, Qi C-L, Zhang S, Zheng Z-H, et al. (2012) Coaction of Spheroid-Derived Stem-Like Cells and Endothelial Progenitor Cells Promotes Development of Colon Cancer. PLoS ONE 7(6): e39069. doi:10.1371/journal.pone.0039069 Editor: Giovanni Camussi, University of Torino, Italy Received December 25, 2011; Accepted May 18, 2012; Published June 26, 2012 Copyright: 2012 Wei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Science Foundation of China (No. 30872462 to Hong-Bo Wei, and No. 81000960 to Bo Wei). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: drweihb@126.com . These authors contributed equally to this work. Introduction Colorectal cancer is the third leading cause of cancer deaths worldwide, and the 5-year relative survival rate is only 53.8–65.2% despite diagnostic and therapeutic advances [1,2]. Tumor recurrence and metastasis are two critical survival-influencing factors of colorectal cancer. Recently, increasing evidence suggests that tumor initiation and metastases are dependent on a small sub-population of tumor cells termed cancer stem cells (CSCs) bearing infinite self-renewal potential and the capacity to differentiate into diverse populations comprising a tumor [3]. According to this model, cancer stem cells were found to sustain carcinogenesis, metastasis, and recurrence of colorectal cancer. The existence of cancer stem cells was first proven in the context of acute myeloid leukemia [4]. This principle was further extended to some solid tumors, including colon cancer, breast cancer, brain tumors, and lung cancer [5,6,7,8,9,10]. Cell sorting of a sub- population on the basis of cell surface markers and confirmation of their tumor-initiating activity in xenograft transplantation assays are commonly used procedures for isolation and identification of CSCs from tumor tissues or cell lines [11]. Although CD133, CD44, EpCAM were used extensively as cell surface markers for colon cancer stem cells, there were still doubts on these cell surface markers [12,13]. Side population (SP) cells isolation were once used to enrich the cancer stem cells. But several studies showed evidence against an association between the SP cells and cancer stem cells [14,15,16,17]. Recently, increasing researches have reported that the non-adherent, three-dimensional (3D) tumor spheres under serum-free conditions could efficiently enrich cancer stem cells [18,19,20] in vitro. Therefore, this method was applied to enrich colon cancer stem cells in the present study. Endothelial progenitor cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, originate mainly from the bone marrow-derived cells with the ability to differentiate into mature endothelial cells [21,22]. EPCs leave the bone marrow and recruit to sites requiring vascular repair following gradients of growth factors and cytokines that are released into the circulation by injured tissues or tumors, and then contribute to blood vessel formation [23,24]. Recent evidences demonstrated that circulating EPCs play an important role in tumor neovascularization, tumor growth, and metastasis [25,26,27,28,29]. Although the contribution of EPCs to tumor neovascularization has been demonstrated in several types of cancer, the interaction between EPCs and colon cancer stem cells have not been reported. The aim of this study was to determine the bionomics of colon PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39069