Enrichment and molecular characterization of single disseminated tumor cells (DTCs) and proof of circulating tumor cell (CTC)- and DTC-mediated tumorigenicity in vitro and in vivo

多年来，已经进行了各种尝试来开发能够从实体癌患者的血液和骨髓中检测和分离循环(CTC)或播散性肿瘤细胞(DTC)的技术(见综述：Pantel K等人，NAT Rev Cancer 2008)。重要的是，我们小组最近的一项系统回顾和荟萃分析证实了结直肠癌(CRC)中CTC的潜在预后相关性(Rahbari，Aigner等人，手稿准备中)。然而，分析揭示了主要的研究间变异性，这可能反映在这样一个事实上，即这些细胞构成一个具有部分类似癌症干细胞(CSC)的异质细胞库，即启动癌症的特性。CSCs是一种罕见的能够自我更新、分化和增殖的肿瘤成分(Reya T等人，《自然》2001年)；在异种移植到免疫缺陷小鼠后，这些细胞重建了原始肿瘤S的形态(O Brien CA等人，《自然》2007年；Ricci-Vitiani L等人。《自然》2007)。因此，这一分项目的重点和目标是更好地确定四氯化碳和二氯甲烷的表型特性。因此，我们首先计划从结直肠癌患者中分离出CTC和DTC，然后从两个方面对其进行表型特征：(I)与CRC干性相关的标记(例如，GD133[O Brien CA等，自然2007；和(Ii)参与上皮-间充质转化的标记物(如E-钙粘蛋白、N-钙粘蛋白、波形蛋白、纤维连接蛋白[Mani SA等人，细胞2008]和扭转[Ansieau S等人，2008]。癌症细胞2008；杨明华等人，NAT Cell Biol 2008；Valdes-Mora F等人，Ann Surg Oncol2008])。此外，新分离的DTC注射到免疫缺陷小鼠的肾被膜中的生长应该作为我们的假设的证据，即CTC和DTC组分包含具有致癌能力的癌症干细胞。总而言之，我们的数据不仅应该揭示转移形成的早期步骤的新见解，而且我们的结果与接受检查的患者的临床随访(参见C-Project)的相关性也可能有助于评估循环中的癌症启动细胞(CIC)的预后相关性。

For many years, various attempts have been made to develop techniques enabling to detect and isolate circulating (CTC) or disseminated tumor cells (DTC) from the blood and bone marrow of patients with solid cancer (see review: Pantel K et al., Nat Rev Cancer 2008). Importantly, a recent systematic review and meta-analysis from our group corroborate the potential prognostic relevance of CTC in colorectal cancer (CRC) (Rahbari, Aigner et al., manuscript in preparation). The analysis, however, reveals major inter-study variability, which may be reflected by the fact that these cells constitute a heterogeneous cell pool with partly cancer stem cell (CSC)-like, i. e. cancer-initiating, properties. CSCs constitute a rare tumor fraction which are able to self-renew, to differentiate and to proliferate (Reya T et aL, Nature 2001); following xenotransplantation into immunodeficient mice, these cells re-establish the original tumor s morphology (O Brien CA et al., Nature 2007; Ricci-Vitiani L et al.. Nature 2007). Thus, the focus and aim of this subproject is to better define the phenotypic properties of CTC and DTC. Therefore, we first plan to isolate and then to phenotypically characterize CTC and DTC from patients with CRC with regard to both (i) markers associated with CRC stemness (e. g. GD133 [O Brien CA et al., Nature 2007; Ricci-Vitiani L et al., Nature 2007], CD166 [Dalerba P et al., PNAS 2007], CD44 [Du L et al., Clin Cancer Res 2008]) and (ii) markers involved in epithelial-mesenchymal transition (EMT, e. g. E-cadherin, N-cadherin, vimentin, fibronectin [Mani SA et al., Cell 2008] and Twist [Ansieau S et al.. Cancer Cell 2008; Yang MH et al., Nat Cell Biol 2008; Valdes-Mora F et al., Ann Surg Oncol 2008]). Furthermore, the outgrowth of freshly isolated DTC injected into the renal capsule of immunodeficient mice should serve as a proof for our hypothesis that the CTC and DTC fraction contains cancer stem cells with cancer-initiating capacities. In conclusion, our data should not only reveal new insights into the early steps of metastasis formation, but correlation of our results with the clinical follow-up of the examined patients (see also C-Project) may also help to assess the prognostic relevance of circulating cancer-initiating cells (CIC).