Abstract 4587: In vitro and In vivo characterization of circulating tumor cells from breast cancer patients.
Authors: Jalla Sanjoo , Chen Xiaoru , Burke Patricia , Shi Xiaoqing , Czapiga Meggan , Datta Vivekananda , Brohawn Philip , Huang Jiaqi , Brown Charles , Hurt Elaine , Richman Laura , Hollingsworth Robert , LaVallee Theresa , Bao Haifeng 
Published: August 2014

Abstract
Circulating tumor cells (CTCs) are an independent prognostic marker associated with poor clinical outcome in patients with metastatic diseases. However, CTC biology, especially the tumorigenic and metastatic activity of CTCs, is not yet well understood. In this study, we hypothesized that CTCs contain a population of cancer stem cells (CSCs) that are the seed cells responsible for metastases. To test this hypothesis, we sought to assess subpopulations of CTCs in breast cancer patients and to characterize CTCs for CSC properties and tumorigenic potential. For assessment of CTC subsets, blood samples from breast cancer patients were enriched for CTCs by magnetic separation. The enriched CTC samples were immunostained for various cell surface markers and then analyzed by FACS. The phenotypes of CTCs were further confirmed by imaging analysis. To evaluate tumorigenicity of CTCs, CTC samples from patients were cultured in vitro and then implanted into immunodeficient mice by orthotopic injection. Different phenotypes of CTCs were found in breast cancer patients, including EpCAM+CD44−CD24−/dimCD45−, EpCAM+CD44+CD24−/dimCD45−, EpCAM+CD44+CD24+CD45−, and EpCAM+CD44+CD24+CD45dim. Of these phenotypes, EpCAM+CD44+CD24−/dim cells have been reported as CSCs in breast cancer. This subset of CSC-like CTCs constituted 4.6% to 71% of the total CTC population. In vitro culture of CTCs resulted in the generation of mammospheres, as is typical of CSCs. During in vitro culture, some of CTCs showed staining positive for Ki-67, indicating that these cells were proliferating. To assess tumorigenic potential of CTCs, cultured CTCs were injected into the mouse mammary fat pads of immunodeficient mice. Human tumors were developed in these mice. Further analysis of the CTC-derived tumor xenografts demonstrated a heterogenous phenotype where EpCAM+CD44+CD24dim/− CSCs accounted for less than 3% of the total tumor cells. Only this subset of cells isolated from CTC-derived xenograft tumors could form secondary tumors after being reimplanted into mice. Our results support the hypothesis that CTCs contain a CSC-like population that may initiate new tumors. As a result, CTCs may be a potential therapeutic target for the prevention of metastasis in addition to serving as a readily accessible source for biomarker evaluation of new therapies in cancer patients.
Citation Format: Sanjoo Jalla, Xiaoru Chen, Patricia Burke, Xiaoqing Shi, Meggan Czapiga, Vivekananda Datta, Philip Brohawn, Jiaqi Huang, Charles Brown, Elaine Hurt, Laura Richman, Robert Hollingsworth, Theresa LaVallee, Haifeng Bao. In vitro and In vivo characterization of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4587. doi:10.1158/1538-7445.AM2013-4587